The sonic hedgehog homologue (SHH)/<em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) signalling pathway is involved in the morphogenesis of many organ systems. This study was designed to investigate the expression of SHH/BMP4 pathway components in human anorectal malformations (ARMs) and the relationship between expression of their genes and the occurrence of ARMs. Expression of SHH, GLI family zinc finger 2 (GLI2) and BMP4 mRNA in the posterior wall of the terminal rectum in 40 patients with ARMs (15 high-type and 25 low-type) and <em>10</em> normal controls was assessed by reverse transcription-polymerase chain reaction. Levels of SHH, GLI2 and BMP4 <em>proteins</em> were assessed by immunohistochemistry and Western blotting. The expressions of SHH, GLI2 and BMP4 were significantly lower in patients with high-type compared with those with low-type anomalies and controls. Patients with low-type ARMs differed from controls only in the expression of GLI2 mRNA. It is concluded that down-regulation of the SHH/BMP4 signalling pathway may be related to the occurrence of high-type ARMs, but the mechanism responsible for low-type ARMs remains unclear.

OBJECTIVE

Bone morphogenetic protein (BMP)-2 and -7 are used in patients with long-bone fractures, nonunions and spinal fusions. It is unknown whether their potential systemic bioavailability following local bone administration might affect skeletal metabolism. To answer this question, we examined effects of systemically administered BMP-2 and -7 on bone in a newly developed rat model with a low level of calciotropic hormones.

METHODS

Removal of thyroid and parathyroid glands (TPTx) in rats resulted in a decreased level of calciotropic hormones and subsequent bone loss assessed by micro computed tomography (micro-CT) and measurement of serum bone formation and resorption markers, including osteocalcin, C-telopeptide, osteoprotegerin and receptor activator of nuclear factor kappa-B ligand. Results were complemented with in vitro studies on osteoblast and osteoclast activity by both BMP-2 and -7. The doses used were calculated from published pharmacodynamic studies and bioavailability results from preclinical BMP-2 and -7 studies.

RESULTS

TPTx resulted in bone loss, which was restored by systemic administration of 10-70 μg/kg of BMP-2 and 10-250 μg/kg of BMP-7. BMP-2 showed a higher capacity for enhancing trabecular microarchitecture, whereas BMP-7 augmented trabecular thickness. In vitro experiments revealed that BMP-2 and -7 when uncoupled increased the number and activity of both osteoblasts and osteoclasts.

CONCLUSIONS

Surprisingly, both BMP-2 and -7 showed an increased bone volume in an in vivo environment of low calciotropic hormones. Locally administered BMP-2 and -7 from bone devices might become partially available in circulation but will not mediate systemic bone loss.

Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about <em>10</em> ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.

OBJECTIVE

The authors have previously reported on their early experience with sublaminar polyester bands in spine surgery. In this paper, the authors describe the use of sublaminar polyester bands in long-segment posterior instrumented spinal fusions from the upper thoracic spine to the ilium in 21 children and transitional adults with progressive neuromuscular scoliosis. Transitional adults were patients older than 18 years of age with a spinal disorder of pediatric onset, such as spina bifida. This dedicated study represents the first reported use of polyester bands in spine surgery for neuromuscular scoliosis in this patient population in the US.

METHODS

The authors retrospectively reviewed the demographics and procedural data of patients who underwent posterior instrumented fusion using sublaminar polyester bands for neuromuscular scoliosis.

