Atelopeptide type I collagen derived from fresh porcine skin was evaluated as a carrier for ectopic osteoinduction by recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) in rats. Four treatment groups (N = 5) were examined: a control group in which only atelopeptide type I collagen was implanted, and groups II, III and IV in which atelopeptide type I collagen with 2, <em>10</em>, 50 micrograms of rhBMP-2 was implanted in to the calf muscles of <em>10</em>-week-old Wistar rats, respectively. Four weeks after the implantation, soft X-ray and light microscopic examinations were performed. In addition, calcium (Ca) content and alkaline phosphatase (ALP) activity were evaluated. New <em>bone</em> formation in the implanted regions (groups II, III and IV) were revealed. <em>Bone</em> formation was induced in the implanted region of even 2 micrograms of rhBMP-2 (group II), and its degree was dependent on the dose of rhBMP 2. These results indicate that atelopeptide type I collagen is an effective carrier for ectopic osteo-induction by rhBMP-2 and may act as a carrier for rhBMP in reconstructive surgery for bony defect and augmentation.

Nontraumatic osteonecrosis is related to alcohol and glucocorticoid with unknown pathogenesis. Increased adipogenesis decreases <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) gene expression after glucocorticoid treatment. Lovastatin enhances BMP2 gene expression in rodents, reverses the effects of glucocorticoids on <em>bone</em>, and prevents glucocorticoid-induced osteonecrosis in chickens and humans. We hypothesized patients with osteonecrosis are more susceptible to glucocorticoid treatment than patients without osteonecrosis. Marrow stromal cell cultures from 14 patients with osteonecrosis, and <em>10</em> patients without osteonecrosis were treated with dexamethasone (0.1 micromol/L), lovastatin (1 micromol/L), or combined treatment. BMP2 and osteocalcin gene expression were evaluated by reverse-transcriptase polymerase chain reaction and real-time polymerase chain reaction. The suppression of BMP2 by dexamethasone was more pronounced and the enhancement by lovastatin was less pronounced in the osteonecrosis group. Dexamethasone suppressed osteocalcin in the osteonecrosis group. Among the subgroups of osteonecrosis, suppression of BMP2 and osteocalcin by dexamethasone occurred in glucocorticoid-induced osteonecrosis group. Our data suggest individuals who are more susceptible to a glucocorticoid-induced decreases in BMP2 and osteocalcin gene expression are more likely to have osteonecrosis, especially glucocorticoid-induced osteonecrosis.

OBJECTIVE

Bone morphogenetic protein (BMP-) and Wnt-signalling play crucial roles in cartilage homeostasis. Our objective was to investigate whether activation of the BMP-pathway or stimulation of Wnt-signalling cascades effectively enhances cartilage-specific extracellular matrix (ECM) accumulation and functional biomechanical parameters of chondrocyte-seeded tissue engineering (TE)-constructs.

METHODS

Articular chondrocytes were cultured in collagen-type-I/III-matrices over 6 weeks to create a biomechanical standard curve. Effects of stimulation with 100 ng/mL BMP-4/-7 heterodimer or 10 mM lithium chloride (LiCl) on ECM-deposition was quantified and characterized histologically. Biomechanical parameters were determined by the Very Low Rubber Hardness (VLRH) method and under confined compression stress relaxation.

RESULTS

BMP-4/-7 treatment resulted in stronger collagen type-II staining and significantly enhanced glycosaminoglycan (GAG) deposition (3.2-fold; *P < 0.01) correlating with improved hardness (∼1.7-fold; *P = 0.001) reaching 83% of native cartilage values after 28 days, a value not reached before 9 weeks without stimulation. LiCl treatment enhanced VLRH slightly, but significantly (∼1.3-fold; *P = 0.016) with a trend to more ECM-deposition. BMP-4/-7 treatment significantly enhanced the E Modulus (105.7 ± 34.1 kPa; *P = 0.000001) compared to controls (8.0 ± 4.2 kPa). Poisson's ratio was significantly improved by BMP-4/-7 treatment (0.0703 ± 0.0409; *P = 0.013) vs controls (0.0432 ± 0.0284) and a significantly lower permeability (5.8 ± 2.1056 × 10(-14) m4/N.s; *P = 0.00001) was detected compared to untreated scaffolds (4.4 ± 3.1289 × 10(-13) m4/N.s).

CONCLUSIONS

While Wnt-activation is less effective, BMP-4/-7 heterodimer stimulation approximated native cartilage features in less than 50% of standard culture time representing a promising strategy for functional cartilage TE to improve biomechanical parameters of engineered cartilage.

