The purpose of this study was to investigate the influence of nano-sized β-tricalcium phosphate (β-TCP) on the biological performance of poly (lactic acid) (PLA) composite scaffolds by using in vitro degradation and an in vivo model of heterotopic <em>bone</em> formation. Nano-sized β-TCP (nβ-TCP) was prepared with a wet grinding method from micro-sized β-TCP (mβ-TCP), and composite scaffolds containing 0, <em>10</em>, 30, or 50 wt% nβ-TCP or 30 wt% mβ-TCP were generated using a freeze-drying method. Degradation was assessed by monitoring changes in microstructure, pH, weight, and compressive strength over a 26-week period of hydrolysis. Composite scaffolds were processed into blocks, and implanted into muscular pockets of rabbits after loading with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2). New <em>bone</em> formation was evaluated based on histological and immunohistochemical analysis 2, 4, and 8 weeks after implantation. The in vitro results indicated that the buffering effect of nβ-TCP was stronger than mβ-TCP, which was positively correlated with the content of nβ-TCP. The in vivo findings demonstrated that nβ-TCP enhanced the osteoconductivity of the scaffolds. Although composite scaffolds containing 30% nβ-TCP exhibited similar osteoconductivity to 50% nβ-TCP, they had better mechanical properties than the 50% nβ-TCP scaffolds. This study supports the potential application of a composite scaffold containing 30% nβ-TCP as a promising scaffold for <em>bone</em> regeneration.

Idiopathic pulmonary arterial hypertension (IPAH) is characterized by smooth muscle cell, endothelial cell, and fibroblast hypertrophy and an increase in extracellular matrix volume in pulmonary precapillary arterioles. These features lead to a gradual increase of pulmonary vascular resistance, right-heart failure, and premature death. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> receptor type 2 (BMPR-2) gene mutations have been identified to cause IPAH. BMPR-2 receptor mutation results in BMP signalling pathway termination and leads to disturbed growth and differentiation of pulmonary circulation cells. Transforming growth factor (TGF)-beta1 inhibits the migration and proliferation of endothelial and smooth muscle cells, and stimulates their differentiation, thus it has antiinflammatory and immunosuppressive properties, inhibiting vascular remodeling and is responsible for extracellular matrix production. The aim of this study was to analyse the profile of TGF-beta1 and the expression of its receptor (TbetaR I, TbetaR II and TbetaR III-betaglycan) genes in IPAH and in secondary forms of pulmonary arterial hypertension [Eisenmenger's syndrome (ES) patients]. Twenty-one patients with IPAH (2 men), 12 ES patients, and <em>10</em> healthy controls were enrolled in the study. QRT-PCR analysis of the transcriptive activity of TGF-beta1 and its receptor genes was performed with each patient. There were differences in receptor gene expression among the patient groups. The highest expression was observed in Eisenmenger syndrome patients (approximately 5-to 8-fold increase). There was a negative correlation between the gene expression of TGF-beta1 and that of its receptors, and a positive correlation between TbetaR II and TbetaR III in healthy controls. In IPAH patients a positive correlation between TGF-beta1 and TbetaR I was found. There was a difference in expression of TGF-beta1/receptor gene ratios and expression of receptor gene ratios between the examined groups. The differences in expression between IPAH and ES patients might suggest the role of these cytokines in IPAH pathogenesis. A disturbed proportion of expression of TGF-beta1 and receptor genes in IPAH patients might be one of the pathogenetic factors of the disease.

BACKGROUND

Bone morphogenetic proteins (BMPs) are biologically active molecules capable of eliciting new bone formation. In combination with biomaterials, these proteins can be used in a clinical setting as bone-graft substitutes to promote bone repair. Collagen from animal sources has previously been the preferred carrier material in animal experiments. More recently, synthetic biodegradable polymers have been tested as a delivery vehicle for osteoinductive agents. In earlier studies performed in our laboratory, it was found that the polylactic acid homopolymers (PLA650) and poly-D,L-lactic acid-polyethylene glycol block copolymers (PLA650-PEG200) are viscous liquids that can be used as BMP delivery systems.

METHODS

To obtain new PLA-PEG polymers that exhibit greater plasticity, the molecular sizes of PLA and PEG segments in the copolymer chains were increased. Plastic PLA-PEG polymers with various molecular sizes and PLA/PEG ratios were synthesized, mixed with recombinant human (rh) BMP-2, and implanted into the dorsal muscles of mice for 3 weeks to evaluate their capacity to elicit new bone formation. To compare the plastic PLA-PEG polymer with the liquid PLA650-PEG200 polymer, these two polymers were combined with rhBMP-2, implanted, and harvested after 3 weeks. Bone mineral content (BMC), bone area, and bone mineral density (BMD) of the ectopic new bone were measured by means of single energy X-ray absorptiometry (SXA).

