Although there is considerable interest in the use of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) to promote periodontal regeneration, little is known of its effects on the early stages of wound healing. The aim of this study was to investigate the effects of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (rhBMP-2) on an early stage of post-operative wound healing and following complete healing (<em>10</em> and 38 days, respectively) in a rat model of periodontal regeneration. The buccal aspects of molar roots were carefully denuded of their periodontal ligament through a bony window created in the mandibles of Wistar rats under general anesthesia. After the root surfaces were acid-conditioned, a <em>10</em>-microL quantity of 50 microg/mL rhBMP-2 in a collagen gel solution was placed into the surgically created defect in test animals; in controls, either a <em>10</em>-microL quantity of only collagen gel was received, or the defect was untreated. Animals were killed <em>10</em> days or 38 days after surgery and the tissues processed for histological examination. Transverse 5-microm sections were stained for the identification of new <em>bone</em>, cementum, and collagen fiber formation. In the <em>10</em>-day study groups, new <em>bone</em> formation over the second molar and beyond the defect was significantly increased in the test group (p < 0.02), although there was no evidence of increased ankylosis. RhBMP-2 stimulated more than twice the area of cementum growth coronally compared with controls (712 +/- 286 microm2 and 258 +/- 57 microm2, respectively). Connective tissue attachment, including the number and width of collagen bundles, was similar in both test and controls. Complete healing without any evidence of ankylosis had occurred in all animals 38 days post-operatively, and no significant differences were observed between test and control groups. In conclusion, a single dose of rhBMP-2 increased the rate of normal intramembranous <em>bone</em> formation and selectively enhanced cementum formation coronally during early wound healing. However, the finding that rhBMP-2 induced <em>bone</em> formation at some distance from the defect suggests the importance of developing a suitable delivery system to maintain the concentration of BMP-2 at the site of implantation for potential therapeutic use.

OBJECTIVE

Vascular calcification is an active deposition process of calcium phosphate which resembles bone formation and is highly regulated by osteoblast-like cells. Existing studies demonstrate that advanced glycation end-products (AGEs) may play a pathogenic role in the vascular calcification process. However, their mechanism remains poorly understood. The aim of our current study is to investigate how non-cross-link and non-fluorescent N(ε)-carboxymethyl-Lysine (CML), a major immunogen of AGEs, affect the progression of atherosclerotic calcification in diabetes.

METHODS

The present study consisted of an in vivo investigation and two in vitro investigations. In study I, male apoE(-/-) mice were first rendered diabetic by the administration of 5 daily intraperitoneal injections of streptozotocin (STZ, 40 mg/kg), and then given a semi-synthetic high-fat diet (HFD) plus daily injections of CML (10mg/kg/day). The mice were euthanized and analyzed at 0 month (group 0M, n = 10), 2 months (group 2M, n = 10), and 4 months (group 4M, n = 10) after the triple administrations of STZ-CML-HFD. In study II, the effects of CML on the apoptosis in macrophages were investigated. RAW264.7 cells were incubated with or without 50 μg/mL oxLDL plus various concentrations of CML for 48 h. In study III, we investigated whether A7r5 aortic smooth muscle cells were induced into osteoblast-like phenotypes by incubation with or without 80 μg/mL of RAW264.7-derived-apoptotic bodies and 50 μg/mL of oxLDL plus various concentrations of CML (or high-glucose) for 7 days. Related analyses (i.e., H&E staining, Masson staining, von Kossa staining, TUNEL staining, immunohistochemical staining, calcium content assay, annexin V-FITC/PI double-staining, and Western blot) were performed.

RESULTS

Morphological analysis showed that early atherosclerotic plaques appeared 2 months after the triple administrations of STZ-CML-HFD, and that typically advanced plaques with extensive calcification lesions, abundant cholesterol crystals, and proliferative collagen were formed 4 months after the triple administrations of STZ-CML-HFD. Furthermore, CML deposition signals and the expression of receptor for advanced glycation end-products (RAGE) in the aortic wall were mainly restricted in the atherosclerotic plaques. After the incubation of A7r5 smooth muscle cells with 10 μmol/L CML plus 50 μg/mL oxLDL, and 80 μg/mL apoptotic bodies (ABs) for 7 days, semi-quantitative analysis of bone morphogenetic protein 2 (BMP-2), core-binding factor α1 (cbfα1), and alkaline phosphatase (ALP) expression showed 5.0-, 2.0-, and 2.9-fold increases, respectively, compared with those in 50 μg/mL oxLDL and 80 μg/mL ABs. Subsequently, a similar trend was observed in the calcium deposition of the cell layer. However, high-glucose had no effects on the ALP activity and calcium deposition of A7r5 cell layer under high-lipid, apoptosis-coexisting conditions. Both animal and cell studies consistently demonstrated that the CML/RAGE axis may first initiate the apoptosis of macrophages in atherosclerotic lesions and then induce BMP-2-cbfα1-ALP-calcification cascade in a high-lipid, apoptosis-coexisting environment.

CONCLUSIONS

The CML/RAGE axis may play an important role in atherosclerotic calcification of diabetes through the mechanism that induces the apoptosis of macrophages followed by the osteogenic differentiation of aortic smooth muscle cells.