BACKGROUND

We have previously shown that platelet aggregation has higher heritability in African Americans than European Americans. However, a genome-wide association study (GWAS) of platelet aggregation in African Americans has not been reported. We measured platelet aggregation in response to arachidonic acid, ADP, collagen, or epinephrine by optical aggregometry. The discovery cohort was 825 African Americans from the GeneSTAR study. Two replication cohorts were used: 119 African Americans from the Platelet Genes and Physiology Study and 1221 European Americans from GeneSTAR. Genotyping was conducted with Illumina 1 M arrays. For each cohort, age- and sex-adjusted linear mixed models were used to test for association between each SNP and each phenotype under an additive model.

RESULTS

Six SNPs were significantly associated with platelet aggregation (P<5×<em>10</em>(-8)) in the discovery sample. Of these, three SNPs in three different loci were confirmed: 1) rs12041331, in PEAR1 (platelet endothelial aggregation receptor 1), replicated in both African and European Americans for collagen- and epinephrine-induced aggregation, and in European Americans for ADP-induced aggregation; 2) rs11202221, in BMPR1A (<em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type1A), replicated in African Americans for ADP-induced aggregation; and 3) rs6566765 replicated in European Americans for ADP-induced aggregation. The rs11202221 and rs6566765 associations with agonist-induced platelet aggregation are novel.

CONCLUSIONS

In this first GWAS of agonist-induced platelet aggregation in African Americans, we discovered and replicated, novel associations of two variants with ADP-induced aggregation, and confirmed the association of a PEAR1 variant with multi-agonist-induced aggregation. Further study of these genes may provide novel insights into platelet biology.

The physiological effects of the flavone, apigenin on <em>bone</em> cells were studied. We first show that apigenin inhibits tumor necrosis factor alpha (TNFalpha)- and interferon gamma (IFNgamma)-induced secretion of several osteoclastogenic cytokines from MC3T3-E1 mouse calvarial osteoblast cell line. Ligands of the TNF receptor family constitute the most potent osteoclastic cytokines. In MC3T3-E1 cells, apigenin dose-dependently (from 5 to 20 microM) inhibits TNFalpha-induced production of the osteoclastogenic cytokines, IL-6 (interleukin-6), RANTES (regulated upon activation, normal T cell-expressed and -secreted), monocyte chemoattractant <em>protein</em>-1 (MCP-1) and MCP-3. In addition, apigenin inhibits IFNgamma-stimulated secretion of monokines, CXCL-9, and -<em>10</em> in MC3T3-E1 cells. Next, we show that apigenin strongly inhibits differentiation of 3T3-L1 preadipocytes to adipocytes with attendant inhibition of adipocyte differentiation-induced IL-6, MCP-1, and leptin production. Inhibition of adipogenic differentiation by apigenin could be due to induction of osteogensis as it robustly upregulates mRNA levels of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-6 (BMP-6). Finally, the presence of apigenin inhibited osteoclast differentiation from the RAW 264.7 cell line by reducing receptor activator of nuclear factor kappa ligand (RANKL)-induced expression of tartrate-resistant acid phosphatase (TRAP), RANK, and calcitonin receptor but not CCR1, resulting in the inhibition of multinucleated osteoclast formation. Similarly, apigenin inhibited expression of the osteoclast differentiation markers TRAP, RANK, and c-Fms in osteoclast precursor cells obtained from mouse <em>bone</em> marrow following treatment with RANKL and macrophage colony stimulating factor (MCSF). Furthermore, apigenin induced apoptosis of mature osteoclasts obtained from rabbit long <em>bone</em> and inhibited <em>bone</em> resorption. In all instances, a structurally related compound, flavone had no significant effect. These data suggest that apigenin has multiple effects on all three <em>bone</em> cells that could prevent <em>bone</em> loss in vivo.