RESULTS

All of the PLA6,500-PEG3,000 implants with 10 or 20 microg of rhBMP-2 showed new bone formation. In contrast, little or no bone formation was seen in other plastic PLA-PEG implants with rhBMP-2. Control implants that lacked rhBMP-2 did not show new bone formation. Radiographic and histologic examinations showed that the PLA6,500-PEG3,000 implants with rhBMP-2 harvested 3 weeks after implantation had normal bone characteristics with hematopoietic marrow and osseous trabeculae. SXA analysis showed that the values for bone mineral content (BMC), bone area, and bone mineral density (BMD) of new bone resulting from the use of plastic PLA6,500-PEG3,000 polymers with rhBMP-2 were significantly higher than those obtained with the liquid PLA650-PEG200 polymers (p < 0.001 for each of the three values).

CONCLUSIONS

These results indicate that the PLA6,500-PEG3000 block copolymer with plastic properties works effectively as a BMP delivery system. These data suggest that the total molecular size and ratio of PLA size to PEG size is an essential factor in determining the efficacy of a BMP delivery system. After implantation, it is possible that the PLA6,500-PEG3,000 pellets might have absorbed tissue fluids and become swollen, resulting in bone formation that exceeded the size of the original implants. This expansion characteristic is a potentially beneficial property, given the intended practical application of the polymer in the repair of bone defects.

CONCLUSIONS

New synthetic biodegradable delivery systems will play an important role in the clinical applications of rhBMPs in which local formation of bone via an osteoinductive graft material is needed. Further pre-clinical and clinical work is necessary to establish the safety of these implants before they are adopted for widespread clinical use.

Grebe syndrome is a recessively inherited acromesomelic dysplasia. We studied, clinically and radiographically, <em>10</em> affected individuals, originating from Bahia, Brazil. The phenotype is characterized by a normal axial skeleton and severely shortened and deformed limbs, with a proximo-distal gradient of severity. The humeri and femora were relatively normal, the radii/ulnae and tibiae/fibulae were short and deformed, carpal and tarsal <em>bones</em> were fused, and several metacarpal and metatarsal <em>bones</em> were absent. The proximal and middle phalanges of the fingers and toes were invariably absent, while the distal phalanges were present. Postaxial polydactyly was found in several affected individuals. Several joints of the carpus, tarsus, hand, and foot were absent. Heterozygotes presented with a variety of skeletal manifestations including polydactyly, brachydactyly, hallux valgus, and metatarsus adductus. Grebe syndrome is caused by a missense mutation in the gene encoding cartilage-derived <em>morphogenetic</em> <em>protein</em>-1.

BACKGROUND

Surgical implantation of recombinant human bone morphogenetic protein 2 (rhBMP-2) in an absorbable collagen sponge carrier (ACS) significantly enhances bone regeneration in horizontal alveolar defects; however, sufficient quantities of bone for implant dentistry are not routinely obtained.

OBJECTIVE

The objective of this proof-of-principle study was to evaluate the potential of a space-providing macroporous expanded polytetrafluoroethylene (ePTFE) device to control volume and geometry of rhBMP-2/ACS-induced alveolar bone augmentation.

METHODS

Bilateral critical-size supra-alveolar periimplant defects were created in four Hound-Labrador mongrel dogs. Two turned and one surface-etched 10 mm titanium dental implants were placed 5 mm into the surgically reduced alveolar ridge creating 5 mm supra-alveolar defects. rhBMP-2/ACS (0.4 mg rhBMP-2) was placed around the exposed dental implants. Additionally, one jaw quadrant in each animal was randomly assigned to receive the dome-shaped macroporous ePTFE device. Mucoperiosteal flaps were advanced for primary wound closure. The animals were euthanized at 8 weeks post surgery for histometric analysis.

RESULTS

The space-providing macroporous ePTFE device defined the volume and geometry of rhBMP-2/ACS-induced bone formation, whereas bone formation at sites receiving rhBMP-2/ACS alone varied considerably. Vertical bone gain at turned dental implants averaged (+/-SD) 4.7 +/-0.2 mm at sites receiving rhBMP-2/ACS and the ePTFE device compared with 3.5 +/-0.9 mm at sites receiving rhBMP-2/ACS only. The corresponding values for rhBMP-2/ACS-induced bone area were 9.6 +/- 0.7 mm2 and 7.5 +/-6.2 mm2. There was a highly significant correlation between induced bone area and the space provided by the ePTFE device (p <.001). There was no difference in induced bone density or bone-implant contact between the two technologies. These observations were consistent with those observed at surface-etched dental implants.

CONCLUSIONS

The data from this study suggest that a space-providing macroporous ePTFE device defines rhBMP-2/ACS-induced alveolar augmentation to provide adequate bone quantities for implant dentistry. The dental implant surface technology does not appear to substantially influence bone formation.