Tooth-supporting periodontium forms a complex with multiple tissues, including cementum, periodontal ligament (PDL), and alveolar <em>bone</em>. In this study, we developed multiphase region-specific microscaffolds with spatiotemporal delivery of bioactive cues for integrated periodontium regeneration. Polycarprolactione-hydroxylapatite (90:<em>10</em> wt%) scaffolds were fabricated using three-dimensional printing seamlessly in three phases: <em>10</em>0-μm microchannels in Phase A designed for cementum/dentin interface, 600-μm microchannels in Phase B designed for the PDL, and 300-μm microchannels in Phase C designed for alveolar <em>bone</em>. Recombinant human amelogenin, connective tissue growth factor, and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 were spatially delivered and time-released in Phases A, B, and C, respectively. Upon 4-week in vitro incubation separately with dental pulp stem/progenitor cells (DPSCs), PDL stem/progenitor cells (PDLSCs), or alveolar <em>bone</em> stem/progenitor cells (ABSCs), distinctive tissue phenotypes were formed with collagen I-rich fibers especially by PDLSCs and mineralized tissues by DPSCs, PDLSCs, and ABSCs. DPSC-seeded multiphase scaffolds upon in vivo implantation yielded aligned PDL-like collagen fibers that inserted into <em>bone</em> sialo<em>protein</em>-positive <em>bone</em>-like tissue and putative cementum matrix <em>protein</em> 1-positive/dentin sialophospho<em>protein</em>-positive dentin/cementum tissues. These findings illustrate a strategy for the regeneration of multiphase periodontal tissues by spatiotemporal delivery of multiple <em>proteins</em>. A single stem/progenitor cell population appears to differentiate into putative dentin/cementum, PDL, and alveolar <em>bone</em> complex by scaffold's biophysical properties and spatially released bioactive cues.

BACKGROUND

Mesenchymal stem cells derived from human liposuction aspirates, termed processed lipoaspirate cells, have been utilized as cellular delivery vehicles for the induction of bone formation in tissue engineering and gene therapy strategies. In this study, we sought to evaluate the efficacy of bone morphogenetic protein (BMP)-2-producing adipose-derived stem cells in inducing a posterolateral spine fusion in an athymic rat model.

METHODS

Single-level (L4-L5) intertransverse spinal arthrodesis was attempted with use of a type-I collagen matrix in five groups of athymic rats, with eight animals in each group. Group I was treated with 5 x 10(6) adipose-derived stem cells transduced with an adenoviral vector containing the BMP-2 gene; group II, with 5 x 10(6) adipose-derived stem cells treated with osteogenic media and 1 microg/mL of recombinant BMP-2 (rhBMP-2); group III, with 10 microg of rhBMP-2; group IV, with 1 microg of rhBMP-2; and group V, with 5 x 10(6) adipose-derived stem cells alone. The animals that showed radiographic evidence of healing were killed four weeks after cell implantation and were examined with plain radiographs, manual palpation, microcomputed tomography scanning, and histological analysis.

RESULTS

All eight animals in group I demonstrated successful spinal fusion, with a large fusion mass, four weeks postoperatively. Furthermore, group-I specimens consistently revealed spinal fusion at the cephalad level (L3 and L4), where no fusion bed had been prepared surgically. In contrast, despite substantial BMP-2 production measured in vitro, group-II animals demonstrated minimal bone formation even eight weeks after implantation. Of the groups treated with the application of rhBMP-2 alone, the one that received a relatively high dose (group III) had a higher rate of fusion (seen in all eight specimens) than the one that received the low dose (group IV, in which fusion was seen in four of the eight specimens). None of the group-V animals (treated with adipose-derived stem cells alone) demonstrated successful spine fusion eight weeks after the surgery.

CONCLUSIONS

Adipose-derived stem cells show promise as gene transduction targets for inducing bone formation to enhance spinal fusion in biologically stringent environments.

Segmental defect regeneration has been a clinical challenge. Current tissue-engineering approach using porous biodegradable scaffolds to delivery osteogenic cells and growth factors demonstrated success in facilitating <em>bone</em> regeneration in these cases. However, due to the lack of mechanical property, the porous scaffolds were evaluated in non-load bearing area or were stabilized with stress-shielding devices (<em>bone</em> plate or external fixation). In this paper, we tested a scaffold that does not require a <em>bone</em> plate because it has sufficient biomechanical strength. The tube-shaped scaffolds were manufactured from poly(propylene) fumarate/tricalcium phosphate (PPF/TCP) composites. Dicalcium phosphate dehydrate (DCPD) were used as <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) carrier. Twenty-two scaffolds were implanted in 5mm segmental defects in rat femurs stabilized with K-wire for 6 and 15 weeks with and without <em>10</em> microg of rhBMP-2. Bridging of the segmental defect was evaluated first radiographically and was confirmed by histology and micro-computer tomography (microCT) imaging. The scaffolds in the BMP group maintained the <em>bone</em> length throughout the duration of the study and allow for bridging. The scaffolds in the control group failed to induce bridging and collapsed at 15 weeks. Peripheral computed tomography (pQCT) showed that BMP-2 does not increase the <em>bone</em> mineral density in the callus. Finally, the scaffold in BMP group was found to restore the mechanical property of the rat femur after 15 weeks. Our results demonstrated that the load-bearing BMP-2 scaffold can maintain <em>bone</em> length and allow successfully regeneration in segmental defects.

