It was hypothesized that monocyte treatment with <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP7) would significantly enhance monocyte polarization into M2 macrophages as well as increasing the levels of anti-inflammatory cytokines. In a cell culture system using monocytes (human acute monocytic leukemia cell line THP-1), we studied the effects of BMP7 on monocytes polarizing into M2 macrophages. The data demonstrate that THP-1 cells contain a BMP type II receptor (BMPR2), and that its activation is significantly (p < 0.05) increased following treatment with BMP7. Furthermore, there was an increase of M2 macrophages, BMPR2, and anti-inflammatory cytokines interleukin (IL)-<em>10</em> and IL-1ra compared with the respective controls. Moreover, treatment with BMP7 caused a significant (p < 0.05) decrease in the levels of pro-inflammatory cytokines IL-6, tumour necrosis factor (TNF-α), and monocyte chemotactic <em>protein</em>-1 (MCP-1), compared with the controls. In conclusion, we suggest for the first time that BMP7 has a unique potential to polarize monocytes into M2 macrophages, required for tissue repair, which will have significant applications for the treatment of atherosclerosis.

Treatment of critical size <em>bone</em> defects is challenging. Recent studies showed that the cytokine stromal cell-derived factor 1 alpha (SDF-1α) has potential to improve the <em>bone</em> regenerative effect of low <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) concentrations. The goal of this study was to demonstrate the combined effect of SDF-1α and BMP-2 on <em>bone</em> regeneration and stem cell recruitment using a critical size femoral <em>bone</em> defect model. A total of 72 mice were randomized to six groups. External fixators were implanted onto the right femur of each mouse and 3 mm defects were created. Depending on the group affiliation, adenovirally activated fat tissue grafts expressing SDF-1α or/and BMP-2 were implanted at the defect site. One day after operation, 1×<em>10</em>⁶ murine mesenchymal stromal cells (MSCs), lentivirally transduced to express the gene enhanced green fluorescent <em>protein</em> (eGFP), firefly luciferase, and CXCR4 were injected systemically in selected groups. Migration of the injected MSCs was observed by bioluminescence imaging on days 0, 2, 4, 6, 8, <em>10</em>, 12, 14, 21, 28, and 42. After 6 weeks, animals were euthanized and 80 μm CT-scans were performed. For histological investigations, hematoxylin and eosin-, tartrate-resistant acid phosphatase-, alkaline phosphatase-, and anti-eGFP-stained sections were prepared. BMP-2 and SDF-1α combined at the defect site increased <em>bone</em> volume (BV) (2.72 mm³; 95% CI 1.95-3.49 mm³) compared with the negative control group (1.80 mm³; 95% CI 1.56-2.04 mm³; p<0.05). In addition, histological analysis confirmed a higher degree of <em>bone</em> healing in the BMP-2 and SDF-1α combined group compared with the negative control group. Bioluminescence imaging demonstrated higher numbers of migrated MSCs toward the defect site in the presence of both BMP-2 and SDF-1α at the defect site. Furthermore, eGFP-labeled migrated MSCs were found in all defect areas, when cells were injected. The ratio of osteoblasts to osteoclasts, assessed by immunohistological staining, was higher and thus showed a trend toward more <em>bone</em> formation for the combined use of BMP-2 and SDF-1α compared with all other groups. This study demonstrated that SDF-1α enhanced BMP-2 mediated <em>bone</em> healing in a critical size segmental <em>bone</em> defect model. Notably, both <em>proteins</em> alone also provided a cumulative effect on MSC attraction toward the site of injury.

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-β3 and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, <em>10</em> µg·L⁻¹ TGF-β3 or <em>10</em>0 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-β3 and 220% by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-β3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and <em>bone</em> fracture in the field of cell therapy.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) signaling is critical for germline establishment during mouse embryogenesis. To exploit its importance for induction of germline precursors in vitro, mouse embryonic stem cells (mESCs) were cultured as embryoid body (EB) aggregates with combinations of BMP2, BMP4, and BMP8B for 3-<em>10</em> days. At Day <em>10</em> of culture, well-delineated clusters of POU5F1-positive (POU5F1+) cells were visible in BMP4-treated and BMP2-treated EBs; these were rarely detected in untreated and BMP8B-treated cultures. Quantitative mRNA analysis revealed that a significant elevation of markers associated with primordial germ cell development had occurred in the presence of BMP4 by Day <em>10</em>, including late germline markers such as Ddx4 (Mvh). Reasoning that germline specification was established by Day <em>10</em>, we surveyed earlier time points for altered levels of germline marker mRNAs. A peak of early markers, Prdm1 (Blimp1), Ifitm3 (Fragilis), and Dppa3 (Stella), was measured in Day 3 to Day 4 EBs grown in BMP4, followed by a decrease at Day 5. In contrast, other markers, Pou5f1, Nanog, Dazl, and Ddx4, progressively increased from Day 3 to Day 5. Transforming growth factor beta superfamily signaling components Acvr1 (ALK2), Smad1, and Smad5 remained relatively constant. Isolated POU5F1+ cells from BMP4-treated Day 5 EBs contained significantly elevated germline markers compared with POU5F1-negative cells, with a transcript profile differing from mESCs, verifying their unique identity. These results demonstrate that signaling by BMP2 and BMP4, but not BMP8B, enhances germline marker expression within EBs and identify Day 3 to Day 5 in EB differentiation as a window for specification of germ cells in vitro.