The aims of these studies were to determine the abilities of antisera against different regions of ovine <em>bone</em> <em>morphogenetic</em> <em>protein</em> 15 (BMP15) and growth differentiation factor 9 (GDF9) to inhibit ovarian follicular activity, estrus (mating), and ovulation in sheep. The 9-15-mer peptides were conjugated to keyhole limpet hemocyanin (KLH) and used to generate antibodies against the flexible N-terminal regions of the mature <em>protein</em> as well as against regions in which dimerization of the <em>protein</em> or interaction with a type 1 BMP or a type 2 TGFB or BMP receptor was predicted to occur. Ewes (n = <em>10</em> per treatment group) were vaccinated with KLH or the KLH-BMP15 (n = 9 different peptides) or KLH-GDF9 (n = <em>10</em>) peptides in Freund adjuvant at five consecutive monthly intervals. Overall, antisera generated against peptides that corresponded to amino acid residues 1-15 of the N-terminus of the BMP15 or GDF9 mature <em>protein</em> or GDF9 amino acid residues 21-34 were the most potent at inhibiting ovulation following primary and single booster vaccination. Several other BMP15 (8/9) or GDF9 (6/<em>10</em>) treatment groups, but not KLH alone, also produced significant reductions in the numbers of animals that ovulated, although 2, 3 or 4 booster vaccinations were required. Anovulation was commonly associated with the inhibition of normal ovarian follicular development and anestrus. The in vitro neutralization studies with IgG from the BMP15 or GDF9 immunized ewes showed that the mean inhibition of BMP15 plus GDF9 stimulation of (3)H-thymidine uptake by rat granulosa cells was approximately 70% for animals without corpora lutea (CL), whereas for animals with one to three CL or more than three CL, the inhibition was 24%-33% or 27%-42%, respectively. In summary, these data suggest that reagents that block the biological actions of BMP15 or GDF9 at their N-termini have potential as contraceptives or sterilizing agents.

The possibility of using adipose tissue-derived stromal cells (ATSC) as alternatives to <em>bone</em> marrow-derived stromal cells (BMSC) for <em>bone</em> repair has garnered interest due to the accessibility, high cell yield, and rapid in vitro expansion of ATSC. For clinical relevance, their <em>bone</em> forming potential in comparison to BMSC must be proven. Distinct differences between ATSC and BMSC have been observed in vitro and comparison of osteogenic potential in vivo is not clear to date. The aim of the current study was to compare the osteogenesis of human xenofree-expanded ATSC and BMSC in vitro and in an ectopic nude mouse model of <em>bone</em> formation. Human MSC were implanted with biphasic calcium phosphate biomaterials in subcutis pockets for 8 weeks. Implant groups were: BMSC, ATSC, BMSC and ATSC mixed together in different ratios, as well as MSC primed with either osteogenic supplements (250 μM ascorbic acid, <em>10</em> mM β-glycerolphosphate, and <em>10</em> nM dexamethasone) or 50 ng/ml recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 prior to implantation. In vitro results show osteogenic gene expression and differentiation potentials of ATSC. Despite this, ATSC failed to form ectopic <em>bone</em> in vivo, in stark contrast to BMSC, although osteogenic priming did impart minor osteogenesis to ATSC. Neovascularization was enhanced by ATSC compared with BMSC; however, less ATSC engrafted into the implant compared with BMSC. Therefore, in the content of <em>bone</em> regeneration, the advantages of ATSC over BMSC including enhanced angiogenesis, may be negated by their lack of osteogenesis and prerequisite for osteogenic differentiation prior to transplantation. Stem Cells Translational Medicine 2017;6:2160-2172.

Obesity is a serious and costly issue to the medical welfare worldwide. Probiotics have been suggested as one of the candidates to resolve the obesity-associated problems, but how they combat obesity is not fully understood. Herein, we investigated the effects of Lactobacillus reuteri 263 (L. reuteri 263) on antiobesity using four groups of Sprague-Dawley rats (n=<em>10</em>/group), namely, C (normal diet with vehicle treatment), HE [high-energy diet (HED) with vehicle treatment], 1X (HED with 2.1×<em>10</em>9 CFU/kg/day of L. reuteri 263) and 5X (HED with 1.05×<em>10</em><em>10</em> CFU/kg/day of L. reuteri 263), for 8 weeks. L. reuteri 263 improved the phenomenon of obesity, serum levels of proinflammatory factors and antioxidant enzymes. More importantly, L. reuteri 263 increased oxygen consumption in white adipose tissue (WAT). The mRNA expressions of thermogenesis genes uncoupling <em>protein</em>-1, uncoupling <em>protein</em>-3, carnitine palmitoyltransferase-1 and cell death-inducing DFFA-like effector-a were up-regulated in WAT of the 5X group. Moreover, L. reuteri 263 might induce browning of WAT due to the higher mRNA levels of browning-related genes peroxisome proliferator-activated receptor-γ, PR domain containing-16, Pparγ coactivator-1α, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 and fibroblast growth factor-21 in the 1X and 5X groups compared to the HE group. Finally, L. reuteri 263 altered the expressions of genes involved in glucose and lipid metabolisms in WAT, including increasing the levels of glucose transporter type 4 and carbohydrate-responsive element-binding <em>protein</em> and decreasing the expression of Acetyl-CoA carboxylase-1. The results suggest that L. reuteri 263 may treat obesity through energy metabolism remodeling of WAT in the high-energy-diet-induced obese rats.