RESULTS

Twenty-one pediatric and adult transitional patients, ranging in age from <em>10</em> to 20 years (mean 14 years), underwent posterior instrumented fusion for progressive neuromuscular scoliosis. The average coronal Cobb angle measured 66° before surgery (range 37°-125°). Immediately after surgery, the mean coronal Cobb angle was 40° (range 13°-85°). At last follow-up, the average coronal Cobb angle was maintained at 42° (range 5°-112°). Regarding sagittal parameters, thoracic kyphosis was restored by 8%, and lumbar lordosis improved by 20% after surgery. Mean follow-up duration was 17 months (range 2-54 months). One patient with an aborted procedure due to loss of intraoperative evoked potentials was excluded from the analysis of radiographic outcomes. Mean surgical time was 7 hours 43 minutes (range 3 hours 59 minutes to <em>10</em> hours 23 minutes). All patients received either a 12- or 24-mg dose of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2. Average estimated blood loss was 976 ml (range 300-2700 ml). Complications directly related to the use of sublaminar instrumentation included transient proprioceptive deficit (1 patient) and prolonged paraparesis (1 patient). Other complications noted in this series included disengagement of the rod from an iliac screw (1 patient), proximal junctional kyphosis (1 patient), noninfected wound drainage (2 patients), and perioperative death (1 patient). The lessons learned from these complications are discussed.

CONCLUSIONS

Pedicle screws, laminar/pedicle/transverse process hooks, and sublaminar metal wires have been incorporated into posterior spinal constructs and widely reported and used in the thoracic and lumbar spines and sacrum with varying success. This report demonstrates the satisfactory radiological outcomes of hybrid posterior spinal constructs in pediatric and adult neuromuscular scoliosis that include sublaminar polyester bands that promise the technical ease of passing sublaminar instrumentation with the immediate biomechanical rigidity of pedicle screws and hooks. However, the high neurological complication rate associated with this technique (2/21, or <em>10</em>%) tempers the acceptable radiographic outcomes.

OBJECTIVE

As mesenchymal stem cells (MSCs) can be isolated easily from adipose tissues while retaining their self-renewal and multi-potential differentiation capacities, they hold promising possibilities for being applied extensively in tissue engineering. Bone morphogenetic protein (BMP) family members have been reported to provide instructive signals to MSCs for them to differentiate into several different cell lineages. The study described here aims to investigate whether BMP-4 could promote adipose-derived stem cell (ASC) differentiation into adipocytes under various concentrations.

METHODS

ASCs were isolated from mouse inguinal adipose pads and cultured in vitro. 10 ng/ml and 50 ng/ml BMP-4 were added to adipogenic media for 8 days. Oil red-O staining, reverse transcription/polymerase chain reaction and immunocytofluorescence staining were performed to examine differentiation of the ASCs.

RESULTS

As indicated by increased expression of adipogenic and lipogenic genes (PPAR-γ, APN and LPL) and proteins, 50 ng/ml BMP-4 seemed to induce mASCs to differentiate into the adipo-lineage compared to 10 ng/ml BMP-4, and control groups. In addition, lipid droplets accumulated within the adipocytes under 50 ng/ml BMP-4 stimulation, as shown by oil red-O staining.

CONCLUSIONS

Our present study suggests that BMP-4, as an adipo-inducing factor, promoted adipogenesis of ASCs at higher concentrations (50 ng/ml) and can perhaps be considered as a candidate for use in adipose tissue engineering.

The goal of this study was to determine the effectiveness of using polyethyleneimine (PEI) and a polyethylene glycol (PEG) tether to bind human recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) to hydroxyapatite (HAp) to enhance rhBMP-2 loading, alter its release properties, and enhance cellular interaction with the material. By using a branched PEI that was derived to express free thiols, rhBMP-2 was coated onto dense HAp surfaces at ~43 ng/cm(2). Using this novel attachment methodology, it was observed that the PEI-SH coating did not change the morphology of the HAp surfaces and that the amount of rhBMP-2 loaded was comparable to a direct adsorption method. In addition, it was also observed that the PEI and PEG tether significantly retained the rhBMP-2 to the HAp surface, inhibiting the burst release effect. Using human fetal osteoblast cells, the PEI- and PEG-tethered BMP-2 was also observed to increase cellular attachment by <em>10</em>-fold when compared with uncoated HAp and adsorbed rhBMP-2. It was concluded from this study that PEI and PEG tether significantly reduce the initial burst release effect of rhBMP-2. It was also concluded that the rhBMP-2 conjugation to PEI and PEG tether promoted an increase in cellular attachment to the HAp surface.