Previous studies have demonstrated that (at least) matrix metallo<em>protein</em>ase (MMP)-2, -8, -9, -14 and -20 are expressed by human odontoblasts. Here, we analysed the expression of 19 MMPs and their specific tissue inhibitors (TIMP)-1, -2 and -3) -1, -2 and -3 in mature human odontoblasts and pulp tissue. Since MMP-20 is almost exclusively expressed by the dentin-pulp complex cells, we further analysed the effect of transforming growth factor (TGF)-beta1 and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMPs)-2 on its expression. Matrix metallo<em>protein</em>ase-9 served as a positive control for growth factor responsiveness. It was found that MMP-1, -2, -9, -<em>10</em>, -11, -13, -14, -15, -16, -17, -19, -20 and -23, in addition to TIMP-1, -2 and -3 were expressed by both odontoblasts and pulp tissue. Neither MMP-3 nor MMP-12 were expressed in odontoblasts or pulp tissue, and MMP-7, -8, -24 and -25 were expressed only in the odontoblasts; MMP-2, -<em>10</em>, -11, -14 and -20 were expressed more abundantly by odontoblasts, whereas pulp tissue expressed more MMP-13 and MMP-17. Transforming growth factor-beta1 (1 ng ml(-1)) and BMP-2 (<em>10</em>0 ng ml(-1)) did not markedly affect MMP-20 mRNA expression. In contrast, TGF-beta1 alone and with BMP-2 significantly upregulated MMP-9 mRNA by 2.4-fold and by 2.6-fold, respectively, in odontoblasts, while in pulp tissue no effects could be detected. The wide-scale expression of MMPs and TIMPs by mature human odontoblasts and pulp tissue suggests that they may participate in dentin matrix organization prior to mineralization, and that growth factors may further control dentin matrix modeling by differentially regulating individual MMPs.

METHODS

This is a prospective cohort study examining the results and radiographic characteristics of anterior lumbar interbody fusion (ALIF) using femoral ring allografts (FRAs) and recombinant human bone morphogenetic protein-2 (rhBMP-2). This was compared to a historical control ALIF using FRAs with autologous iliac crest bone graft (ICBG).

OBJECTIVE

To determine whether the use of rhBMP-2 can enhance fusion ALIF with stand-alone FRAs.

BACKGROUND

ALIF is a well-accepted procedure in reconstructive spine surgery. Advances in spinal surgery have produced a multitude of anterior interbody implants. The rhBMP-2 has promoted fusion in patients undergoing ALIF with cages and threaded allograft dowels. The FRA still remains a traditional alternative for anterior support. However, as a stand-alone device, the FRA has fallen into disfavor because of high rates of pseudarthrosis. With the advent of rhBMP-2, the FRA may be more attractive because of its simplicity and remodeling potential. It is important to understand the implications when rhBMP-2 is used with such structural allografts.

METHODS

A total of 36 consecutive patients who underwent ALIF with stand-alone FRAs by a single surgeon (E.G.D.) at 1 institute were included. A cohort of 9 consecutive patients who received FRAs filled with rhBMP-2 was followed prospectively. After noticing suboptimal results, the senior author terminated this method of lumbar fusion. A total of 27 prior consecutive patients who received FRAs filled with autogenous ICBG were used for comparison. Analyzing sequential radiographs, flexion-extension radiographs, and computerized tomography with multiplanar reconstructions determined nonunions. Minimum follow-up was 24 months.

RESULTS

Pseudarthrosis was identified in 10 of 27 (36%) patients who underwent stand-alone ALIF with FRAs and ICBG. Nonunion rate was higher among patients who received FRAs with rhBMP-2 (i.e., 5 of 9 [56%]). Statistical significance was not established because of the early termination of the treatment group (P>> 0.3). Of interest, radiographs and computerized tomography revealed early and aggressive resorption of the FRAs when used with rhBMP-2. This preceded graft fracture and even disintegration, resulting in instability and eventual nonunion.

CONCLUSIONS

The use of rhBMP-2 did not enhance the fusion rate in stand-alone ALIF with FRAs. In fact, the trend was toward a higher nonunion rate with rhBMP-2, although this was not significant with the numbers available. This result appears to be caused by the aggressive resorptive phase of allograft incorporation, which occurs before the osteoinduction phase.

Members of the transforming growth factor-beta superfamily, including <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP-4), have been implicated as regulators of neuronal and glial differentiation. To test for a possible role of BMP-4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse embryonic stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron-specific antibody TuJ1, aggregates maintained for 8 days in serum-free medium containing BMP-4 generated 5- to <em>10</em>-fold fewer neurons. The action of BMP-4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP-4 contained fewer cells that were immunoreactive for glial fibrillary acidic <em>protein</em> or the HNK-1 neural antigen. Furthermore, under phase contrast, cultures prepared from BMP-4-treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament <em>protein</em> vimentin; they were rare or absent in control cultures. Treatment with BMP-4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP-4 antagonist noggin counteracted the effect of exogenous BMP-4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP-4 can overcome the neuralizing action of retinoic acid to enhance mesodermal differentiation of murine ES cells.