BACKGROUND

We previously demonstrated embryotrophic actions of maternal (oocyte-derived) follistatin during bovine early embryogenesis. Classical actions of follistatin are attributed to inhibition of activity of growth factors including activins and bone morphogenetic proteins (BMP). However, temporal changes in BMP mRNA in early bovine embryos and the effects of exogenous BMP on embryo developmental progression are not understood. The objectives of present studies were to characterize mRNA abundance for select BMP, BMP receptors and BMP receptor associated SMADs during bovine oocyte maturation and early embryogenesis and determine effects of addition of exogenous BMP protein on early development.

METHODS

Relative abundance of mRNA for BMP2, BMP3, BMP7, BMP10, SMAD1, SMAD5, ALK3, ALK6, ALK2, BMPR2, ACVR2A and ACVR2B was determined by RT-qPCR analysis of germinal vesicle (GV) and in vitro matured metaphase II (MII) oocytes and in vitro produced embryos collected at pronuclear, 2-cell (C), 4C, 8C, 16C, morula and blastocyst stages. Effects of addition of recombinant human BMP2 (0, 1, 10 and 100 ng/ml) during initial 72 h of embryo culture on early cleavage (within 30 h post insemination), total cleavage, development to 8C-16C and blastocyst stages and blastocyst mRNA abundance for markers of inner cell mass (NANOG) and trophectoderm (CDX2) were also determined.

RESULTS

Abundance of mRNA for BMP2, BMP10, SMAD1, SMAD5, ALK3, ALK2, BMPR2 and ACVR2B was elevated in MII oocytes and/or pronuclear stage embryos (relative to GV) and remained elevated through the 8C -16C stages, whereas BMP3, BMP7 and ALK2 mRNAs were transiently elevated. Culture of embryos to the 8C stage in the presence of α-amanitin resulted in increased abundance for all of above transcripts examined relative to untreated 8C embryos. Effects of addition of exogenous BMP2 on early cleavage rates and rates of development to 8C-16C and blastocyst stages were not observed, but BMP2 treatment increased blastocyst mRNA for CDX2 and NANOG.

CONCLUSIONS

Abundance of maternally derived mRNAs for above BMP system components are dynamically regulated during oocyte maturation and early embryogenesis. Exogenous BMP2 treatment does not influence progression to various developmental endpoints, but impacts characteristics of resulting blastocysts. Results support a potential role for BMPs in bovine early embryogenesis.

Silicon is essential for <em>bone</em> formation. A low-silicon diet leads to <em>bone</em> defects, and numerous animal models have demonstrated that silicon supplementation increases <em>bone</em> mineral density (BMD) and reduces <em>bone</em> fragility. However, the exact mechanism of this action has not been characterized. In this study, we aimed to determine the role of biological silicon in the induction of osteoblast differentiation and the possible underlying mechanism. We examined whether orthosilicic acid promotes collagen type 1 (COL-1) and osteocalcin synthesis through the <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2)/Smad1/5/runt-related transcription factor 2 (RUNX2) signaling pathway by investigating its effect in vitro at several concentrations on COL-1 and osteocalcin synthesis in human osteosarcoma cell lines (MG-63 and U2-OS). The expression of relevant <em>proteins</em> was detected by Western blotting following exposure to noggin, an inhibitor of BMP-2. In MG-63 cells, immunofluorescence methods were applied to detect changes in the expression of BMP-2, phosphorylated Smad1/5 (P-Smad1/5), and RUNX2. Furthermore, rat <em>bone</em> mesenchymal stem cells (BMSCs) were used to determine the effect of orthosilicic acid on osteogenic differentiation. Exposure to <em>10</em> μM orthosilicic acid markedly increased the expression of BMP-2, P-Smad1/5, RUNX2, COL-1, and osteocalcin in osteosarcoma cell lines. Enhanced ALP activity and the formation of mineralized nodules were also observed under these conditions. Furthermore, preconditioning with noggin inhibited the silicon-induced upregulation of P-Smad1/5, RUNX2, and COL-1 expression. In conclusion, the BMP-2/Smad1/5/RUNX2 signaling pathway participates in the silicon-mediated induction of COL-1 and osteocalcin synthesis, and orthosilicic acid promotes the osteogenic differentiation of rat BMSCs.