OBJECTIVE

To characterize and evaluate the validity of a novel coculture system for studying human B-lymphocyte developmental biology.

METHODS

We developed a long-term culture system to produce B lymphocytes from human CD34(+) cells purified from umbilical cord blood using human mesenchymal stem cells (hMSC) as stroma. We evaluated the effects of several low molecular weight inhibitors, recombinant proteins, and neutralizing antibodies (Abs) as potential regulators of B-lymphocyte development.

RESULTS

Our cocultures of 2000 CD34(+) cells in the presence of stem cell factor and Flt3-ligand produced 1-5 x 10(5) CD10(+) cells after 4 weeks of culture. Surface IgM(+) immature B cells began to appear after 4 weeks. We evaluated the negative-regulatory effects of the transforming growth factor (TGF)-beta superfamily on human B lymphopoiesis, and found that adding an anti-activin A antibody enhanced generation of CD10(+) cells two- to three-fold. As well, the proportion of CD10(+) cells in the generated cells increased markedly, indicating that activin A downregulated B lymphopoiesis more efficiently than myelopoiesis. Addition of TGF-beta1 suppressed B-lymphocyte production by 20% to 30%, while addition of an anti-bone morphogenetic protein (BMP)-4 antibody or recombinant BMP-4 had no effect. Therefore, the strength of ability to suppress human B lymphopoiesis seemed to be activin A>> TGF-beta1>> BMP-4. None of these three factors influenced the emergence of IgM(+) cells.

CONCLUSIONS

hMSC coculture supported human B lymphopoiesis. Activin A selectively suppressed B lymphocyte production.

Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) induced <em>bone</em> regeneration and osseointegration was evaluated in supraalveolar peri-implant defects in 5 beagle dogs. Alveolar <em>bone</em> was reduced 5 mm in height in mandibular premolar areas and the premolars were extracted. Three <em>10</em> mm titanium fixtures were placed 5 mm into each reduced alveolar crest, leaving 5 mm in a supraalveolar position. Alternate quadrants in consecutive animals received a surgical implant consisting of 2 ml rhBMP-2 (0.43 mg/ml) in a type I bovine collagen carrier, or the carrier alone (control). Fixtures and surgical implants were submerged under the gingival flaps. The healing interval was 16 weeks. Histometric and radiographic evaluations were made. Defect height averaged (+/- SD) 5.3 +/- 0.2 and 5.1 +/- 0.2 mm for rhBMP-2 and control defects, respectively (P>> 0.05, n = 5, paired, t-test) <em>Bone</em> regeneration (height) averaged 4.2 +/- 1.0 and 0.5 +/- 0.3 mm for rhBMP-2 and control defects, respectively (P < 0.05). <em>Bone</em> regeneration (area) averaged 6.1 +/- 6.3 and 0.2 +/- 0.2 mm2 for rhBMP-2 and control defects, respectively (P>> 0.05). Osseointegration within the confines of the defect averaged 19.1 +/- <em>10</em>.1% and 8.2 +/- 4.6% for rhBMP-2 and control defects, respectively (P>> 0.05). Osseointegration within regenerated <em>bone</em> averaged 29.1 +/- 9.8% and 65.3 +/- 15.3% for rhBMP-2 and control defects, respectively (P < 0.05). Osseointegration within the retained alveolar <em>bone</em> averaged 67.1 +/- 4.6% and 68.1 +/- 3.6% for rhBMP-2 and control defects, respectively (P>> 0.05). The results suggest that there is a potential for rhBMP-2 induced <em>bone</em> regeneration and osseointegration in surgical peri-implant defects.