Gene therapy using constitutively active viral promoters to drive expression of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) has been extensively evaluated as a strategy for inducing <em>bone</em> regeneration. However, this approach offers little control over the concentration, timing, or duration of BMP synthesis. To gain greater control over BMP kinetics, we developed a new inducible system for the controlled expression of BMP2 using a two-component transcription factor that is dimerized with rapamycin (Rap). This approach provided stringent control over BMP2 synthesis with no BMP expression detected in the uninduced state. Rapamycin or the less immunosuppressive analogue, AP21967, rapidly and reversibly induced BMP2 in a dose-dependent manner (range 0.1-<em>10</em> nM). Subcutaneous implants of fibroblasts containing the Rap-inducible system in syngeneic C57BL/6 mice were highly responsive to ip Rap injection (0.1-1 mg/kg). Peak BMP2 levels were detected within 24 h of a single Rap injection and declined to undetectable levels after 8-<em>10</em> days. Alternate-day Rap injections (1 mg/kg) for 6 weeks induced subcutaneous ectopic <em>bone</em> formation. Rap-dependent healing of a critical-sized cranial defect was also achieved using this system. This regulated BMP2 expression system will be extremely useful for examining the role of timing and sequence of BMP delivery on <em>bone</em> regeneration.

This study investigated the effects of imposed optical defocus on the expression patterns of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 and 7 (BMP4, BMP7) in chick retinal pigment epithelium (RPE), as indicators of roles in postnatal eye growth regulation. BMP4 and BMP7 gene and <em>protein</em> expression patterns were characterized for retina, RPE and choroid tissues of young normal White-Leghorn chickens. The effects of short-term (2 and 48 h) exposure to monocular +<em>10</em> and -<em>10</em> diopter (D) lenses on RPE gene expression of BMP4 and BMP7 were also examined. Tissues from both treated and fellow eyes as well as from eyes of age-matched untreated birds were included in the latter experiment. Of ocular tissues comprising the posterior wall of the chick eye, RPE showed the highest expression of BMP4 and BMP7 mRNA, compared to retina and choroid. Western blots and immunohistochemistry confirmed the expression of BMP4 and BMP7 <em>protein</em> in all layers - retina, RPE, choroid and sclera. With imposed defocus, both BMP4 and BMP7 showed bidirectional changes in expression in RPE, however, with different temporal patterns. With +<em>10</em> D lenses, BMP4 gene expression was up-regulated after both 2 and 48 h of treatment, while BMP7 expression was up-regulated only after 48 h of lens wear. With -<em>10</em> D lenses, both BMP4 and BMP7 showed down-regulation of gene expression for both 2 and 48 h treatment durations. With the -<em>10</em> D lens treatment applied for 48 h, gene expression for both BMP4 and BMP7 was also down-regulated in contralateral fellows of treated eyes compared to eyes of untreated chicks. The rapid changes in gene expression in chick RPE observed for both BMP4 and BMP7, up or down according to the sign of imposed optical defocus, resemble similar trends reported for BMP2. Further studies are needed to confirm the roles of BMPs as ocular growth modulators, as suggested by these data. The data also suggest a role for the RPE as a conduit for relaying growth modulatory retinal signals.

OBJECTIVE

Calcific aortic valve disease (CAVD) is a significant cardiovascular disorder, and controversy exists as to whether it is primarily a dystrophic or osteogenic process in vivo. In this study, we sought to clarify the mechanism of CAVD by assessing a genetic mutation, Notch1 heterozygosity, which leads to CAVD with <em>10</em>0% penetrance in humans.

RESULTS

Murine immortalized Notch1(+/-) aortic valve interstitial cells (AVICs) were isolated and expanded in vitro. Molecular signaling of wild-type and Notch1(+/-) AVICs were compared to identify changes in pathways that have been linked to CAVD-transforming growth factor-β1/<em>bone</em> <em>morphogenetic</em> <em>protein</em>, mitogen-activated <em>protein</em> kinase, and phosphoinositide 3-kinase/<em>protein</em> kinase B-and assessed for calcification potential. Additionally, AVIC mechanobiology was studied in a physiologically relevant, dynamic mechanical environment (<em>10</em>% cyclic strain) to investigate differences in responses between the cell types. We found that Notch1(+/-) AVICs resembled a myofibroblast-like phenotype expressing higher amounts of cadherin-11, a known mediator of dystrophic calcification, and decreased Runx2, a known osteogenic marker. We determined that cadherin-11 expression is regulated by Akt activity, and inhibition of Akt phosphorylation significantly reduced cadherin-11 expression. Moreover, in the presence of cyclic strain, Notch1(+/-) AVICs exhibited significantly upregulated phosphorylation of Akt at Ser473 and smooth muscle α-actin expression, indicative of a fully activated myofibroblast. Finally, these Notch1-mediated alterations led to enhanced dystrophic calcific nodule formation.

CONCLUSIONS

This study presents novel insights in our understanding of Notch1-mediated CAVD by demonstrating that the mutation leads to AVICs that are fully activated myofibroblasts, resulting in dystrophic, but not osteogenic, calcification.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-<em>10</em>]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis.

OBJECTIVE

To explore different roles of TGF-beta (transforming growth factor beta) and bone morphogenetic proteins (BMPs) in hepatic stellate cell proliferation and trans-differentiation.

METHODS

Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).

RESULTS

The results indicated that TGF-beta1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGF-beta1 and BMPs. However, BMPs was more potent at trans-differentiation of hepatic stellate cells than TGF-beta1. In addition, we observed that TGF-beta1 transient reduced the abundance of SMA in hepatic stellate cells.