OBJECTIVE

Bone morphogenetic proteins (BMP) are recombinant osteoinductive proteins with their primary role being to promote bone formation. The off-label use of BMP in orthopaedic surgery has dramatically increased. However, reports of complications with BMP have emerged, and the safety of these proteins in orthopaedics is questioned. The purpose of this review was to evaluate safe situations in which BMP should be used and situations in which their use should be restricted.

METHODS

We recorded all studies from PubMed database from 2002 (date of first authorisation for both BMPs) until January 2014 using "BMP" or "bone morphogenetic protein". Then we screened and extracted all studies dealing with orthopaedic surgery. All situations in which BMP were used, even cases reports, were considered, and complications reported were then listed.

RESULTS

Situations in which it seems safe and efficient to use BMP are long-bone nonunions, or arthrodesis as an alternative or combined to autograft in small-bone loss. Surgeons and patients should be aware of transient aseptic wound swelling when BMP is located superficially. The use of BMP in spine surgery for intersomatic fusion is efficient but should be restricted to approaches that respect the vertebral canal to avoid neurological complications.

CONCLUSIONS

This review is an off-label map of BMP use in orthopaedics during the past 10 years. Our results could provide a useful tool to help decisions around when to use a BMP in a specific complex, and sometimes off-label, situation.

Demineralized <em>bone</em> matrix (DBM) reproducibly induces extraskeletal <em>bone</em> formation in rodents, but its effects in dogs and primates are negative or uncertain. In previous studies on the squirrel monkey, DBM did not induce <em>bone</em>, although the same implants were effective in rats. DBM implants augmented with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) induced intramuscular <em>bone</em> formation in squirrel monkeys. However, the amount of induced <em>bone</em> was often minimal and sometimes absent. One explanation of this weak and unpredictable effect could be reactions to cellular components of the allogeneic DBM that was used as carrier. Therefore, we now repeated the experiment, using bovine type 1 collagen as carrier. 48 collagen discs (<em>10</em> mm diameter), containing 0, <em>10</em>, 40 or 200 microgram rhBMP-2, were implanted in 6 monkeys and 6 rats. The BMP-2 implants induced dose-dependent amounts of intramuscular <em>bone</em> in rats whereas, in squirrel monkeys, macroscopic <em>bone</em> induction occurred in less than half of the BMP-2 implants. There was no dose-dependency. Whether <em>bone</em> was formed or not was significantly influenced by individual variation among the monkeys, and by implant location within the muscle. Implants close to the muscle aponeurosis more often induced <em>bone</em> than did purely intramuscular ones. In this small series, we could not demonstrate a significant effect of BMP-2, as compared to control implants. Presumably there were too few cells expressing BMP-2 receptors in the only minimally traumatized muscle of these monkeys.

METHODS

Osteoblastic cells derived from vertebral lamina and iliac crest were isolated and cultured under the same conditions (osteogenic medium, pH, temperature, and CO2 levels).

OBJECTIVE

To compare proliferation and expression of osteoblastic phenotype of cells derived from vertebral lamina and iliac grafting.

BACKGROUND

Many factors play a role in the success of bone graft in spinal fusion including osteoblastic cell population. Two common sources of graft are vertebral lamina and iliac crest, however, differences in proliferation and osteoblastic phenotype expression between cells from these sites have not been investigated.

METHODS

Cells obtained from cancellous bone of both vertebral lamina and iliac crest were cultured and proliferation was evaluated by direct cell counting and viability detected by Trypan blue. Alkaline phosphatase (ALP) activity was evaluated by thymolphthalein release from thymolphthalein monophosphate and matrix mineralization by staining with alizarin red S. Gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, osteoprotegerin, and receptor activator of NF-kB ligand was analyzed by real-time PCR. All comparisons were donor-matched.