Osteoblasts are mechanosensitive cells, which respond to biomechanical stimuli to regulate the <em>bone</em> structure through anabolic and catabolic gene regulation. To examine the effects of mechanical forces on the osteogenic responses through the SMAD signaling in osteoblasts, the cells were cultured in well-characterized mechanoresponsive 3-D scaffolds and exposed to <em>10</em>% dynamic compressive strain (Cmp) at 1 Hz. Subsequently, SMAD phosphorylation and osteogenic gene induction was examined. Osteoblasts cultured in 3-D scaffolds exhibited increased constitutive SMAD 1/5/8 phosphorylation, as compared to monolayers cultures. This SMAD 1/5/8 phosphorylation was further upregulated after <em>10</em>, 30 and 60 min in response to Cmp, exhibiting a peak activation at 30 min. No significant changes in SMAD2 phosphorylation were observed, suggesting signals generated by Cmp may not activate the Transforming Growth Factor-β signaling cascade. Subsequently, biomechanical stimulation-induced SMAD 1/5/8 phosphorylation upregulated the expression of osteogenic genes such as Osteoprotegrin, Msx2 and Runx2. Dorsomorphin, a selective inhibitor of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) receptor type 1 (BMPR1), blocked Cmp-induced SMAD 1/5/8 phosphorylation, as well as Osteoprotegrin, Msx2 and Runx2 gene expression. Collectively, the present findings demonstrate that biomechanical stimulation of osteoblasts activates SMAD 1/5/8 in the BMP signaling pathway through BMPR1 and may enhance osteogenesis by upregulating SMAD-dependent osteogenic genes.

Pediatric craniofacial surgery is complicated by a shortage of autologous <em>bone</em>. Children between 2 and <em>10</em> years of age are especially problematic, as the dura has lost its potential to spontaneously heal large calvarial defects by approximately 2 years of age, and split calvarial grafts are often unavailable because of the underdeveloped diploic space until later childhood. We demonstrate the efficacy of a BMP-2-based system in repairing large-scale cranial defects in a rabbit model. Calvarial defects, 15 mm, were created in 18 adult New Zealand white rabbits, treated as follows: group 1, no repair (n = 6); group 2, absorbable collagen sponge (ACS) (n = 4); and group 3, recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 delivered on ACS (rhBMP-2/ACS) (n = 8). <em>Bone</em> regeneration 6 weeks postoperatively was evaluated by 2- and 3-dimensional standard computed tomography, micro-computed tomography. Analysis of variance was performed using SPSS. The generated <em>bone</em> was also evaluated histologically. After 6 weeks, group 1 defects were on average 32.8% (SD, 8.8%) ossified. Group 2 defects were on average 34.4% (SD, 17.1%) ossified. Defects in group 3 were on average 96.9% (SD, 3.7%) ossified, significantly (P < 0.005) more than the defects in groups 1 and 2. rhBMP-2-induced <em>bone</em> was histologically and radiographically consistent with native <em>bone</em>. This study demonstrates the efficacy of rhBMP-2/ACS for the repair of calvarial defects in the rabbit model. rhBMP-2/ACS may offer a viable treatment option for craniofacial surgeons facing a shortage of <em>bone</em>, with the potential to replace autologous <em>bone</em> grafts and render their attendant morbidities obsolete.

BACKGROUND

Currently, more than 20 bone morphogenetic proteins (BMPs) have been identified, and many trials have been carried out using recombinant human BMPs (rhBMPs) for bone tissue engineering. However, comparative analyses on bone formative activities of rhBMP using a preclinical model have been limited. Therefore, the aim of this study was to evaluate and compare the osteogenic potential of rhBMP-2, -4, and -7 delivered with absorbable collagen sponge (ACS) upon early (2 weeks) and complete (8 weeks) wound healing phases in a critical sized rat calvarial defect model.

METHODS

Eight-millimeter critical sized calvarial defects were created in 30 male Sprague-Dawley rats. The animals were divided into three groups of 10 animals each. The defects were treated with 0.025 mg/ml rhBMP-2/ACS, rhBMP-4/ACS, or rhBMP-7/ACS. The rats were sacrificed at either 2 (five rats) or 8 (five rats) weeks after surgery, and the results were evaluated histologically, histomorphometrically, and immunohistometrically.

RESULTS

The surgical implantation of rhBMP-2/ACS, rhBMP-4/ACS, or rhBMP-7/ACS resulted in enhanced local bone formation in the rat calvarial defect model at both 2 and 8 weeks. The amount of defect closure, new bone area, and bone density were similar in the three groups at each time point (P>> 0.05). In terms of bone density and new bone area, there were statistically significant differences between results obtained at 2 weeks and those obtained at 8 weeks in all groups (P < 0.05). Two-way analysis of variance (ANOVA) revealed that there was no correlation between the time and conditions (P>> 0.05), but time was found to have a strong influence on defect closure, new bone area, and bone density (P < 0.05). Irrespective of rhBMP type, positive immunoreactions of osteopontin (OPN) and osteocalcin (OCN) were evident at 2 and 8 weeks. Intense OPN and OCN staining was observed near the newly formed bone as well as in some cells within the new bone.