CONCLUSIONS

TGF-beta may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell trans-differentiation.

Idiopathic pulmonary arterial hypertension (formerly primary pulmonary hypertension) can affect more than one member of the same family. In the past 25 years scientists have exposed the inheritance pattern and a major element of the molecular basis for inherited pulmonary arterial hypertension. Familial pulmonary arterial hypertension is inherited as an autosomal dominant trait with incomplete penetrance (i.e., several individuals inherit a predisposition to the disease, but never express it). Mutations in the gene that codes for <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type II (BMPR-II) are a major predisposition for the development of pulmonary arterial hypertension. These mutations are present in at least half of familial cases of pulmonary arterial hypertension and <em>10</em> to 25% of idiopathic pulmonary arterial hypertension patients. Mutations in the gene that codes for activin receptor-like kinase (ALK 1), another transforming growth factor beta (TGF-beta) cell surface receptor, appear responsible for the rare occurrence of pulmonary arterial hypertension in patients with hereditary hemorrhagic telangiectasia. These discoveries coupled with other basic investigations offer opportunities for advances in the management of pulmonary arterial hypertension.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) induce cartilage and <em>bone</em> differentiation in vivo and promote osteoblast differentiation from calvarial and marrow stromal cell preparations. Functional differences between BMP-2, -4, and -6 are not well understood. Recent investigations find that these three closely related osteoinductive <em>proteins</em> may exert different effects in primary rat calvarial cell cultures, suggesting the possibility of unique functions in vivo. In this study, we use a fetal rat secondary calvarial cell culture system to examine the differential effects of BMP-2, -4, and -6 on early osteoblast differentiation. These cells do not spontaneously differentiate into osteoblasts, as do cells in primary calvarial cultures, but rather require exposure to a differentiation initiator such as glucocorticoid or BMP. We determined that BMP-6 is a 2- to 2.5-fold more potent inducer of osteoblast differentiation than BMP-2 or -4. BMP-6 induced the formation of more and larger <em>bone</em> nodules as well as increased osteocalcin secretion. The effects of all three of these BMPs were potentiated up to <em>10</em>-fold by cotreatment or pretreatment with the glucocorticoid triamcinolone (Trm). The Trm effects were synergistic with those of BMP-2 or -4, suggesting that this glucocorticoid may increase the cell responsiveness to these BMPs. Finally, BMP-6 did not require either cotreatment or pretreatment with Trm to achieve greater amounts of osteoblast differentiation than seen with BMP-2 or BMP-4 treatment, suggesting that BMP-6 may act at an earlier stage of cell differentiation.

BACKGROUND

Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial sutures and the factors regulating its expression in vitro.

METHODS

Microarray analysis was performed on rat posterior frontal and sagittal cranial sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonsuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction.

RESULTS

BMP3 expression in the posterior frontal suture decreased over the time course analyzed, whereas it increased in the sagittal suture. Notably, BMP3 expression was higher in the patent sagittal suture during the window of posterior frontal suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01).

CONCLUSIONS

BMP3 is expressed in rat cranial sutures in a temporal pattern suggesting involvement in cranial suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial suture biology.

Ficolins are oligomeric lectins comprising a collagen-like and a fibrinogen-like domain, with a binding specificity for N-acetylglucosamine. It has been reported recently that L-ficolin/P35 associates with mannan-binding lectin (MBL)-associated serine proteases (MASP-1 and -2) and MBL-associated <em>protein</em> 19 (MAp19) in serum and forms complexes able to activate complement. Using surface plasmon resonance spectroscopy we have shown that recombinant MASP-1 and -2, their N-terminal CUB1 (module originally found in complement <em>proteins</em> C1r/C1s, Uegf, and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-1)-epidermal growth factor (EGF)-CUB2 and CUB1-EGF segments, and MAp19 bind to immobilized L-ficolin/P35 in the presence of Ca(2+) ions. Comparable K(d) values were obtained for the full-length proteases and their CUB1-EGF-CUB2 segments (9.2 and <em>10</em> nM for MASP-1 and 4.6 and 5.4 nM for MASP-2, respectively), whereas higher values were obtained for the CUB1-EGF segments (26.7, 15.6, and 14.3 nM for MASP-1, MASP-2, and MAp19). These values are in the same range as those determined for the interaction of these <em>proteins</em> with MBL. Binding was Ca(2+) dependent and was only partly sensitive to EDTA for MASP-1, MASP-2, and MASP-2 CUB1-EGF-CUB2. Half-maximal binding was obtained at comparable Ca(2+) concentrations for MASP-1 and MASP-2 (0.45 and 0.47 micro M, respectively), their CUB1-EGF-CUB2 segments (0.37 and 0.72 micro M), and their CUB1-EGF segments (0.31 and 0.79 micro M). These values are lower than those determined in the case of MBL, indicating a difference between MBL and L-ficolin/P35 with respect to the Ca(2+) dependence of their interaction with the MASPs. Preincubation of the MASPs with soluble MBL inhibited subsequent binding to immobilized L-ficolin/P35 and, conversely, suggesting that these lectins compete with each other for binding to the MASPs in vivo.