Growth factors contained in platelet-rich plasma (PRP) have recently been proposed to enhance maturation of <em>bone</em> grafts and, in combination with anorganic bovine <em>bone</em>, to support repair in the treatment of small <em>bone</em> defects in maxillofacial surgery. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP) carried in a matrix may be able to replace the autologous <em>bone</em> graft in the treatment of critical size defects. However, no studies have compared the <em>bone</em> stimulating capacity of PRP and BMP. Likewise there is no data comparing the effects of PRP in either an autologous <em>bone</em> graft or in anorganic bovine <em>bone</em>. We augmented the mandible of Wistar rats (n = 28) on both sides with either anorganic bovine <em>bone</em> (Bio-Oss) or autologous rib <em>bone</em>. On the test side we applied either 20 microl of autologous PRP or <em>10</em> microl of rhBMP-7 (4 groups, n = 7). In addition, <em>bone</em> induction was evaluated in an extraskeletal site (n = 14). A polychrome sequential labeling was performed. The animals were sacrificed by intra-vital perfusion on day 50. Undecalcified ground sections were evaluated by microradiography, digitized histomorphometry and under fluorescent light. The qualitative analysis of fluorochrome labels suggested that PRP and rhBMP-7 accelerated <em>bone</em> growth. However, histomorphometric analysis revealed no significant differences in the area of newly mineralized <em>bone</em> under either the influence of PRP or rhBMP-7 on autologous <em>bone</em> graft. Likewise, the addition of PRP to anorganic bovine <em>bone</em> showed no statistical difference to the control group. The strongest <em>bone</em> stimulating effect was seen for the combination of rhBMP-7 with anorganic bovine <em>bone</em> (p = 0.028). In the extraskeletal model, newly formed <em>bone</em> was evident in the presence of rhBMP-7, but not of PRP. In conclusion, according to the histomorphometry, the addition of platelet-rich plasma failed to enhance <em>bone</em> formation on anorganic bovine <em>bone</em> and on autologous <em>bone</em> grafts.

BACKGROUND

Bone morphogenetic proteins (BMPs) are now being used as bone-graft substitutes to enhance spinal fusion. However, the large doses of BMP required to induce a spinal fusion in humans suggests that the delivery of these proteins should be improved. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone marrow cells, and these autologous cells were found to induce a posterolateral fusion of the spine in syngeneic rats.

METHODS

Intertransverse spinal arthrodesis (L4 and L5) was attempted in ten groups of Lewis rats with 5 x 10 (6) BMP-2-producing rat bone marrow cells (Ad-BMP-2 cells), created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group I); 5 x 10 (6) Ad-BMP-2 cells on a collagen sponge carrier (Group II); 10 micro g of recombinant BMP-2 (rhBMP-2) in a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group III); 10 micro g of rhBMP-2 in a collagen sponge carrier (Group IV); autogenous iliac crest bone-grafting (Group V); 5 x 10 (6) beta-galactosidase-producing rat bone marrow cells, created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group VI); decortication of the transverse processes alone (Group VII); 5 x 10 (6) uninfected rat bone marrow cells with a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group VIII); guanidine hydrochloride-extracted demineralized bone matrix only (Group IX); or a collagen sponge alone (Group X). Each specimen underwent plain radiography, manual palpation, and histological analysis.

RESULTS

All spines in Groups I and II (BMP-2-producing bone marrow cells) and all spines in Groups III and IV were fused at four weeks postoperatively. In contrast, none of the spines in the other groups had fused at a minimum of eight weeks after implantation. Histological analysis of the specimens revealed that the spines that had received BMP-2-producing bone marrow cells (Groups I and II) were filled with coarse trabecular bone postoperatively, whereas those that had received rhBMP-2 (Groups III and IV) were filled with thin, lace-like trabecular bone. All of the other spines, including those that had been treated with autogenous iliac crest bone-grafting (Group V), produced little or no new bone.

CONCLUSIONS

BMP-2-producing bone marrow cells, created by adenoviral gene transfer, produce sufficient BMP to induce an intertransverse fusion in the rat spine model.

An ideal mesenchymal stem cell (MSC) source for <em>bone</em> tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34-, and CD45-), which surround capillaries and microvessels, and (2) adventitial cells (CD146-, CD34+, and CD45-), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic <em>bone</em> model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated <em>bone</em> formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated <em>protein</em>, Nel-like molecule I (NELL-1). Implants consisting of the demineralized <em>bone</em> matrix putty combined with NELL-1 (3 μg/μL), hPSC (2.5×<em>10</em>(5) cells), or hPSC+NELL-1, were inserted in the bicep femoris of SCID mice. <em>Bone</em> growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on <em>bone</em> formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2. Next, hPSCs induced greater implant vascularization than the unsorted stromal vascular fraction from patient-matched samples. Finally, we observed an additive effect on implant vascularization with hPSC+NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/<em>protein</em> combination therapy hPSC+NELL-1 in the development of strategies for vascularized <em>bone</em> regeneration.

MicroRNAs (miRNAs) are short, single-stranded non-coding RNAs that repress their target genes by binding their 3' UTRs. These RNAs play critical roles in myogenesis. To gain knowledge about miRNAs involved in the regulation of myogenesis, porcine longissimus muscles were collected from 18 developmental stages (33-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, <em>10</em>0- and <em>10</em>5-day post-gestation fetuses, 0 and <em>10</em>-day postnatal piglets and adult pigs) to identify miRNAs using Solexa sequencing technology. We detected 197 known miRNAs and 78 novel miRNAs according to comparison with known miRNAs in the miRBase (release 17.0) database. Moreover, variations in sequence length and single nucleotide polymorphisms were also observed in 1<em>10</em> known miRNAs. Expression analysis of the 11 most abundant miRNAs were conducted using quantitative PCR (qPCR) in eleven tissues (longissimus muscles, leg muscles, heart, liver, spleen, lung, kidney, stomach, small intestine and colon), and the results revealed that ssc-miR-378, ssc-miR-1 and ssc-miR-206 were abundantly expressed in skeletal muscles. During skeletal muscle development, the expression level of ssc-miR-378 was low at 33 days post-coitus (dpc), increased at 65 and 90 dpc, peaked at postnatal day 0, and finally declined and maintained a comparatively stable level. This expression profile suggested that ssc-miR-378 was a new candidate miRNA for myogenesis and participated in skeletal muscle development in pigs. Target prediction and KEGG pathway analysis suggested that <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) and mitogen-activated <em>protein</em> kinase 1 (MAPK1), both of which were relevant to proliferation and differentiation, might be the potential targets of miR-378. Luciferase activities of report vectors containing the 3'UTR of porcine BMP2 or MAPK1 were downregulated by miR-378, which suggested that miR-378 probably regulated myogenesis though the regulation of these two genes.

Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml fibroblast growth factor-2 (FGF-2), were cultured on three-dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without <em>10</em> ng/ml <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2). Chondrocytes expanded without FGF-2 exhibited high intensity immunostaining for smooth muscle alpha-actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile fibroblasts. Chondrocytes expanded in the presence of FGF-2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7-fold higher cell number, 4.2-fold higher wet weight, and 2.8-fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF-2. Chondrocytes expanded with FGF-2 and cultured on PGA scaffolds in the presence of BMP-2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5-fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP-2. The presence of BMP-2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF-2. In summary, the presence of FGF-2 during 2D expansion reduced chondrocyte expression of fibroblastic molecules and induced responsiveness to BMP-2 during 3D cultivation on PGA scaffolds.

The <em>bone</em> <em>morphogenetic</em> <em>proteins</em>, BMP-2 and OP-1, are candidates for growth factors that control renal branching morphogenesis. We examined their effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting duct morphogenesis (mIMCD-3 cells are derived from the terminal inner medullary collecting duct of the SV40 mouse). Osteogenic <em>protein</em>-1 (OP-1), at a dose of 0.25 nM, increased explant growth by 30% (P = 0.001). In contrast, <em>10</em>0-fold greater concentrations of OP-1 (28 nM) decreased explant growth by <em>10</em>% (P < 0.001). BMP-2 was entirely inhibitory (maximum inhibition of 7% at 5 nM, P < 0.0004). In an in vitro model for branching morphogenesis utilizing the kidney epithelial cell line, mIMCD-3, low doses of OP-1 (< 0.5 nM) increased the number of tubular structures formed by 28 +/- 5% (P = 0.01), whereas concentrations>> 0.5 nM decreased that number by 22 +/- 8% (P = 0.02). All concentrations of BMP-2 (0.05-<em>10</em> nM) were inhibitory (maximum inhibition at <em>10</em> nM of 88 +/- 3%, P < 0.0001). Stimulatory doses of OP-1 increased tubular length (P = 0.003) and the number of branch points/structure (3.2-fold increase, P = 0.0005) compared with BMP-2. To determine the molecular basis for these effects, we demonstrated that BMP-2 is bound to mIMCD-3 cells by the type I serine/threonine kinase receptor, ALK-3, and that OP-1 bound to an approximately 80-kDa <em>protein</em> using ligand-receptor affinity assays. To demonstrate that OP-1 can exert both stimulatory and inhibitory effects within a developing kidney, embryonic explants were treated with agarose beads saturated with 2 microM OP-1. OP-1 decreased the number of ureteric bud/collecting duct branches adjacent to the beads by 58 +/- 1% (P < 0.0001). In contrast, the number of branches in tissue distal to the OP-1 beads was enhanced, suggesting a stimulatory effect at lower doses of OP-1. We conclude that OP-1 and BMP-2 directly control branching morphogenesis and that the effects of OP-1 are dependent on its local concentration within developing kidney tissue.

BACKGROUND

Successful anterior cruciate ligament reconstruction requires secure healing between tendon and bone.

OBJECTIVE

Bone morphogenetic protein-signaling plays an important role in tendon-to-bone healing. rhBMP-2, a powerful osteoinductive agent, can improve tendon-bone interdigitation.

METHODS

Controlled laboratory study.

METHODS

The study was designed in 2 phases: Phase I consisted of a dose-response study where 21 New Zealand White rabbits underwent bilateral anterior cruciate ligament reconstructions. Rabbits received either rhBMP-2 (11.5, 50, or 115 microg) or noggin (10, 15, 30, or 100 ng) (a potent bone morphogenetic proteins inhibitor) delivered in an injectable calcium phosphate matrix. Animals were sacrificed at 2 weeks and histomorphometric analyses were performed. In phase II, 60 rabbits underwent bilateral anterior cruciate ligament reconstructions and were assigned to 3 groups: rhBMP-2 (115 microg), noggin (30 ng) in a calcium phosphate carrier, and calcium phosphate carrier alone. Animals were sacrificed at 2, 4, and 8 weeks and histomorphometric and biomechanical analyses were performed.

RESULTS

rhBMP-2 treatment led to a significant increase in the width of new bone formation at the tendon-bone interface in a dose-dependent fashion (0.24-0.35 mm vs 0.13-0.16 mm in controls). All dosages of noggin inhibited new bone formation (0.06-0.1 mm vs 0.15-0.16 mm in controls); however, there was no dose-dependent effect in the concentrations studied. In the phase II study, rhBMP-2 resulted in a significant increase in new bone formation (81%, 89%, and 113%) at increasing time periods compared with controls. Tunnel diameters in the rhBMP-2 group were significantly smaller (15%-45%) than in the carrier group. The negative effect of noggin was not sustained, as new bone formation increased with time. The rhBMP-2 group demonstrated significantly increased stiffness at 8 weeks, while there was no significant difference in ultimate tensile load when compared with the other 2 groups.