RESULTS

Proliferation was greater at days 7 and 10 in cells from vertebral lamina compared with ones from iliac crest without difference in cell viability. ALP activity was higher in cells from vertebral lamina compared with cells from iliac crest at days 7 and 10. At 21 days, mineralized matrix was higher in cells derived from vertebral lamina than from iliac crest. At day 7, gene expression of ALP, osteocalcin, runt-related transcription factor 2, Msh homeobox 2, bone morphogenetic protein 7, intercellular adhesion molecule 1 precursor, receptor activator of NF-kB ligand, and osteoprotegerin was higher in cells derived from vertebral lamina compared with iliac crest.

CONCLUSIONS

Cell proliferation and osteoblastic phenotype development in cells derived from cancellous bone were more exuberant in cultures of vertebral lamina than of iliac crest.

The aim of the present study was to investigate the effects of quercetin on the mitogen-activated <em>protein</em> kinase (MAPK) signaling pathway in the osteogenic differentiation of rat mesenchymal stem cells (MSCs). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and an alkaline phosphatase (ALP) assay were used to determine the effects of quercetin (concentrations of 0.1, 1 and <em>10</em> µmol/l) on the proliferation and differentiation of MSCs and the expression of ALP, respectively. In addition, through the introduction of inhibitors of p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and c-Jun NH2-terminal kinase (JNK), the effects of quercetin on the <em>proteins</em>, ALP, collagen type I (COL I) and <em>bone</em> γ-carboxyglutamate <em>protein</em> (BGP), which are indicators of osteogenic differentiation, were investigated. Immunoblotting was performed to determine the phosphorylation levels of p38 MAPK, ERK1/2 and JNK, while fluorescent quantitative polymerase chain reaction was used to determine the mRNA expression levels of transforming growth factor (TGF)-β1, <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2 and core binding factor (CBF)α1. At all the concentrations tested, the concentrations of <em>10</em>, 1 and 0.1 µmol/l quercetin were shown to promote the differentiation of MSCs and the expression of ALP, in which the concentration of <em>10</em> µmol/l was optimal. When compared with the control group, the phosphorylation levels of p38 MAPK, ERK1/2 and JNK, the <em>protein</em> expression levels of ALP, COL I and BGP, and the mNRA expression levels of TGF-β1, BMP-2 and Cbfα1 were increased in the quercetin-treated group. However, with the introduction of inhibitors, the levels of phosphorylated p38 MAPK, ERK1/2 and JNK, and the <em>protein</em> expression levels of ALP, COL I and BGP decreased. Furthermore, the mRNA expression levels of TGF-β1, BMP-2 and CBFα1 decreased in the quercetin + SP600125 (inhibitor of JNK) and quercetin + PD98059 (inhibitor of ERK1/2) groups. Therefore, quercetin was demonstrated to promote the osteogenic differentiation of MSCs by activating the MAPK signaling pathway. The ERK1/2 and JNK signaling pathways regulate the expression of TGF-β1, BMP-2 and CBFα1. Thus, activation of the ERK1/2 and JNK signaling pathways may play a leading role in the quercetin-promoted osteogenic proliferation and differentiation of MSCs.

This study used the mRNA differential display technique to identify differentially expressed genes during the process of adipogenesis in the preadipocyte cell line, 3T3-L1. 3T3-L1 cells were treated with dexamethasone, isobutyl-1-methylxanthine, and insulin to induce differentiation into mature adipocytes. Cells were collected at three time-points during differentiation: Day 0 (d0), or nondifferentiated; Day 3 (d3), during differentiation; and Day <em>10</em> (d<em>10</em>), >90% of the cells had differentiated into mature adipocytes. Initial studies yielded 18 potentially differentially regulated cDNA candidates (8 down-regulated and <em>10</em> up-regulated). Reverse Northern and Northern blots confirmed differential expression of six of the candidates. Four of the candidates up-regulated on d3 and d<em>10</em> were identified by sequence analysis to be lipo<em>protein</em> lipase, a well-known marker of adipocyte differentiation. A fifth candidate that was expressed in d0, but not d3 or d<em>10</em>, was identified as DRM/gremlin, a <em>bone</em> <em>morphogenetic</em> <em>protein</em> antagonist. Finally, a sixth candidate that was increased at d3 and d<em>10</em> was identified as the peripheral benzodiazepine receptor, which has been implicated in proliferation, differentiation, and cholesterol transport in cells. This study is the first to show that peripheral benzodiazepine receptor and DRM/gremlin are expressed in preadipocyte cell lines and that they are differentially regulated during adipogenesis.