CONCLUSIONS

Within the rhBMP types used, rhBMP concentration, and the observation interval, there appears to be no specific differences in bone regenerative potential. All rhBMPs used in this study may be considered effective factors for inducing bone formation.

BACKGROUND

Familial pulmonary arterial hypertension (FPAH) is a rare, autosomal-dominant, inherited disease with low penetrance. Mutations in the <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 2 (BMPR2) have been identified in at least 70% of FPAH patients. However, the lifetime penetrance of these BMPR2 mutations is <em>10</em>% to 20%, suggesting that genetic and/or environmental modifiers are required for disease expression. Our goal in this study was to identify genetic loci that may influence FPAH expression in BMPR2 mutation carriers.

METHODS

We performed a genome-wide linkage scan in 15 FPAH families segregating for BMPR2 mutations. We used a dense single-nucleotide polymorphism (SNP) array and a novel multi-scan linkage procedure that provides increased power and precision for the localization of linked loci.

RESULTS

We observed linkage evidence in four regions: 3q22 ([median log of the odds (LOD) = 3.43]), 3p12 (median LOD) = 2.35), 2p22 (median LOD = 2.21), and 13q21 (median LOD = 2.09). When used in conjunction with the non-parametric bootstrap, our approach yields high-resolution to identify candidate gene regions containing putative BMPR2-interacting genes. Imputation of the disease model by LOD-score maximization indicates that the 3q22 locus alone predicts most FPAH cases in BMPR2 mutation carriers, providing strong evidence that BMPR2 and the 3q22 locus interact epistatically.

CONCLUSIONS

Our findings suggest that genotypes at loci in the newly identified regions, especially at 3q22, could improve FPAH risk prediction in FPAH families. We also suggest other targets for therapeutic intervention.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are biologically active molecules capable of eliciting new <em>bone</em> formation. In combination with biomaterials, these <em>proteins</em> can be used in a clinical setting as <em>bone</em>-graft substitutes to promote <em>bone</em> repair. To find new synthetic absorbable polymers with plastic nature that can be used as BMP-carrier materials, six types of poly-D,L-lactic acid-polyethylene glycol block copolymer (PLA-PEG) with various molecular weights of PLA and PEG were synthesized. These were PLA6, 500-PEG3,000 (P-1), PLA11,500-PEG3,000 (P-2), PLA17,500-PEG3,000 (P-3), PLA6,500-PEG1,000 (P-4), PLA15,000-PEG8,000 (P-5), and PLA8, 500-PEG1,000 (P-6). Fifty milligrams of these polymers was mixed with 0 microg (control) or 5, <em>10</em>, or 20 microg of recombinant human BMP-2 (rhBMP-2). These pellets were implanted into the dorsal muscle pouches of 144 mice (six pellets consisting of the same polymer and dose of rhBMP-2 for a specific group). Three weeks after surgery, the pellets were harvested and examined by radiographic and histological methods. All P-1 pellets with <em>10</em> or 20 microg of rhBMP-2 showed <em>bone</em> formation with hematopoietic marrow and bony trabeculae, as did one third of those with 5 microg of rhBMP-2. The incidence of new <em>bone</em> formation with P-2 pellets or that of P-5 pellets was lower than that of P-1 pellets. No <em>bone</em> was formed in any other type of pellet. These results indicated that the PLA6, 500-PEG3,000 polymer with plastic properties was found to work well as a BMP carrier.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are also implicated in the commitment of mesenchymal stem cells (MSCs) toward adipocytes. We tested that stretching of cells may downregulate BMP4 induction of MSC adipogenesis. C3H<em>10</em>T1/2 MSCs were pretreated with BMP4 and induced to differentiate to adipocytes using adipogenic hormonal inducers. To test the stretch effect on BMP4 function, cells were exposed to cyclic tensile stretch (<em>10</em>% strain, 0.25Hz, 120min/day) during the BMP4 pretreatment period. BMP4 induced MSC adipocytic commitment. Stretching during the BMP4 exposure could suppress BMP4 induction of MSC adipogenesis, as assessed by downregulated adipogenic transcription factors (PPARγ, C/EBPα, aP2) and decreased lipid accumulation. BMP4 signaled through Smad1/5/8 and p38MAPK, whereas cell stretch did not affect BMP4-induced activation in Smad or p38. On the other hand, cell stretch triggered significant ERK1/2 phosphorylation relative to BMP4 treatment alone cells. Further, stretch suppression of BMP4-induced MSC adipogenesis was significantly deteriorated if cells were stretched with ERK blocked by PD98059. Combined, these suggest that cell stretch suppresses the BMP4 induction of MSC adipogenesis potentially via upregulating ERK but not through the downregulation of Smad or p38. Our data on inhibiting MSC adipogenesis will be of significant interest for obesity and developmental mechanobiology studies.