CONCLUSIONS

rhBMP-2 demonstrated a strong, positive dose-dependent effect on osteointegration at the tendon-bone junction. In contrast, noggin decreased osteointegration. No tunnel widening was detected with rhBMP-2 using the calcium phosphate carrier.

CONCLUSIONS

Further studies are needed to investigate the potential clinical application of enhancing healing and decreasing recovery time using bone morphogenetic proteins in soft tissue ligament reconstruction.

Osteoblast phenotypic expression in monolayer culture depends on surface microtopography. Here we tested the hypothesis that mineralized <em>bone</em> nodule formation in response to osteotropic agents such as <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and dexamethasone is also influenced by surface microtopography. Fetal rat calvarial (FRC) cells were cultured on Ti implant materials (PT [pretreated], Ra = 0.6 microm; SLA [course grit blasted and acid etched], Ra = 4.0 microm; TPS [Ti plasma sprayed], Ra = 5.2 microm) in the presence of either BMP-2 (20 ng/ml) or <em>10</em>(-8) M dexamethasone (Dex). At 14 days post-confluence, a homogenous layer of cells covered the surfaces, and stacks of cells that appeared to be nodules emerging from the culture surface were present in some areas on all three Ti surfaces. Cell proliferation decreased while alkaline phosphatase specific activity (ALPase) and nodule number generally increased with increasing surface roughness in both control and treated cultures. There was no difference in cell number between the control and Dex-treated cultures for a particular surface, but BMP-2 significantly reduced cell number compared with control or Dex-treated cultures. Treatment with Dex or BMP-2 further increased ALPase on all surfaces except for PT cultures with Dex. Dex had no effect on nodule area in cultures grown on PT or SLA disks, yet increased nodule number by more than <em>10</em>0% in cultures on PT disks. Though the effect of BMP-2 on nodule number was the same as Dex, BMP-2 increased nodule area on all surfaces except TPS, where area was decreased. Ca and P content of the cell layers in control cultures did not vary with surface roughness. However, cultures treated with Dex had increased Ca content on all surfaces, but the greatest increase was seen on SLA and TPS. BMP-2 increased Ca content in cultures on all surfaces, with the greatest increase on the PT surface. BMP-2 treatment increased P content on all surfaces, whereas Dex only increased P on rough surfaces. Of all cultures examined, the Ca/P weight ratio was 2:1 only on rough surfaces with BMP-2, indicating the presence of <em>bone</em>-like apatite. This was further validated by Fourier transform infrared (FTIR) imaging showing a close association between mineral and matrix on TPS and SLA surfaces with BMP-2-treated cells, and individual spectra indicated the presence of an apatitic mineral phase comparable to <em>bone</em>. In contrast, mineral on the smooth surface of BMP-2-treated cultures and on all surfaces where cultures were treated with Dex was not associated with the matrix and the spectra, not typical of <em>bone</em> apatite, implying dystrophic mineralization. This demonstrates that interactions between growth factor or hormone and surface microtopography can modulate <em>bone</em> cell differentiation and mineralization.

There is increasing evidence that TGF-beta family member cytokine <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-4 plays different pathophysiological roles in the pulmonary and systemic circulation. Upregulation of BMP-4 has been linked to atherosclerosis and hypertension in the systemic circulation, whereas disruption of BMP-4 signaling is associated with the development of pulmonary hypertension. To test the hypothesis that BMP-4 elicits differential effects in the pulmonary and systemic circulation, we compared the prooxidant and proinflammatory effects of BMP-4 in cultured human coronary arterial endothelial cells (CAECs) and pulmonary arterial endothelial cells (PAECs). We found that BMP-4 (from 0.3 to <em>10</em> ng/ml) in CAECs increased O(2)(*-) and H(2)O(2) generation, induced NF-kappaB activation, upregulated ICAM-1, and induced monocyte adhesiveness to ECs. In contrast, BMP-4 failed to induce oxidative stress or endothelial activation in PAECs. Also, BMP-4 treatment impaired acetylcholine-induced relaxation and increased O(2)(*-) production in cultured rat carotid arteries, whereas cultured rat pulmonary arteries were protected from these adverse effects of BMP-4. Thus, we propose that BMP-4 exerts prooxidant, prohypertensive, and proinflammatory effects only in the systemic circulation, whereas pulmonary arteries are protected from these adverse effects of BMP-4. The vascular bed-specific endothelial effects of BMP-4 are likely to contribute to its differential pathophysiological role in the systemic and pulmonary circulation.