Combinations of calcium phosphate scaffolds and bioactive factors are promising niche-mimetic solutions for repairing large-sized <em>bone</em> defects. However, the importance of compatibility between scaffolds and bioactive factors on their osteogenic outcomes has been largely ignored. This study aimed to investigate the compatibility of calcinated antler cancellous <em>bone</em> (CACB) scaffolds with various bioactive factors including icariin (ICA), velvet antler polypeptides (VAP) or recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) as well as their combinational osteogenic potential in vitro and in vivo. Scanning electron microscopy and fourier transform infrared spectroscopy confirmed the uniform distribution and chemical stability of the reagents on CABC. In vitro release profiles showed relative steady release of ICA from ICA/CACB, burst VAP release from VAP/CACB, and minimal rhBMP-2 release from rhBMP-2/CACB composites. When compared with VAP and rhBMP-2, incorporation of ICA within CACB resulted in most increased cell attachment, proliferation, alkaline phosphatase activity, osteogenic gene expression, and mineralization of rat <em>bone</em> marrow mesenchymal stem cells. In rabbit mandible critical-sized defects, the most extensive osteogenesis and neovascularization were observed in the ICA/CACB group. Differences between the VAP/CACB and rhBMP-2/CACB groups were not apparent. Interestingly, low pro-inflammatory (TNF-α, IL-6) and high anti-inflammatory (IL-<em>10</em>) mRNA levels were observed at scaffold implantation sites which were in close association with amount of new <em>bone</em> formation. These findings highlight that the compatibility between scaffolds and bioactive factors should been taken into account when considering the formula of optimized <em>bone</em> defect repair.

BACKGROUND

Bone morphogenetic protein (BMP) is a member of the superfamily of transforming growth factor-beta. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP (rhBMP)-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells (MSCs) and increase the levels of hematopoietic cytokines in MSCs.

METHODS

2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino) carbonyl)-2H-tetrazolium hydroxide (XTT), real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted.

RESULTS

The XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation (P < 0.001, P < 0.001, P < 0.001, and P = 0.001, respectively) and intravenous transplantation (P < 0.001, P < 0.001, and P < 0.001 respectively). The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF.

CONCLUSIONS

The treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 15 (BMP15) and fibroblast growth factor <em>10</em> (FGF<em>10</em>) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF<em>10</em>, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF<em>10</em> increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease <em>10</em> (ADAM<em>10</em>), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced <em>protein</em> 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF<em>10</em> did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF<em>10</em> and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF<em>10</em> stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM<em>10</em>, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.

With the aim of discovering new molecules for induction of <em>bone</em> formation and biomineralization, combination of bioinformatics and simulation methods were used to design the structure of artificial peptides based on proline-rich domains of enamel matrix <em>proteins</em>. In this study, the effect of such peptides on the differentiation toward the osteogenic lineage of human umbilical cord mesenchymal stem cells (hUCMSCs) was evaluated with or without osteogenic supplements (hydrocortisone, β-glycerol phosphate, and ascorbic acid) and compared to the effect of the commercially available enamel matrix derivative (EMD). It was hypothesized that the differentiation toward the osteogenic lineage of hUCMSCs would be promoted by the treatment with the synthetic peptides when combined with differentiation media, or it could even be directed exclusively by the synthetic peptides. Osteoinductivity was assessed by cell proliferation, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 secretion, and gene expression of osteogenic markers after 1, 3, and 14 days of treatment. All peptides were safe with the dosages used, showing lower cell toxicity. P2, P4, and P6 reduced cell proliferation with growing media by <em>10</em>%-15%. Higher expression of early osteoblast markers was found after 3 days of treatment with EMD in combination with osteogenic supplements, while after 14 days of treatment, cells treated by the different synthetic peptides in combination with osteogenic supplements showed higher osteocalcin mRNA levels. We can conclude that osteogenic differentiation of hUCMSCs is promoted by short-term EMD treatment in combination with osteogenic supplements and by long-term treatment by the synthetic peptides in combination with osteogenic supplements, showing similar results for all the peptide variants analyzed in this study.