In spite of previous reports, the precise role of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) on cardiomyocyte differentiation, especially in the absence or presence of minimum amount of serum in culture medium is still unclear. So, the aim of the present study was to investigate the effect of BMP-4 on mouse embryonic stem cells (ESCs)-derived cardiomyocyte differentiation in serum-free and low-serum media. The mouse ESCs differentiation to cardiomyocytes was induced by embryoid bodies' (EBs') development through hanging drop, suspension and plating stages. Different models of differentiation were designed according to addition of fetal bovine serum (FBS) or knockout serum replacement (KoSR) to the medium of three stages. <em>10</em> ng/ml BMP-4 was added throughout the suspension period. Up to 30 days after plating, contraction and beating frequency were monitored and evaluated daily. The growth characteristics of cardiomyocytes were assessed by cardioactive drugs, immunocytochemistry, transmission electron microscopy (TEM) and reverse transcription-polymerase chain reaction (RT-PCR). In the complete absence of serum, neither control nor BMP-4 treated groups resulted in cardiac differentiation. Addition of FBS to hanging drop stage resulted in the appearance of beating cardiac clusters in some BMP-4 treated EBs. In the best designed differentiation model in which only hanging drop and the first 24 h of plating stage was carried out at the presence of FBS, the BMP-4 treatment resulted in cardiac differentiation in EBs characterized by positive immunostaining for the applied antibodies, chronotropic response to the cardioactive drugs and cardiac-specific genes expression at different developmental stages. These cardiomyocytes showed immature myofibrils and numerous intercellular junctions. In conclusion, BMP-4 is unable to induce cardiomyocyte differentiation from mouse ESCs in serum-free models, and at least small amount of FBS in hanging drop stage is necessary. Furthermore, serum factors are not strictly necessary after the initial activation, but they do favor a better differentiation of cardiomyocytes.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (<em>10</em>(-)<em>10</em> - <em>10</em>(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 <em>protein</em> production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on <em>bone</em> and cartilage may be mediated by increased production of BMP-6 by osteoblasts.

METHODS

A non-human primate lumbar intertransverse process arthrodesis model was used to evaluate the effectiveness of different formulations of recombinant human bone morphogenetic protein-2 (rhBMP-2) to induce consistent bone formation.

OBJECTIVE

To determine if the combination of rhBMP-2/absorbable collagen sponge (ACS) wrapped around a bulking agent, consisting of a biphasic calcium phosphate/collagen composite, could achieve posterolateral spine fusion with a dose of rhBMP-2 (3.0 mg/side) that previously failed to induce posterolateral fusion in rhesus monkeys with other carriers.

BACKGROUND

Successful bone induction in both human and non-human primates has required higher concentrations of BMP than were required in lower order models. The Food and Drug Administration approved concentration of rhBMP-2 for interbody fusion (1.5 mg/mL) when delivered on the ACS alone without a bulking agent (doses 3-9 mg/side) has failed to induce clinically relevant amounts of bone formation in a posterolateral spine fusion model in rhesus monkeys. Previously, a higher concentration of 2.0 mg/mL of rhBMP-2 delivered on stacked sheets of a biphasic calcium phosphate ceramic/collagen compression resistant matrix (CRM) was required to achieve fusion in the rhesus monkey and was the basis for this study (doses of 6-12 mg/side).

METHODS

Nine skeletally mature, rhesus macaque monkeys underwent single level posterolateral arthrodesis at L4-L5. Two different rhBMP-2 doses were evaluated in 3 delivery configurations. The first 3 monkeys received 10 mg/side (2.5 mL at 4.0 mg/mL) of rhBMP-2 loadeddirectly onto a CRM carrier (15% hydroxyapatite/85%beta-tricalcium phosphate ceramic/collagen matrix), resulting in a final concentration of 2.0 mg/mL. The second 3 monkeys received 3 mg/side (2.0 mL at 1.5 mg/mL) of rhBMP-2 loaded directly on the CRM carrier, resulting in a 0.6 mg/mL final concentration. Three additional monkeys also received the 3 mg/side (2.0 mL at 1.5 mg/mL) of rhBMP-2 delivered on an ACS, which was then wrapped around the dry CRM matrix used as a bulking agent, yielding a 1.5 mg/mL final concentration of rhBMP-2 on the sponge wrapped around the bulking agent. The monkeys were euthanized at 24 weeks after surgery. Manual palpation, plain radiographs, computerized tomography, and nondecalcified histology were used to evaluate fusion in a blinded fashion.