<em>Bone</em>-<em>morphogenetic</em> <em>proteins</em> (BMPs) play an important role in development and many cellular processes. However, their functional role in the development and progression of breast cancer is not clearly understood. In the present study, we performed a systematic expression analysis of the 14 types of BMPs in <em>10</em> human breast cancer cell lines. We found that <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) was one of the most frequently expressed BMPs. Furthermore, the expression level of BMP4 was maybe correlated with the metastatic potential of the cancer lines. Accordingly, overexpression of BMP4 in the breast cancer cell lines MCF-7 and MBA-MD-231 promoted the migration and invasion phenotypes of the cancer cells, whereas RNAi-mediated knockdown of BMP4 expression inhibited the migration and invasion activities of the cancer cells. To identify the important factors that may mediate the BMP4 functions in breast cancer cells, we analyzed a panel of cancer-related genes, and found that the expression of matrix metallo<em>protein</em>ase-1 (MMP-1) and C-X-C chemokine receptor type 4 (CXCR4) sharply increased at both the mRNA and <em>protein</em> levels in the breast cancer cells overexpressing BMP4. Interestingly, when breast cancer cells MDA-MB-231 or MCF-7 were co-cultured with the osteoblast-like cells MG63 to mimic a <em>bone</em> metastasis microenvironment, BMP4 did not exhibit any significant effect on the expression of OPG or RANKL, two important factors in <em>bone</em> remodeling. BMPs antagonists, Noggin, parallel inhibited breast cancer cell migration and invasion and induced <em>bone</em> remodeling. Taken together, our results strongly suggest that BMP4 may promote the migration and invasion of breast cancer cells, at least in part by up-regulating the expressions of MMP-1 and CXCR4. It is conceivable that novel therapeutics for breast cancer may be developed by targeting BMP4 signaling pathway and/or its important downstream mediators in breast cancer cells.

Pulmonary arterial hypertension (PAH) is associated with mutations of <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 2 (BMPR2), and BMPR2 expression decreases with the development of experimental PAH. Decreased BMPR2 expression and impaired intracellular BMP signaling in pulmonary artery (PA) smooth muscle cells (PASMC) suppresses apoptosis and promotes proliferation, thereby contributing to the pathogenesis of PAH. We hypothesized that overexpression of BMPR2 in resistance PAs would ameliorate established monocrotaline PAH. Human BMPR2 was inserted into a serotype 5 adenovirus with a green fluorescent <em>protein</em> (GFP) reporter. Dose-dependent transgene expression was confirmed in PASMC using fluorescence microscopy, quantitative RT-PCR, and immunoblots. PAH was induced by injecting Sprague-Dawley rats with monocrotaline (60 mg/kg ip) or saline. On day 14, post-monocrotaline (MCT) rats received 5 x <em>10</em>(9) plaque-forming units of either Ad-human BMPR2 (Ad-hBMPR2) or Ad-GFP. Transgene expression was confirmed by fluorescence microscopy, quantitative RT-PCR of whole lung samples, and laser-capture microdissected resistance PAs. Invasive hemodynamic and echocardiographic end points of pulmonary hypertension were assessed on day 24. Endogenous BMPR2 mRNA levels were greatest in resistance PAs, and expression declined with MCT PAH. Despite robust hBMPR2 expression in all lung lobes and within resistance PAs of treated rats, hBMPR2 did not lower mean PA pressure, pulmonary vascular resistance index, right ventricular hypertrophy, or remodeling of resistance PAs. Nebulized intratracheal adenoviral gene therapy with hBMPR2 reliably distributed hBMPR2 to resistance PAs but did not ameliorate PAH. Depressed BMPR2 expression may be a marker of PAH but is not central to the pathogenesis of this model of PAH.

Estrogens exert their physiological effects on target tissues by interacting with the estrogen receptors, ERalpha and ERbeta. Estrogen replacement is one the most common and effective strategies used to prevent osteoporosis in postmenopausal women. Whereas it was thought that estrogens work exclusively by inhibiting <em>bone</em> resorption, our previous results show that 17beta-estradiol (E2) increases mouse <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2 mRNA, suggesting that estrogens may also enhance <em>bone</em> formation. In this study, we used quantitative real-time RT-PCR analysis to demonstrate that estrogens increase BMP-2 mRNA in mouse mesenchymal stem cells. The selective ER modulators, tamoxifen, raloxifene, and ICI-182,780 (ICI), failed to enhance BMP-2 mRNA, whereas ICI inhibited E2 stimulation of expression. To investigate if estrogens increase BMP-2 expression by transcriptional mechanisms and if the response is mediated by ERalpha and/or ERbeta, we studied the effects of estrogens on BMP-2 promoter activity in transient transfected C3H<em>10</em>T1/2 cells. E2 produced a dose-dependent induction of the mouse -2712 BMP-2 promoter activity in cells cotransfected with ERalpha and ERbeta. At a dose of <em>10</em> nM E2, ERalpha induced mouse BMP-2 promoter activity 9-fold, whereas a 3-fold increase was observed in cells cotransfected with ERbeta. Tamoxifen and raloxifene were weak activators of the mouse BMP-2 promoter via ERalpha, but not via ERbeta. ICI blocked the activation of BMP-2 promoter activity by E2 acting via both ERalpha and ERbeta, indicating that mouse BMP-2 promoter activation is ER dependent. In contrast to E2 and selective ER modulators, the phytoestrogen, genistein was more effective at activating the mouse BMP-2 promoter with ERbeta, compared with ERalpha. Using a deletion series of the BMP-2 promoter, we determined that AP-1 or Sp1 sites are not required for E2 activation. A mutation in a sequence at -415 to -402 (5'-GGGCCActcTGACCC-3') that resembles the classical estrogen-responsive element abolished the activation of the BMP-2 promoter in response to E2. Our studies demonstrate that E2 activation of mouse BMP-2 gene transcription requires ERalpha or ERbeta acting via a variant estrogen-responsive element binding site in the promoter, with ERalpha being the more efficacious regulator. Estrogenic compounds may enhance <em>bone</em> formation by increasing the transcription of the BMP-2 gene.