Previous studies suggest that icariin has anabolic effects on <em>bone</em>, but the mechanisms are unknown. We aimed to investigate the osteogenic effects of icariin in an undifferentiated osteoblast cell line by detecting cell morphology, viability, cell cycling and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) expression. We treated pre-osteoblastic MC3T3-E1 cells with different concentrations of icariin [0 (as a control), <em>10</em>, 20 and 40 ng/ml] for 48, 72 and 96 h. Cell morphology, viability and the cell cycle were examined and measured using microscopy, the MTT assay or flow cytometry, respectively. BMP-2-positive cells and BMP-2 <em>protein</em> expression levels in icariin-treated MC3T3-E1 cells were examined using immunohistochemistry staining with fluorescence optical density analysis and Western blotting. MC3T3-E1 cells showed typical characteristics of osteoblasts in response to treatment with icariin. Cells treated with all concentrations of icariin had increased percentages of S-phase cells and decreased percentages of G1-phase cells, especially in the <em>10</em> and 20 ng/ml icariin groups. The number of BMP-2-positive cells and BMP-2 <em>protein</em> expression levels in the <em>10</em> and 20 ng/ml icariin treatment groups were greater compared to the 0 and 40 ng/ml groups. Treatment of icariin promotes osteoblast MC3T3-E1 proliferation and differentiation in vitro, potentially owing to its role in increasing BMP-2 <em>protein</em> expression. Icariin potentially can be used as a drug in clinical settings to treat osteoporosis.

Secreted phospho<em>protein</em> 24 kDa (spp24) is a <em>bone</em> matrix <em>protein</em>. It contains a TGF-beta receptor II homology 1 (TRH1) domain. A cyclic, synthetic 19 amino acid peptide (<em>bone</em> <em>morphogenetic</em> <em>protein</em> binding peptide or BBP) based on the sequence of the TRH1 domain enhances BMP-2 induced osteogenesis. Many observations suggest that different size forms of this <em>protein</em> have very different effects (inhibiting or enhancing) on BMP-2 induced osteogenesis. Using the stable recombinant Met(His)(6)-tagged secretory form of full-length (fl) bovine spp24 [Met(His)(6)-spp24 (residues 24-203)] and transgenic (TG) mice expressing fl bovine spp24 (residues 1-203), we have demonstrated that spp24 inhibits BMP-2 induced <em>bone</em> formation. The effects of Met(His)(6)-spp24 (24-203) were determined in the ectopic <em>bone</em>-forming bioassay in male mice. Implantation of 5 microg of BMP-2 stimulated <em>bone</em> formation, assessed densitometrically as <em>bone</em> area and mineral content. When Met(His)(6)-spp24 (24-203) was implanted with BMP-2, it elicited a dose-dependent decrease in BMP-2-medicated ectopic <em>bone</em> formation. When added at a 50-fold excess (w/w), Met(His)(6)-spp24 (24-203) completely ablated the effects of BMP-2, while addition of a <em>10</em>-fold excess had no effect. Constitutive expression of fl bovine spp24 (1-203) under the control of the osteocalcin promoter in TG female mice reduced femoral and vertebral <em>bone</em> mineral density at 3 months of age and reduced femoral BMD at 8 months of age, but had no effects in male mice, which can exhibit less osteocalcin-promoter driven gene transcription than females. Histomorphometric analysis demonstrated that <em>bone</em> volume and trabecular thickness were lower in TG female mice at 3 months of age than in sex- and age-matched wild type (WT) controls. Thus, fl spp24 and its secretory isoform (Met(His)(6)-spp24 [24-203]), which contain a BMP-binding or TRH1 motif, inhibit ectopic <em>bone</em> formation in male mice and adversely affects BMD and histological parameters related to <em>bone</em> mass and formation in female mice expressing the human transgene. Under these conditions, fl spp24 acts as a BMP antagonist in vivo.