RESULTS

The 3 monkeys with 10 mg rhBMP-2 placed directly on the CRM carrier (2.0 mg/mL final concentration) achieved solid fusion. The 3 monkeys that underwent fusion with 3 mg of rhBMP-2 placed directly on the CRM carrier (0.6 mg/mL final concentration) failed to achieve fusion. In contrast, the 3 monkeys that underwent fusion with the same 3 mg dose of rhBMP-2 dispensed only on an ACS that was wrapped around the CRM achieved solid bilateral fusion.

CONCLUSIONS

This study shows the importance of carrier optimization and final implant protein concentration for the successful delivery of rhBMP-2. By combining the properties of the ACS with the CRM, the required dosage of rhBMP-2 was diminished by more than 3-fold in the non-human primate model. This finding suggests that the currently available concentration of rhBMP-2 (1.5 mg/mL) could be successful for achieving posterolateral spine fusion when combined with an osteoconductive bulking agent that can support the induced new bone formation.

BACKGROUND

Adipose-derived stromal cells are a potential cell source for the successful healing of skeletal defects. In this study, the authors sought to investigate the potential for cranial suture-derived mesenchymal cells to promote the osteogenic differentiation of adipose-derived stromal cells. Various reports have previously examined the unique in vitro attributes of suture-derived mesenchymal cells; this study sought to extend those findings.

METHODS

Suture-derived mesenchymal cells were isolated from wild-type mice (n = 30) from both fusing posterofrontal and patent sagittal sutures. Cells were placed in Transwell inserts with human adipose-derived stromal cells (n = 5 patients) with osteogenic differentiation medium with or without recombinant Noggin (<em>10</em> to 400 ng/ml). Specific gene expression of osteogenic markers and Hedgehog pathway were assayed; standard osteogenic assays (alkaline phosphatase and alizarin red staining) were performed. All assays were performed in triplicate.

RESULTS

Both posterofrontal and sagittal suture-derived mesenchymal cells induced osteogenic differentiation of adipose-derived stromal cells (p < 0.05). Posterofrontal suture-derived mesenchymal cells induced adipose-derived stromal cell osteogenesis to a greater degree than sagittal suture-derived mesenchymal cells (p < 0.05). This was accompanied by an increase in bone morphogenetic protein expression (p < 0.05). Finally, recombinant Noggin mitigated the pro-osteogenic effects of co-culture accompanied by a reduction in Hedgehog signaling (p < 0.05).

CONCLUSIONS

Suture-derived mesenchymal cells secrete paracrine factors that induce osteogenic differentiation of multipotent stromal cells (human adipose-derived stromal cells). Cells derived from the fusing posterofrontal suture do this to a significantly greater degree than cells from the patent sagittal suture. Enhanced bone morphogenetic protein and Hedgehog signaling may underlie this paracrine effect.

The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (rhBMP-7) on the collagen types and the quantity of the collagen cross-links synthesized in a three-dimensional culture on various biomaterials for <em>bone</em> replacement. Trabecular <em>bone</em> chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP-7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and <em>10</em>0 microl osteoblasts (at a density of 3.3 x <em>10</em>(5)) were transferred onto nine blocks of either Bio-Oss, Tutoplast, or PepGen p-15. Blocks incubated with cells that were not treated with rhBMP-7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the collagen cross-link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized <em>bone</em> specific LP and collagen type I. However, in rhBMP-7-stimulated samples, the amount of HP and LP found was increased by 45% compared to non-stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP-7. In vitro stimulation of osteoblasts on Bio-Oss, Tutoplast, or PepGen p-15 with rhBMP-7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo.

BACKGROUND

The critical event in heart formation is commitment of mesodermal cells to a cardiomyogenic fate, and cardiac fate determination is regulated by a series of cytokines. Bone morphogenetic proteins (BMPs) and fibroblast growth factors have been shown to be involved in this process, however additional factors needs to be identified for the fate determination, especially at the early stage of cardiomyogenic development.

RESULTS

Global gene expression analysis using a series of human cells with a cardiomyogenic potential suggested Gremlin (Grem1) is a candidate gene responsible for in vitro cardiomyogenic differentiation. Grem1, a known BMP antagonist, enhanced DMSO-induced cardiomyogenesis of P19CL6 embryonal carcinoma cells (CL6 cells) 10-35 fold in an area of beating differentiated cardiomyocytes. The Grem1 action was most effective at the early differentiation stage when CL6 cells were destined to cardiomyogenesis, and was mediated through inhibition of BMP2. Furthermore, BMP2 inhibited Wnt/beta-catenin signaling that promoted CL6 cardiomyogenesis.