The two marine inorganic polymers, biosilica (BS), enzymatically synthesized from ortho-silicate, and polyphosphate (polyP), a likewise enzymatically synthesized polymer consisting of <em>10</em> to>><em>10</em>0 phosphate residues linked by high-energy phosphoanhydride bonds, have previously been shown to display a <em>morphogenetic</em> effect on osteoblasts. In the present study, the effect of these polymers on the differential differentiation of human multipotent stromal cells (hMSC), mesenchymal stem cells, that had been encapsulated into beads of the biocompatible plant polymer alginate, was studied. The differentiation of the hMSCs in the alginate beads was directed either to the osteogenic cell lineage by exposure to an osteogenic medium (mineralization activation cocktail; differentiation into osteoblasts) or to the chondrogenic cell lineage by incubating in chondrocyte differentiation medium (triggering chondrocyte maturation). Both biosilica and polyP, applied as Ca²⁺ salts, were found to induce an increased mineralization in osteogenic cells; these inorganic polymers display also <em>morphogenetic</em> potential. The effects were substantiated by gene expression studies, which revealed that biosilica and polyP strongly and significantly increase the expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) and alkaline phosphatase (ALP) in osteogenic cells, which was significantly more pronounced in osteogenic versus chondrogenic cells. A differential effect of the two polymers was seen on the expression of the two collagen types, I and II. While collagen Type I is highly expressed in osteogenic cells, but not in chondrogenic cells after exposure to biosilica or polyP, the upregulation of the steady-state level of collagen Type II transcripts in chondrogenic cells is comparably stronger than in osteogenic cells. It is concluded that the two polymers, biosilica and polyP, are <em>morphogenetic</em>ally active additives for the otherwise biologically inert alginate polymer. It is proposed that alginate, supplemented with polyP and/or biosilica, is a suitable biomaterial that promotes the growth and differentiation of hMSCs and might be beneficial for application in 3D tissue printing of hMSCs and for the delivery of hMSCs in fractures, surgically created during distraction osteogenesis.

Members of the transforming growth factor-beta (TGF-beta) superfamily, the <em>bone</em> <em>morphogenetic</em> and osteogenic <em>proteins</em> (BMPs/OPs) but not the TGF-beta <em>proteins</em> themselves, induce endochondral <em>bone</em> formation in vivo, when implanted in extraskeletal heterotopic sites of rodents. Here we show that recombinant human TGF-beta2 (hTGF-beta2) induces endochondral <em>bone</em> formation 30 days after implantation in heterotopic intramuscular sites of the baboon (Papio ursinus) at doses of 1, 5 and 25 microg per <em>10</em>0 mg of guanidinium-inactivated collagenous <em>bone</em> matrix as carrier. On day 90 there was generation of large radiopaque and corticalized intramuscular ossicles. Five and 25 microg hTGF-beta2 induced large ossicles in the rectus abdominis of the primate as evaluated by key parameters of <em>bone</em> formation, including generated tissue area, mineralized <em>bone</em> and osteoid volumes, and tissue alkaline phosphatase activity. On day 30 and 90 after healing, hTGF-beta2 also induced <em>bone</em> formation when implanted in the rectus abdominis in conjunction with a sintered porous hydroxyapatite as carrier. mRNA expression in tissues from heterotopic specimens showed OP-1 (BMP-7) and BMP-3 transcripts in low abundance and with a linear dose-dependent increase both in collagenous matrix and hydroxyapatite samples. Type IV collagen mRNA expression, a marker of angiogenesis, was stronger in collagenous than hydroxyapatite samples. Growth and differentiation factor-<em>10</em> (GDF-<em>10</em>) mRNA transcripts were expressed in ossicles with a distinctly chondrogenic phase, but its expression was greater in ossicles generated in porous hydroxyapatites, in which <em>bone</em> formation is not via a chondrogenic phase, but is rather intramembranous, without expression of type II collagen mRNA. In the same animals, however, <em>10</em> and <em>10</em>0 microg of the recombinant morphogen delivered by identical carriers (collagenous matrix and sintered hydroxyapatite) failed to heal calvarial defects. Thus in the primate, TGF-betas themselves are inducers of endochondral <em>bone</em> formation, although the present data strongly indicate that the <em>bone</em> inductive activity of hTGF-beta2 is site and tissue specific, since a single application of hTGF-beta2, or hTGF-beta1 in previously published experiments, did not induce <em>bone</em> in calvarial defects, but did induce endochondral <em>bone</em> differentiation in heterotopic sites.

OBJECTIVE

To observe the feasibility of using fibrin gel as a scaffold during rabbit spinal fusion with rat kidney cells carrying bone morphogenetic protein-2 (BMP-2) expression vector.