Heart disease is among the leading causes of death worldwide, and the limited proliferation of mammalian cardiomyocytes prevents heart regeneration in response to injury. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-<em>10</em> (BMP<em>10</em>) exerts multiple roles in various developmental events; however, the effect of BMP<em>10</em> and the underlying mechanism involved in cardiac repair remains unclear. After stimulation with the recombinant BMP<em>10</em>, an obvious dose-dependent cardiomyocyte proliferation and reentry of differentiated mammalian cardiomyocytes into the cell cycle was observed. Furthermore, BMP<em>10</em> stimulation strikingly enhanced Tbx20 expression. Further analysis demonstrated that T-box 20 (Tbx20) was involved in BMP<em>10</em>-induced proliferation of differentiated cardiomyocytes as preconditioning with Tbx20 siRNA significantly attenuated BMP<em>10</em>-induced DNA synthesis. In vivo, BMP<em>10</em> induced rat cardiomyocyte DNA synthesis and cytokinesis. After myocardial infarction (MI), BMP<em>10</em> stimulated cardiomyocyte cell-cycle reentry and mitosis, resulting in the decrease of infarct size and improvement of cardiac repair. Taken together, these data indicated that BMP<em>10</em> stimulated cardiomyocyte proliferation and repaired cardiac function after heart injury. Consequently, BMP<em>10</em> may be a potential target for innovative strategies against heart failure.

Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> (rhBMP) is viewed as a therapeutic cytokine because of its ability to induce <em>bone</em>. However, the high doses of rhBMP required for <em>bone</em> induction in humans remain a major hurdle for the therapeutic application of this <em>protein</em>. The development of a methodology that would effectively overcome the weak responsiveness to human BMP is highly desired. In the present study, we investigate the ability of a prostaglandin E EP4 receptor selective agonist (EP4A) to augment the <em>bone</em>-inducing ability of BMP in a biodegradable delivery system. A block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG, polymer) was used as the delivery system. Polymer discs containing rhBMP-2 and EP4A were implanted into the left dorsal muscle pouch of mice to examine the dose-dependent effects of EP4A. Fifty mice were divided into 5 groups based on the contents of rhBMP and EP4 in the polymer (group 1; BMP 5 microg EP4A 0 microg, group 2; BMP 5 microg EP4 3 microg, group 3; BMP 5 microg EP4 30 microg, group 4; BMP 5 microg EP4 300 microg, group 5; BMP 0 microg EP4 30 microg, n=<em>10</em> each). All implants were harvested, examined radiologically, and processed for histological analysis 3 weeks after surgery. On dual-energy X-ray absorptiometry (DXA) analysis, the <em>bone</em> mineral content (BMC) of the ossicles was 6.52+/-0.80 (mg), 9.36+/-1.89, 14.21+/-1.27, and 18.75+/-2.31 in groups 1, 2, 3, and 4 respectively. In terms of BMC, the values of groups 3 and 4 were significantly higher than those of group 1. The mean BMC value of group 4 was approximately 3 times higher than that of group 1. No significant difference in body weight was noted among the groups during the experimental period. In summary, the presence of a prostaglandin E EP4 receptor selective agonist in the carrier polymer enhanced the <em>bone</em>-inducing capacity of rhBMP-2 with no apparent systemic adverse effects.

Transforming growth factor beta (TGF-beta) is expressed in the growth plate and is an important regulator of chondrocyte maturation. Loss of function results in premature chondrocyte maturation both in vitro and in vivo. While TGF-beta inhibits chondrocyte maturation in cell cultures, the effect of increased TGF-beta has not been well characterized in an in vivo development model. Addition of Affi-gel agarose beads loaded with TGF-beta1 (<em>10</em> ng/microl) to developing stage 24-25 chick limb buds resulted in limb shortening and altered morphology. In situ hybridization studies showed down regulation of Indian hedgehog (ihh), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 6 (bmp6), and collagen type X (colX) expression, markers of chondrocyte maturation, in TGF-beta1 treated limbs. TGF-beta1 also decreased chondrocyte proliferation in the developing anlage. The findings confirm a critical role for TGF-beta during skeletal development. A more complete understanding of the role of TGF-beta and its down-stream signals will lead to improved understanding and treatment of cartilage diseases.

OBJECTIVE

Gene therapy techniques have the potential to treat numerous diseases, from cancer to diabetes. One promising application is the use of bone morphogenetic protein (BMP) gene transfer to induce bone formation. Previous studies have demonstrated that both direct and ex vivo BMP gene therapy have the capacity to initiate the normal endochondral pathway, leading to rapid mature bone formation. In the present study, computed tomography (CT) and radionuclide imaging was used to assess bone formation induced by BMP gene therapy accurately and noninvasively.