The highly sulfated glycosaminoglycan (GAG) heparin is widely used in the clinic as an anticoagulant, and researchers are now using it to enhance stem cell expansion/differentiation protocols, as well as to improve the delivery of growth factors for tissue engineering (TE) strategies. Growth differentiation factor 5 (GDF5) belongs to the <em>bone</em> <em>morphogenetic</em> <em>protein</em> family of <em>proteins</em> and is vital for skeletal formation; however, its interaction with heparin and heparan sulfate (HS) has not been studied. We identify GDF5 as a novel heparin/HS binding <em>protein</em> and show that HS proteoglycans are vital in localizing GDF5 to the cell surface. Clinically relevant doses of heparin (≥<em>10</em> nM), but not equivalent concentrations of HS, were found to inhibit GDF5's biological activity in both human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures and the skeletal cell line ATDC5. We also found that heparin inhibited both GDF5 binding to cell surface HS and GDF5-induced induction of Smad 1/5/8 signaling. Furthermore, GDF5 significantly increased aggrecan gene expression in chondrocyte pellet cultures, without affecting collagen type X expression, making it a promising target for the TE of articular cartilage. Importantly, this study may explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE and the adverse skeletal effects reported in the clinic following long-term heparin treatment. Our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g., HS derivatives), with better-defined structures and fewer off-target effects.

OBJECTIVE

To investigate the role of bone morphogenetic protein-2 (BMP-2) in patients with urinary tract infection (UTI) and renal stone in relation to Tamm-Horsfall protein (THP) and osteopontin (OPN).

METHODS

ELISA kits were used to determine these markers in serum and urinary samples of 20 patients with UTI, 15 with renal stone and 10 controls.

RESULTS

BMP-2 significantly increased in serum of patients who had UTI (P=0.05) and renal stone (P=0.01). In the case of UTI, serum BMP-2 at cutoff 44 pg/mL had sensitivity and specificity (92%, 80%), while cystatin C at cutoff 525 ng/mL showed sensitivity and specificity (85%, 91%). THP is a good predictor of renal diseases (P<0.001) by regression analysis. It is also the most sensitive urinary marker for UTI with sensitivity and specificity (94%, 75%) at cutoff 305 ng/mL.

CONCLUSIONS

Combination of serum BMP-2 and cystatin C are more sensitive and accurate for early diagnosis of renal infection and damage.

Outgrowths of mesenchyme-type cells from explants of allogeneic rat muscle onto a substratum of <em>bone</em> matrix containing <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) differentiate into cartilage. When BMP is chemically extracted from the <em>bone</em> matrix, the explanted cells develop only into fibrous tissue. When exogenous bovine BMP is introduced into the culture medium, either as a microsuspension or as a layer of particles between the matrix and the muscle cell tissue, cartilage develops at the interface between the matrix and the mesenchymal cell outgrowth. The chondrogenetic response is induced by as little as 2 micrograms of BMP; the optimum dose is <em>10</em> micrograms/40 mg (wet weight) of explant. The endogenous BMP equivalent for a comparable chondrogenetic response is about 0.6 micrograms/mg of allogeneic matrix. The minimum time for transfer of BMP to mesenchymal cell receptors is 1.0 hour, adequate time is 2.5 hours, and optimum time is approximately 5.0 hours. Measured in terms of incorporation of 3H-thymidine into DNA and of 35S sulfate into glycosaminoglycan, there is a latent period of one to three days preceeding the differentiation of mesenchyme-type cells into cartilage. During this latent period BMP-modulated mesenchymal cells disaggregate, migrate, reaggregate, and proliferate on new surfaces and constitute the <em>morphogenetic</em> phase of <em>bone</em> development. By the fourth day cells simultaneously undergo mitotic division, synthesize extracellular cartilage matrix, and establish the cytodifferentiation phase of development.