METHODS

Normal Rattusnorvegicus (rat) kidney (NRK) cells were transfected with pCMV-BMP2 vectors by using a nonviral reagent (FuGENE6), following which the transfected cells (1.5 x 10(7)) were encapsulated and evenly suspended in a fibrin scaffold, and implanted to either side of the L5-L6 intertransverse space for six test rabbits. For the control group (six rabbits), the transfected cells (1.5 x 10(7)) were added on and absorbed in a surgical gelatin sponge, and implanted in an analogous location. Radiographs of the spine of all animals were taken at six, ten, and 12 weeks subsequent to fusion surgery. Gross and histological examination of the fusion masses for all the animals were performed subsequent to animals having been sacrificed at 12 weeks.

RESULTS

Expression of BMP-2 was verified in the pCMV-BMP2 transfected NRK cells which were used for the subsequent rabbit experiment. For all 12 rabbits, no evidence of implanted-tissue rejection was seen during the postoperative course. Neither residual scaffold nor inflammatory granulation tissue was seen in the harvested spinal segments. For the six study-group rabbits, radiographic examinations revealed that four individuals (67%) featured prominent new-bone formation (good fusion), and two (33%) revealed moderate new-bone formation (fair fusion) at the implanted sites, whereas all six control-group rabbits revealed no evidence of new-bone formation (nonfusion) at the implanted sites. For the histological examinations, all animals in the study group revealed the presence of osseous tissue at the sites corresponding to the sites of radiographically demonstrated new-bone formation, whereas for the control group, no osseous tissue was seen at the implanted sites for any animal

CONCLUSIONS

Fibrin gel, being a biocompatible and biodegradable matrix, can encapsulate pCMV-BMP2 transfected NRK cells and adhere to the intertransverse lumbar-spine spaces of test rabbits. With the above characteristics, it plays an important role in the successful delivery of pCMV-BMP2-transfected cells for this rabbit spinal-fusion experiment. Being a natural matrix and able to be obtained readily from patients' own blood, fibrin gel seems to be a promising scaffold for future gene therapy in human trials.

OBJECTIVE

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-beta superfamily that controls multiple functions in a variety of cells. We have previously shown that human optic nerve head (ONH) astrocytes and lamina cribrosa (LC) cells express BMP and BMP receptor mRNA and proteins. The purpose of the present study was to determine whether human ONH tissues express the canonical BMP signaling pathway and whether ONH cells secrete BMP-4 and respond to exogenous BMP-4 through this pathway.

METHODS

Well-characterized human ONH astrocytes (N = 2) and LC cells (N = 3) were treated with exogenous BMP-4 (20 ng/mL) for various times. Western immunoblot analysis was used to detect secreted BMP-4 in serum-free conditioned media of ONH cells and in human ONH tissues (N = 4) and Smad proteins in total cell lysate of ONH cells and tissues. Intracellular colocalization of p-R-Smad1 with Co-Smad4 and localization of inhibitory Smads (e.g., I-Smad6 and I-Smad7) were studied through immunocytochemistry. In addition, coimmunoprecipitation was used to verify the interaction of p-R-Smad1 with Co-Smad4.

RESULTS

ONH astrocytes and LC cells secrete BMP-4 and synthesize R-Smad1, R-Smad5, I-Smad6, I-Smad7, and Co-Smad4 proteins. Exposure to BMP-4 for either 10 or 60 minutes resulted in increased p-R-Smad1 and p-R-Smad1/5/8 protein levels that declined after 12 hours of treatment. Immunocytochemistry and coimmunoprecipitation studies revealed that p-R-Smad1/5/8 and Co-Smad4 interact and colocalize in the nucleus. BMP-4 treatment resulted in increased coprecipitation of p-R-Smad1/5/ 8 and Co-Smad4. I-Smad6 and I-Smad7 are localized in the nucleus and cytoplasm of ONH astrocytes and LC cells. Proteins for BMP-4, p-R-Smad1/5/8, R-Smad1, R-Smad5, R-Smad8, and Co-Smad4 are present in human ONH tissues. In addition, phosphorylated Smad1 and Smad5 colocalize with Smad4 in the nuclei of ONH tissues.

CONCLUSIONS

These results indicate that BMP-4 and Smad signaling proteins are present in human ONH tissues, isolated ONH astrocytes, and LC cells. In addition, exogenous BMP-4 treatment of ONH astrocytes and LC cells results in downstream signaling through the canonical Smad pathway. Thus, cells within the human ONH may respond to locally released BMP through paracrine or autocrine mechanisms.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) play a critical role in early skeletal development. BMPs are also potential mediators of <em>bone</em> response to mechanical loading, but their role in later stages of <em>bone</em> growth and adaptation has yet to be studied. We characterized the postcranial skeletal defects in mature mice with BMP deficiency by measuring hind-limb muscle mass and long <em>bone</em> geometric, material, and torsional mechanical properties. The animals studied were 26-week-old short ear mice (n = <em>10</em>) with a homozygous deletion of the BMP-5 gene and their heterozygous control litter mates (n = 15). Gender-related effects, which were found to be independent of genotype, were also examined. The femora of short ear mice were 3% shorter than in controls and had significantly lower values of many cross-sectional geometric and structural strength parameters (p < 0.05). No significant differences in ash content or material properties were detected. Lower femoral whole <em>bone</em> torsional strength was due to the smaller cross-sectional geometry (16% smaller section modulus) in the short ear mice. The diminished cross-sectional geometry may be commensurate with lower levels of in vivo loading, as reflected by body mass (-8%) and quadriceps mass (-11%). While no significant gender differences were found in whole <em>bone</em> strength or cross-sectional geometry, males had significantly greater body mass (+18%) and quadriceps mass (+15%) and lower tibio-fibular ash content (-3%). The data suggest that adult female mice have a more robust skeleton than males, relative to in vivo mechanical demands. Furthermore, although the <em>bones</em> of short ear mice are smaller and weaker than in control animals, they appear to be biomechanically appropriate for the in vivo mechanical loads that they experience.