METHODS

Athymic nude rodents were treated with 1.25 x 10(10) particles of adenovirus-BMP-2 (Ad-BMP-2) (treatment group) or adenovirus-beta-gal (control group). At various intervals after treatment, the animals underwent CT, planar digital radiography, and planar radionuclide scintigraphic imaging.

RESULTS

Radionuclide scintigraphy clearly demonstrated active bone deposition that began as early as 15 days after treatment and peaked at approximately 36 days, only at the Ad-BMP-2 injection sites. CT clearly demonstrated ectopic bone induction over time at the Ad-BMP-2 treatment sites, in perfect correlation with the scintigraphic findings.

CONCLUSIONS

This study clearly illustrates that gene therapy-induced osteogenesis can be studied with multimodality imaging and supports the use of these approaches in future preclinical and clinical studies.

We investigated whether paracrine signalling between the bovine oocyte and cumulus cells is altered during the course of in vitro maturation (IVM). Bovine COCs were cocultured with denuded oocytes or treated with specific oocyte-secreted factors, namely recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-15 or growth differentiation factor (GDF)-9, beginning from 0 or 9h IVM. To generate a 9-h denuded oocyte (DO) group, COCs were cultured intact for the first 9h of IVM and then denuded. Coculturing intact COCs with DOs denuded immediately after collection or following 9h of maturation did not affect cleavage rate, but improved blastocyst yield (P<0.05) on Day 8 (51 and 61%, respectively; P<0.05) and cell number compared with COCs cultured alone (41%). Significantly, we observed higher levels of endogenous GDF-9 and BMP-15 <em>protein</em> in oocytes of COCs matured for 9h compared with no incubation. The addition of 175 ng mL(-1) GDF-9 or <em>10</em>%v/v BMP-15 from partially purified transfected 293H cell supernatant for 24h IVM significantly enhanced development to the blastocyst stage from 40% (control) to 51 and 47%, respectively (P<0.05). However, treatment of COCs with GDF-9 or BMP-15 between 9 and 24h of IVM did not increase blastocyst yield. These results provide evidence of quantitative and possibly qualitative temporal changes in oocyte paracrine factor production during IVM.

This study tested the hypothesis that mitogen-activated <em>protein</em> kinase inhibitors suppress hypertrophy and enhance chondrogenesis during chondrogenesis of multipotent mesenchymal stromal cells (MSCs). The effects of PD98059 (an extracellular signal-regulated kinase-1/2 inhibitor) and SB203580 (a p38 inhibitor) were tested on <em>bone</em> marrow-derived MSCs (BMMSCs) and adipose tissue-derived MSCs (ATMSCs). In vitro pellet cultures were carried out using 2.5 x <em>10</em>(5) MSCs in a chondrogenic medium containing 5 ng/mL of transforming growth factor-beta(2) (TGF-beta(2)) for BMMSCs, and 5 ng/mL of TGF-beta(2) and <em>10</em>0 ng/mL of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) for ATMSCs. From the 14th day of culture, the pellets were additionally treated with PD98059 or SB203580. After 14 more days of in vitro culture, pellets were harvested for analysis. PD98059 increased DNA content and glycosaminoglycan amount in BMMSCs and ATMSCs, whereas SB203580 had little effect. Collagen type I (COL1A1) mRNA decreased to almost a quarter in BMMSCs treated with PD98059. The mRNA levels of collagen type II (COL2A1) and SRY (sex determining region Y)-box 9 (SOX-9) increased several fold in both cells after PD98059 treatment, whereas SB203580 had only a slight effect. The gene expression of collagen type X (COL<em>10</em>A1) and runt-related transcription factor 2 (Runx-2) decreased by half after PD98059 treatment in BMMSCs, and decreased further in ATMSCs. SB203580 elevated COL<em>10</em>A1 and Runx-2 gene expression in both cell types. Safranin-O staining and immunohistochemistry generally mirrored findings from real-time PCR except for diminished expression of type I collagen in ATMSCS, and more pronounced decrease in type X collagen and Runx-2 in BMMSCs after PD98059 treatment. Our study demonstrated that PD98059 suppressed hypertrophy and promoted chondrogenesis of MSCs, and provides a ground for using them in cartilage tissue engineering.