We investigated the effects of extracellular ATP on TBR31-2 cells established from the <em>bone</em> marrow of transgenic mice harboring the temperature-sensitive simian virus (SV) 40 T-antigen gene. These cells showed the capacity to differentiate toward osteoblasts and could be enhanced by <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2, an inducer of osteoblasts. The intracellular calcium ion level ([Ca(2+)](i)) in differentiating TBR31-2 cells was measured by fluorescence confocal microscopic imaging using the Ca(2+)-sensitive probe, Calcium Green 1/AM. P2 receptor agonists, such as ATP (1 microM), uridine 5'-triphosphate (1 microM), and ADP (1 microM), significantly increased the [Ca(2+)](i) of TBR31-2 cells in 2-d and 5-d cultures, but a potent P2X receptor agonist, alpha,beta-methylene ATP (<em>10</em> microM), did not increase [Ca(2+)](i). The increase in [Ca(2+)](i) induced by ATP in the 2-d culture tended to be higher than in the 5-d culture. The increase in [Ca(2+)](i) of both cultures was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2 receptor antagonist. However, in an external Ca(2+)-free condition ATP-induced increase in [Ca(2+)](i) was unchanged at either stage. U73122, phospholipase C inhibitor and Thapsigargin, a calcium-pump inhibitor, significantly inhibited the increase in [Ca(2+)](i) at both stages. Reverse transcription-polymerase chain reaction analysis showed that the expression of P2Y receptor mRNA was higher in the 2-d culture than in the 5-d culture. These results indicate that ATP induces the increase in [Ca(2+)](i) from the calcium store through activating P2Y receptors in TBR31-2 cells and that the 2-d culture can respond to ATP more than the 5-d culture due to the higher expression of P2Y receptors. This suggests that the physiological role of ATP in osteoblasts is altered during differentiation.

OBJECTIVE

To repair large-size bone defects, most bone-defect-filling materials in clinic need to obtain osteoinductivity either by mixing them with particulate autologous bone or adsorbing bone morphogenetic protein 2 (BMP2). However, both approaches encounter various limitations. In this study, we hypothesized that our novel particles of biomimetic BMP2-coprecipitated calcium phosphate (BMP2-cop.BioCaP) could serve as an independent and biodegradable osteoinducer to induce bone formation efficiently for these bone-defect-filling materials, for example, deproteinized bovine bone (DBB).

METHODS

We alternately layer-by-layer assembled amorphous and crystalline CaP triply to enable a "bamboo-like" growth of the particles. We functionalized BioCaP by coprecipitating BMP2 into the most outer layer of BioCaP. We monitored the degradation, osteoinductivity, and foreign-body reaction of either BMP2-cop.BioCaP or its combination with DBB in an ectopic site in rats.

RESULTS

After 5 weeks, the BMP2-cop.BioCaP significantly induced new bone formation not only alone but also when mixed with DBB. Its osteoinductive efficiency was 10-fold higher than the adsorbed BMP2. Furthermore, BMP2-cop.BioCaP also reduced significantly the host foreign-body reaction to DBB in comparison with the adsorbed BMP2. After a 5-week implantation, more than 90% of BMP2-cop.BioCaP degraded.

CONCLUSIONS

These findings indicate a promising clinical potential for BMP2-cop.BioCaP in the repair of large-size bone defects.

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related <em>proteins</em> also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase <em>protein</em> array (RPPA), were used to measure the expression of 20 <em>proteins</em> linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven <em>proteins</em> differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related <em>10</em>-<em>protein</em> panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related <em>proteins</em> and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-<em>10</em>0 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (<em>10</em> ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 1<em>10</em>.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (<em>10</em> ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.