OBJECTIVE

Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations.

METHODS

We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls.

RESULTS

We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)).

CONCLUSIONS

We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Nonunion is a challenging problem that may occur following certain <em>bone</em> fractures. However, there has been little investigation of the molecular basis of nonunions. <em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) play a significant role in osteogenesis. However, little is known about the expression patterns of BMPs in abnormal <em>bone</em> healing that results in nonunion formation. These facts prompted us to investigate and compare the gene expression patterns of BMPs and their antagonists in standard healing fractures and nonunions using rat experimental models. Standard closed healing fractures and experimental atrophic nonunions produced by periosteal cauterization at the fracture site were created in rat femurs. At postfracture days 3, 7, <em>10</em>, 14, 21, and 28, total RNA was extracted from the callus of standard healing fracture and fibrous tissue of nonunion (n=4 per each time point and each group). Gene expression of BMPs, BMP antagonists, and other regulatory molecules were studied by methods including Genechip microarray and real-time quantitative RT-PCR. Gene expression of BMP-2, 3, 3B, 4, 6, 7, GDF-5, 7, and BMP antagonists noggin, drm, screlostin, and BAMBI were significantly lower in nonunions compared to standard healing fractures at several time points. Downregulation in expression of osteogenic BMPs may account for the nonunions of fracture. The balance between BMPs and their endogenous antagonists is critical for optimal fracture healing.

BACKGROUND

Recombinant human bone morphogenetic protein-2 (rhBMP-2) on an absorbable collagen sponge has been shown to be a safe and effective replacement for iliac crest bone graft when used with a threaded fusion device in anterior lumbar interbody arthrodesis. Use of rhBMP-2 on an absorbable collagen sponge in posterolateral lumbar arthrodesis requires the addition of a bulking agent to provide resistance against compression and to serve as an osteoconductive scaffold for new bone formation.

METHODS

We performed a prospective, randomized, multicenter pilot study to investigate the use of rhBMP-2 on an absorbable collagen sponge combined with a ceramic-granule bulking agent as a replacement for autogenous iliac crest bone graft in single-level posterolateral lumbar arthrodesis with instrumentation. The investigational group (twenty-five patients) was treated with a 1.5 mg/mL solution of rhBMP-2 on two strips of absorbable collagen sponge (total dose of rhBMP-2, 12 mg) combined with 10 cm(3) of ceramic granules. The control group (twenty-one patients) received iliac crest bone graft. Clinical outcomes were assessed with use of well-established instruments. Radiographs were reviewed to assess consolidation of fusion.

RESULTS

Eighty-eight percent (twenty-two) of the twenty-five patients in the investigational group and 86% (eighteen) of the twenty-one patients in the control group were considered to have completed the twenty-four-month follow-up. At all follow-up intervals, there were significant improvements in the clinical outcome measures, including the Oswestry Disability Index (ODI) scores, Short Form-36 scores, and back and leg pain scores, in both groups. At twenty-four months, the improvement in the mean ODI score, as compared with the preoperative score, was 28.2 points in the investigational group and 23.0 points in the control group. By twenty-four months, 95% (eighteen) of nineteen patients in the investigational group compared with 70% (fourteen) of twenty in the control group had a radiographically documented fusion. The overall success rate was 81% (seventeen of twenty-one) in the investigational group and 55% (eleven of twenty) in the control group (p = 0.345).

CONCLUSIONS

Compared with an iliac crest bone graft, the combination of an absorbable collagen sponge soaked with rhBMP-2 and ceramic granules resulted in trends toward improvements in clinical outcomes and toward a higher rate of radiographic fusion. This combination of an osteoinductive agent with an osteoconductive matrix may be an effective replacement for autograft in single-level posterolateral lumbar arthrodeses with instrumentation.

Pulmonary arterial hypertension (PAH) is a rare disease characterized by distinctive changes in pulmonary arterioles that lead to progressive elevation of pulmonary artery pressure, pulmonary vascular resistance, right ventricular failure, and a high mortality rate. The etiology of PAH is heterogeneous and incompletely understood. Based on clinical classification, WHO Group 1 PAH includes sporadic disease (idiopathic PAH), inherited PAH (heritable PAH), and association with certain medical conditions (associated PAH). Genes play an important role in idiopathic and heritable PAH. Mutations in <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 2 (BMPR2), a member of the transforming growth factor β (TGFβ) superfamily of receptors, have been identified in 70 % of cases of familial PAH, as well as in <em>10</em>-40 % of cases of idiopathic PAH. Mutations in ALK-1, ENG, SMAD4 and SMAD8, other TGFβ family members, are additional rare causes of PAH. CAV1 regulates SMAD2/3 phosphorylation, and mutations in CAV1 are a rare cause of PAH. KCNK3 is a member of the two-pore domain potassium channels expressed in pulmonary artery smooth muscle cells, and mutations in KCNK3 are a rare cause of both familial and IPAH. The genetics of PAH are complex due to incomplete penetrance and genetic heterogeneity. In addition to rare mutations as a monogenic cause of HPAH, common variants in cerebellin 2 (CBLN2) increase the risk of PAH by approximately twofold. PAH in children is much more heterogeneous than in adults and can be associated with several genetic syndromes, specifically syndromes with congenital heart disease, vascular disease, and hepatic disease. Clinical genetic testing is available for PAH and should be considered in families to allow for more definitive risk stratification and allow for reproductive planning.