To examine the effectiveness of gene transfer of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2 in vivo, we evaluated osteoinduction by an adenoviral vector, AxCAOBMP-2, under transient immunosuppression with an immunosuppression drug (cyclophosphamide), which was given at a dose of 125 mg/kg intraperitoneally the day before vector injection. Twenty-five microliters of AxCAOBMP-2 (8.75 x <em>10</em>(8) pfu, Group I) and AxCALacZ (1.75 x <em>10</em>(8) pfu, control group) and 5 microliter of AxCAOBMP-2 (1.75 x <em>10</em>(8) pfu, Group II) were injected into a right calf muscle. On day 21, induced <em>bone</em> in each group was investigated radiologically, histologically, and biochemically. The finding of osteoinduction was only seen in the AxCAOBMP-2-treated groups with immunosuppression. The activity of osteoinduction in Group I was higher than that in Group II. These results suggest that gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for <em>bone</em> reconstruction.

During recent decades the utilisation of growth factors, especially BMPs, has received an increasing interest in orthopaedic surgery. For clinical implantation the two main options are demineralised <em>bone</em> matrix (DBM) and recombinant <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (rhBMP). Many clinical studies agree on an equivalent osteoinductive effect between DBM, BMPs and autologous <em>bone</em> graft; however, the different origins and processing of DBM and rhBMP may introduce some fluctuations. Their respective characteristics are reviewed and possible interactions with their effectiveness are analysed. The main difference concerns the concentration of BMPs, which varies to an order of magnitude of <em>10</em>(6) between DBM and rhBMPs. This may explain the variability in efficiency of some products and the adverse effects. Currently, considering osteoinductive properties, safety and availability, the DBM seems to offer several advantages. However, if DBM and rhBMPs are useful in some indications, their effectiveness and safety can be improved and more evidence-based studies are needed to better define the indications.

OBJECTIVE

The purpose of this study was to develop a tissue-engineered bone graft model in the shape of a human mandibular condyle.

METHODS

Natural coral with a pore size of 150 to 220 microm and porosity of about 36% was molded into the shape of a human mandibular condyle. Culture-expanded rabbit marrow mesenchymal stem cells were induced by recombinant human bone morphogenetic protein-2 (rhBMP2) to improve osteoblastic phenotype. Then marrow-derived osteoblasts were seeded into natural coral at a density of 2 x 10(8)/mL and incubated in vitro for 3 days before implantation. The cell-coral complexes were implanted subcutaneously into the backs of nude mice and incubated in vivo for 2 months before harvesting. Implantation of coral alone acted as control. The specimens were processed for gross inspection, radiographic examination, and histologic and scanning electronic microscopic observation.

RESULTS

The results showed that new bone grafts in the shape of a human mandibular condyle were successfully developed 2 months after implantation and maintained the initial shape of the natural coral scaffold. New bone could be observed histologically on the surface and in the pores of natural coral in all specimens in the cell-seeding group (6 of 6), whereas in the control group there was no evidence of osteogenesis process (0 of 4).

CONCLUSIONS

This study suggests the feasibility of using porous coral as scaffold material transplanted with marrow-derived osteoblasts to restore bone graft in the shape of human mandibular condyle and shows the potential of using this method for the reconstruction of bone defects.

The objective of this study was to evaluate the effect of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) dose on alveolar ridge augmentation and dental implant osseointegration. Bilateral, 5 mm supraalveolar, peri-implant defects were surgically created in 6 beagle dogs. rhBMP-2 (0.05, 0.1 or 0.2 mg/ml) in an absorbable collagen sponge (ACS) carrier was molded around the fixtures and wounds were closed. Treatment variations were alternated between animals (incomplete block design). Animals were sacrificed at week 8 postsurgery. Nine of twelve jaw quadrants healed uneventfully. Two jaw quadrants exhibited wound failure by week 4 and one by week 8 postsurgery. Radiographic <em>bone</em> regeneration was observed in defects without wound failure from week 4 postsurgery. Radiolucent voids of variable size and shape were observed and regressed over time. In weeks 6 through 8, there was an apparent increase in <em>bone</em> density and trabecular structure, while <em>bone</em> height and volume decreased. Histometric analysis revealed limited differences in <em>bone</em> regeneration between experimental conditions. <em>Bone</em> regeneration area averaged (+/- SD) 1.0 +/- 0.5, 3.5 +/- 1.4 and 2.3 +/- 0.4 mm2 for the 0.05, 0.1 and 0.2 mg/ml dose, respectively. There were no significant differences in osseointegration. Osseointegration in newly formed <em>bone</em> averaged 19 +/- 4%, 18 +/- <em>10</em>% and 21 +/- 6% for the 0.05, 0.1 and 0.2 mg/ml rhBMP-2 sites, respectively. Collectively, the data suggest that there are no dramatic differences in <em>bone</em> induction and osseointegration within the selected dose and observation interval.