CONCLUSIONS

Grem1 enhances the determined path to cardiomyogenesis in a stage-specific manner, and inhibition of the BMP signaling pathway is involved in initial determination of Grem1-promoted cardiomyogenesis. Our results shed new light on renewal of the cardiovascular system using Grem1 in human.

Recent endeavors to use stem cells as trophic factor production sources have the potential to translate into viable therapies for damaged or diseased musculoskeletal tissues. Adipose stem cells (ASCs) can be differentiated into chondrocytes using the chondrogenic medium (CM), but it is unknown if this approach can optimize ASC growth factor secretion for cartilage regeneration by increasing the chondrogenic factor production, while decreasing angiogenic and hypertrophic factor production. The objective of this study was to determine the effects the CM and its components have on growth factor production from ASCs to promote cartilage regeneration. ASCs isolated from male Sprague-Dawley rats and cultured in monolayer or alginate microbeads were treated with either the growth medium (GM) or the CM for 5 days. In subsequent studies, ASC monolayers were treated with either the GM supplemented with different combinations of 50 μg/mL ascorbic acid-2-phosphate (AA2P), <em>10</em>0 nM dexamethasone (Dex), <em>10</em> ng/mL transforming growth factor (TGF)-β1, and <em>10</em>0 ng/mL <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-6 or with the CM excluding different combinations of AA2P, Dex, TGF-β1, and BMP-6. mRNA levels and growth factor production were quantified at 8 and 24 h after the last media change, respectively. The CM increased chondrogenic factor secretion (TGF-β2, TGF-β3, and insulin-like growth factor [IGF]-I) and decreased angiogenic factor production (the vascular endothelial growth factor [VEGF]-A, the fibroblast growth factor [FGF]-2). Microencapsulation in the GM increased production of the chondrogenic (IGF-I, TGF-β2) and angiogenic (VEGF-A) factors. AA2P increased secretion of chondrogenic factors (IGF-I, TGF-β2), and decreased angiogenic factor (VEGF-A) secretion, in addition to decreasing mRNA levels for factors associated with chondrocyte hypertrophy (FGF-18). Dex increased mRNA levels for hypertrophic factors (BMP-2, FGF-18) and decreased angiogenic factor secretion (VEGF-A). TGF-β1 increased angiogenic factor production (FGF-2, VEGF-A) and decreased chondrogenic factor mRNA levels (IGF-I, PTHrP). BMP-6 increased hypertrophic mRNA levels (FGF-18) and chondrogenic factor production (TGF-β2). When ASC microbeads preconditioned with the CM were implanted in a focal cartilage defect and immobilized within an RGD-conjugated hydrogel, tissue infiltration from the edges of the defect and perichondrium was observed. These results show that differentiation media components have distinct effects on ASC's production of angiogenic, chondrogenic, and hypertrophic factors and that AA2P may be the most beneficial CM component for preconditioning ASCs to stimulate cartilage regeneration.

BACKGROUND

Tissue engineering of human nasal septal chondrocytes offers the potential to create large quantities of autologous material for use in reconstructive surgery of the head and neck. Culture with recombinant human growth factors may improve the biochemical and biomechanical properties of engineered tissue. The objectives of this study were to (1) perform a high-throughput screen to assess multiple combinations of growth factors and (2) perform more detailed testing of candidates identified in part I.

METHODS

In part I, human nasal septal chondrocytes from three donors were expanded in monolayer with pooled human serum (HS). Cells were then embedded in alginate beads for 2 weeks of culture in medium supplemented with 2% or <em>10</em>% HS and 1 of 90 different growth factor combinations. Combinations of insulin-like growth factor-I (IGF-1), <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2, BMP-7, BMP-13, growth differentiation factor-5 (GDF-5), transforming growth factor β (TGFβ)-2, insulin, and dexamethasone were evaluated. Glycosaminoglycan (GAG) accumulation was measured. A combination of IGF-1 and GDF-5 was selected for further testing based on the results of part I. Chondrocytes from four donors underwent expansion followed by three-dimensional alginate culture for 2 weeks in medium supplemented with 2% or <em>10</em>% HS with or without IGF-1 and GDF-5. Chondrocytes and their associated matrix were then recovered and cultured for 4 weeks in 12 mm transwells in medium supplemented with 2% or <em>10</em>% HS with or without IGF-1 and GDF-5 (the same medium used for alginate culture). Biochemical and biomechanical properties of the neocartilage were measured.

RESULTS

In part I, GAG accumulation was highest for growth factor combinations including both IGF-1 and GDF-5. In part II, the addition of IGF-1 and GDF-5 to 2% HS resulted in a 12-fold increase in construct thickness compared with 2% HS alone (p < 0.0001). GAG and type II collagen accumulation was significantly higher with IGF-1 and GDF-5. Confined compression modulus was greatest with 2% HS, IGF-1, and GDF-5.