Growth differentiation factor-9 (GDF-9) and <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are crucial factors in follicular growth and development. GDF-9 and BMPs initiate signaling by assembling type I (ALK-3, ALK-5 and ALK-6) and type II (BMPRII) receptors. However, the mechanism regulating the expression of these receptors in the process of bovine follicle development is still unknown. The aim of the present study was to clarify the involvement of receptor systems for GDF-9 and BMPs in follicular selection by examining the effects of FSH and estradiol-17beta (E2) on the regulation of BMPRII, ALK-3, ALK-5 and ALK-6 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression during follicular development, follicles were obtained from heifers and classified into two groups: pre-selection follicles (PRFs) (an average of 7.7 mm follicles with low E2) and post-selection follicles (POFs) (an average of 15 mm follicles with high E2). Theca layer cells (TCs) and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7 mm diameter and cultured in Dulbecco's modified Eagle's/F12 (DMEM/F-12) medium with <em>10</em>% fetal calf serum for 24 h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, <em>10</em>, <em>10</em>0 ng/ml) or FSH (1, 5, <em>10</em> ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses. Total RNA was extracted and the mRNA expression of BMPRII, ALK-3, ALK-5 and ALK-6 was estimated by the quantitative real-time PCR method using a LightCycler. BMPRII and ALK-5 expression was significantly higher in the GCs of POFs than in those of PRFs, whereas ALK-3 expression was significantly lower in the GCs of POFs than in those of PRFs. There was no difference in ALK-6 expression in GCs between PRFs and POFs. The expression of BMPRII, ALK-5, ALK-3 and ALK-6 genes in the TCs was not significantly different between PRFs and POFs. Treatment of GCs with E2 alone increased BMPRII mRNA expression at a concentration of <em>10</em>0 ng/ml and ALK-5 mRNA expression at <em>10</em> ng/ml. BMPRII and ALK-5 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). On the other hand, FSH alone down-regulated the expression of BMPRII and ALK-5 in GCs. The results of the present study provide the first evidence that FSH and E2 regulate the expression of BMPRII and ALK-5 genes in bovine GCs. Thus, our data suggest that the GDF-9/BMPRII/ALK-5 system may be critically involved in the process of selection of bovine follicles.

OBJECTIVE

The treatment of vertebral osteomyelitis includes antibiotics with or without surgical intervention. The decision to place instrumentation into an infected spinal column remains controversial. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in patients with osteomyelitis is also extremely controversial. The authors review their experience in performing corpectomy and fusion with titanium cages and rhBMP-2 in patients with vertebral instability and/or neurological compromise due to vertebral osteomyelitis.

METHODS

Data obtained in 15 patients treated between 2001 and 2005 were included in this analysis. Nine patients presented primarily with axial pain and six with radiculopathy or myelopathy. Seven patients had an associated epidural abscess. The cervical spine was affected in six patients, the thoracic spine in five, and the lumbar spine in four. All patients underwent corpectomy of the involved vertebral bodies; the authors then performed spinal reconstruction, placing a titanium cage-plate system with morcellized allograft/autograft and rhBMP-2. In 10 patients, supplemental posterolateral screw-rod fixation was conducted. A one-level corpectomy was performed in one patient, a two-level corpectomy in 13, and a six-level corpectomy in one. A morcellized allograft and rhBMP-2-filled titanium cage was used in 10 patients, and an autograft and rhBMP-2-filled cage in five patients. The most common pathogen was Staphylococcus aureus. All patients received intravenous antibiotics for at least 6 weeks postoperatively, and life-long antibiotic therapy was required in three patients with coccidiomycoses, candida, and tuberculosis osteomyelitis, respectively. There were no recurrent infections. Radiography demonstrated evidence of fusion in all patients at the last follow-up examination. The mean follow-up period was 20 months.

CONCLUSIONS

Corpectomy followed by titanium cage-plate reconstruction and the placement of rhBMP-2 may be a safe and effective treatment for selected patients with vertebral osteomyelitis. This surgical therapy does not appear, at least based on preliminary results, to lead to recurrent hardware infections. Based on the results obtained in this limited series, the authors found that rhBMP-2 can be used in the setting of active infection with excellent fusion rates and without complication. The morbidity associated with the autograft donor site is avoided when using cages. Antibiotic therapy tailored to the specific organism should be continued for at least 6 weeks after surgery, and life-long therapy is required in cases of fungal or tuberculosis infections.