OBJECTIVE

Traumatized muscle is a complex healing environment containing cells with robust reparative and regenerative potential interacting in a cytokine milieu that influences the function and differentiation of these cells, leading to a spectrum of healing responses. In particular, bone morphogenetic protein-4 (BMP-4) is of interest as a potential modulator of healing because its dysregulation has been associated with fibrosis and heterotopic ossification formation. We propose a descriptive study of altered BMP-4 expression in traumatized muscle tissue and to evaluate its role in the fibroregulatory function of resident mesenchymal progenitor cells (MPCs) at the protein- and gene-expression levels.

METHODS

Protein-level expression of BMP-4 from cells resident in traumatized muscle specimens was evaluated using ELISA and also using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to compare BMP-4 in homogenized muscle tissue specimens. BMP-4, cartilage oligomeric matrix protein (COMP), and osteocalcin expression localization was analyzed via immunohistochemistry. Reverse transcription-polymerase chain reaction was performed to evaluate fibroregulatory gene expression in MPCs after treatment with BMP-4.

RESULTS

BMP-4 was present in all traumatized muscle tissue specimens. Immunohistochemistry demonstrated that traumatized muscle fibers contained greater number of cells expressing BMP-4 in a more disorganized fashion compared with control samples. Reverse transcription-polymerase chain reaction demonstrated that COMP, growth and differentiation factor-10, and integrin beta-2 were up-regulated, whereas tumor necrosis factor-alpha was significantly down-regulated. COMP expression was colocalized in the traumatized muscle tissue with osteocalcin.

CONCLUSIONS

BMP-4 has an effect on MPCs that seems to promote fibrotic tissue formation. These findings suggest that BMP-4, while promoting osteoinduction, may also act on MPCs to promote formation of a fibrotic osteoinductive matrix. Thus, this signaling axis might be a potential target for heterotopic ossification prevention.

The <em>bone</em> <em>morphogenetic</em> <em>protein</em> 5 (BMP5) participates in skeletal development but its direct effects on the function of growth plate chondrocytes during chondrogenesis have not been explored. We have investigated the signaling pathways activated by BMP5 and its effect on chondrogenic differentiation in the ATDC5 growth plate chondrocyte model. BMP5 transiently activated p38 mitogen-activated <em>protein</em> kinase (MAPK) and extracellular signal-regulated kinase signaling after <em>10</em> days of differentiation; sustained Smad and p38 MAPK signaling were seen after 15 days differentiation. All three pathways were activated by BMP5 in human adult articular chondrocytes. BMP5 alone and in combination with the chondrogenic enhancer, insulin, induced proteoglycan synthesis, aggrecan core <em>protein</em> 1 expression, and alkaline phosphatase activity. Upregulation of hypertrophic markers parathyroid receptor 1 and collagen type X alpha 1 occurred in BMP5-treated ATDC5 cultures. BMP5 is clearly chondrogenic and exhibits stage-specific regulation of multiple signaling pathways in this growth plate model. In particular, BMP5 accelerates expression of hypertrophy markers which is of relevance in both development and diseases such as osteoarthritis.

OBJECTIVE

Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs.

METHODS

hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed.

RESULTS

Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and a-myosin heavy chain (aMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and a-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS.

CONCLUSIONS

We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.

We investigated the enhancement of new <em>bone</em> |formation elicited ectopically by recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2), using parathyroid hormone (PTH) and a phosphodiesterase inhibitor (PDEi), pentoxifylline (PTX), in an animal model. Collagen sponge sheet discs containing rhBMP were implanted onto the back muscles of mice. PTX alone (200 mg/kg body weight [BW]), PTH(1-34) (<em>10</em> microg/kg BW), PTX plus PTH (200 mg/kg BW and <em>10</em> microg/kg BW, respectively), or vehicle (control) were injected subcutaneously daily for 3 weeks after implantation. At the end of this period, rhBMP-2-induced ectopic ossicles were harvested from each group of animals. Ossicles from the PTX-treated group were significantly larger in size, with unchanged <em>bone</em> mineral density (BMD), as compared with the ossicles from the controls. In contrast, the ossicles from the PTH-treated group had significantly higher BMD, but showed no difference in size when compared with those from the control animals. The ossicles of the PTX + PTH treatment group were significantly larger than those of the control and PTH treatment groups. In addition, the BMD of the harvested tissues from the PTX + PTH treatment group was signifi-cantly higher than that of tissues from the control and PTX treatment groups. Although the calcium content of ossicles was significantly higher in the PTX-, PTH-, and PTX + PTH-treated groups than in the control group, the Ca content of ossicles from the PTH + PTX-treated group was highest (two times that of controls), followed by the PTH- and PTX-treated groups.