CONCLUSIONS

Supplementation of medium with IGF-1 and GDF-5 during creation of neocartilage constructs results in increased accumulation of GAG and type II collagen and improved biomechanical properties compared with constructs created without the growth factors.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em>-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER(+)) breast cancer cell line MCF-7, but not in ER negative (ER(-)) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5' flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER(-) cell line MDA-MB-231 was relatively lower than that in ER(+) MCF-7 and T47D cell lines. After the treatment with 5-aza-2'-deoxycytidine (5-aza-dC, especially in the concentration of <em>10</em> microM), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (<em>10</em>0%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer.

BACKGROUND

Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17beta-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER+) breast cancer cell line MCF-7.

METHODS

After the treatment of MCF-7 cells with E2 at different concentrations (10(-11) mol/L, 10(-9) mol/L, 10(-7) mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0.7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10(-7) mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half-site (1/2 ERE) element and Sp1 sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (ChIP) assay was also used to confirm the binding of estrogen receptor alpha (ERalpha) on BMP-6 promoter in the presence of E2.

RESULTS

E2 dose dependently increased BMP-6 mRNA expression in human ER+ breast cancer cell line MCF-7. At a dose of 10(-7) mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P < 0.05). Both the 1/2 ERE response element mutant form and the Sp1 site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter's response to E2. Through ChIP assay, the binding of ERalpha on 1/2 ERE response element in BMP-6 promoter was further validated.

CONCLUSIONS

Estrogen induces BMP-6 expression in human ER+ breast cancer cell line MCF-7 through its receptor ERalpha binding on 1/2 ERE element in the BMP-6 promoter.

Adenomatous polyposis coli (APC)-regulated Wnt and transforming growth factor-β (TGFβ) signaling cooperate in the intestine to maintain normal enterocyte functions. Human clinical trials showed that estrogen [17β-estradiol (E2)], the ligand of nuclear receptors estrogen receptor α (ERα) and ERβ, inhibited colorectal cancer (CRC) in women. Consistent with this finding, we reported that E2, ERα and ERβ suppressed intestinal tumorigenesis in the C57BL/6J-Min/+ (Min/+) mouse, a CRC model. Here, we extended our results with further comparisons of colon and small intestine from intact female Apc (+/+) (WT), Min/+ and ER-deficient Min/+ mice. In the colon of ER-deficient Min/+ mice, ER loss reduced TGFβ signaling in crypt base cells as evidenced by minimal expression of the effectors Smad 2, 3 and 4 in these strains. We also found reduced expression of Indian hedgehog (Ihh), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 and hepatocyte nuclear factor 3β or FoxA2, factors needed for paracrine signaling between enterocytes and mesenchyme. In proximal colon, ER loss produced a>><em>10</em>-fold increased incidence of crypt fission, a marker for wound healing and tumor promotion. These data, combined with our previous work detailing the specific roles of E2, ERα and ERβ in the colon, suggest that ER activity helps to maintain the intestinal stem cell (ISC) microenvironment by modulating epithelial-stromal crosstalk in ways that regulate cytokine, Wnt and Ihh availability in the extracellular matrix (ECM).

Mechanosensory hair cells of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Members of several morphogen families are expressed within and surrounding the chick inner ear during stages of SAG axon outgrowth and pathfinding. On the basis of their localized expression patterns, we hypothesized that <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), fibroblast growth factors (FGFs), and sonic hedgehog (Shh) may function as guidance cues for growing axons and/or may function as trophic factors once axons have reached their targets. To test this hypothesis, three-dimensional collagen cultures were used to grow Embryonic Day 4 (E4) chick SAG explants for 24 h in the presence of purified <em>proteins</em> or beads soaked in <em>proteins</em>. The density of neurite outgrowth was quantified to determine effects on neurite outgrowth. Explants displayed enhanced neurite outgrowth when cultured in the presence of purified BMP4, BMP7, a low concentration of Shh, FGF8, FGF<em>10</em>, or FGF19. In contrast, SAG neurons appeared unresponsive to FGF2. Collagen gel cultures were labeled with terminal dUTP nick-end labeling and immunostained with anti-phosphohistone H3 to determine effects on neuron survival and proliferation, respectively. Treatments that increased neurite outgrowth also yielded significantly fewer apoptotic cells, with no effect on cell proliferation. When presented as focal sources, BMP4, Shh, and FGFs -8, -<em>10</em>, and -19 promoted asymmetric outgrowth from the ganglion in the direction of the beads. BMP7-soaked beads did not induce this response. These results suggest that a subset of morphogens enhance both survival and axon outgrowth of otic neurons.