Elevated wall stress by hypertension induces an adaptive myocardial hypertrophy via releasing prohypertrophic hormones such as angiotensin II. In this study, we investigated the involvement of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-<em>10</em> (BMP<em>10</em>) in hypertension-induced cardiac hypertrophy. Expression of BMP<em>10</em> was increased in the hypertrophied ventricles from hypertensive rats. BMP<em>10</em> localized on cell surface and at stretch-sensing Z disc of cardiomyocytes, where BMP<em>10</em> interacted with a <em>protein</em> called titin-cap (Tcap). A rare variant of the human BMP<em>10</em> gene, Thr326Ile, was found to be associated with hypertensive dilated cardiomyopathy. The variant BMP<em>10</em> demonstrated decreased binding to Tcap and increased extracellular secretion. Conditioned medium from cells transfected with wild-type or variant BMP<em>10</em> induced hypertrophy in rat neonatal cardiomyocytes, except that medium from variant BMP<em>10</em>-carrying cells showed an enhanced effect reflecting the increased secretion. These observations suggested that hypertension induced expression of prohypertrophic BMP<em>10</em>, and the hypertrophic effect of BMP<em>10</em> was modulated, at least in part, by its binding to Tcap at the Z disc.

This study was designed to show whether rat liver epithelial cells could undergo epithelial-mesenchymal transition (EMT), thereby directly contributing to liver fibrosis. The role of the ratio of transforming growth factor-β1 (TGF-β1)/<em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) was evaluated in the progression of EMT or mesenchymal-epithelial transition. Primary rat liver epithelial cells were stimulated with different ratios of TGF-β1/BMP-7 and examined for evidence of transition to a mesenchymal or epithelial phenotype. Liver sections were labeled to detect antigens associated with liver epithelial cells [E-cadherin (E-cad)], EMT [fibroblast-specific <em>protein</em>-1 (FSP-1), vimentin], myofibroblasts [α-smooth muscle actin (α-SMA)], and intracellular signal-transduction mediated by forming liver fibrosis undergo EMT, resulting in the formation of invasive fibroblasts; this process may be driven or impeded by a response to local TGF-β1 or BMP-7. BMP-7 downregulated α-SMA and phosphorylated Smad2/3. Stimulation of cultured cells with TGF-β1 induced the expression of pSmad2/3, FSP-1, and α-SMA. Stimulation of cultured cells with BMP-7 induced the expression of E-cad. We demonstrated that the cells upregulated E-cad release compared with untreated cells, but TGF-β1 was different. We found that the equilibrium of the ratio of TGF-β1/BMP-7 was 1/<em>10</em>. In summary, the mechanism for this process was not determined. Demonstration of the contribution of what the ratio of TGF-β1/BMP-7 induced to EMT to the chronic liver diseases would provide a new basis for understanding pathogenesis and potential treatment.

BACKGROUND

Deletions within the short arm of chromosome 7 are observed in approximately 25% of adult and <em>10</em>% of Wilms pediatric renal tumors. Within Wilms tumors, the region of interest has been delineated to a 2-Mb minimal region that includes ten known genes. Two of these ten candidate genes, SOSTDC1 and MEOX2, are particularly relevant to tumor development and maintenance. This finding, coupled with evidence that SOSTDC1 is frequently downregulated in adult renal cancer and regulates both Wingless-Int (Wnt)- and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-induced signaling, points to a role for SOSTDC1 as a potential tumor suppressor.

METHODS

To investigate this hypothesis, we interrogated the Oncomine database to examine the SOSTDC1 levels in adult renal clear cell tumors and pediatric Wilms tumors. We then performed single nucleotide polymorphism (SNP) and sequencing analyses of SOSTDC1 in 25 pediatric and 36 adult renal tumors. Immunohistochemical staining of patient samples was utilized to examine the impact of SOSTDC1 genetic aberrations on SOSTDC1 protein levels and signaling.

RESULTS

Within the Oncomine database, we found that SOSTDC1 levels were reduced in adult renal clear cell tumors and pediatric Wilms tumors. Through SNP and sequencing analyses of 25 Wilms tumors, we identified four with loss of heterozygosity (LOH) at 7p and three that affected SOSTDC1. Of 36 adult renal cancers, we found five with LOH at 7p, two of which affected SOSTDC1. Immunohistochemical analysis of SOSTDC1 protein levels within these tumors did not reveal a relationship between these instances of SOSTDC1 LOH and SOSTDC1 protein levels. Moreover, we could not discern any impact of these genetic alterations on Wnt signaling as measured by altered beta-catenin levels or localization.

CONCLUSIONS

This study shows that genetic aberrations near SOSTDC1 are not uncommon in renal cancer, and occur in adult as well as pediatric renal tumors. These observations of SOSTDC1 LOH, however, did not correspond with changes in SOSTDC1 protein levels or signaling regulation. Although our conclusions are limited by sample size, we suggest that an alternative mechanism such as epigenetic silencing of SOSTDC1 may be a key contributor to the reduced SOSTDC1 mRNA and protein levels observed in renal cancer.