We recently showed that osteogenic <em>protein</em>-1(OP-1), a <em>bone</em> <em>morphogenetic</em> <em>protein</em> member of TGF-beta superfamily, induces endochondral <em>bone</em> formation in vivo, and stimulates growth and differentiation of osteoblasts in rat calvarial-derived cell cultures. In the present study, we examined the effect of OP-1 on cell growth and expression of markers that are characteristic of osteoblast phenotype using the clonal rat osteosarcoma cells (ROS 17/2.8). A comparison of OP-1 and TGF-beta 1 effects on cell growth showed that, both OP-1 and TGF-beta 1 inhibited DNA synthesis up to 90 percent and 60 percent of the controls at concentrations of <em>10</em> ng/ml and 1 ng/ml, respectively, in serum-free medium. In the presence of 5% serum, TGF-beta 1 did not have any significant inhibitory effects while 40 ng OP-1/ml inhibited the DNA synthesis up to 80% of the controls. Examination of collagen synthesis showed that 40 ng OP-1/ml increased the expression of type I collagen mRNA, and thus increased collagen synthesis (4-fold), as examined by collagenase-digestible <em>protein</em>. Evaluation of markers that are characteristic of the osteoblast phenotype demonstrated that OP-1 stimulated cAMP production in response to PTH (<em>10</em>-fold at 200 ng/ml), alkaline phosphatase specific activity (ALPase) (4-fold at 80 ng/ml), and osteocalcin (OC) synthesis (4.5-fold at 40 ng/ml). Northern blot analysis revealed that OP-1 increased mRNA expression for both ALPase and OC in a dose-dependent manner. These data collectively demonstrate that OP-1 suppresses cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat clonal osteoblastic osteosarcoma cells (ROS 17/2.8).

A cDNA clone encoding a <em>protein</em> homologous to human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 1 (huBMP1) was isolated from a sea urchin embryo cDNA library. This sea urchin gene, named suBMP, encodes a <em>protein</em> of M(r) of 72 x <em>10</em>(3). The deduced amino acid sequence of suBMP shares 72% sequence similarity (55% identity) with that of huBMP1. Like huBMP1 it also contains an N-terminal metalloendoprotease domain that shares sequence similarity with the astacin protease from crayfish, a C-terminal domain that is similar to the repeat domain found in C1r or C1s serine proteases, and an EGF-like segment. Although suBMP mRNA was detectable at a low level in the unfertilized egg, maximal expression of mRNA was observed at hatched blastula stage, with only a modest decrease in level at later stages of development. In situ hybridization studies revealed that suBMP mRNA is found in both ectodermal and primary mesenchyme cells in hatched blastula-stage embryos. Maximal expression of suBMP was observed at mesenchyme blastula, just before the onset of primitive skeleton (spicule) formation. SuBMP was found by immunoelectronmicroscopy in all cell types in late gastrula stage embryos. The antibody gold particles appeared in small clusters in the cytoplasm, on the surface of the cells and within the blastocoel. This distribution of suBMP, coupled with the finding that it was associated with membranes but was released by sodium carbonate treatment, suggests that the <em>protein</em> is secreted, and subsequently associates with a cell surface component. Two models for the possible function of suBMP in spiculogenesis in the sea urchin embryo are discussed.

Premature cranial suture fusion, or craniosynostosis, can result in gross aberrations of craniofacial growth. The biology underlying cranial suture fusion remains poorly understood. Previous studies of the Sprague-Dawley rat posterior frontal suture, which fuses at between 12 and 20 days, have suggested that the regional dura mater beneath the cranial suture directs the overlying suture's fusion. To address the dura-suture paracrine signaling that results in osteogenic differentiation and suture fusion, the authors investigated the possible role of insulin-like growth factors (IGF) I and II. The authors studied the temporal and spatial patterns of the expression of IGF-I and IGF-II mRNA and IGF-I peptide and osteocalcin (<em>bone</em> <em>morphogenetic</em> <em>protein</em>-4) <em>protein</em> in fusing posterior frontal rat sutures, and they compared them with patent coronal (control) sutures. Ten Sprague-Dawley rats were studied at the following time points: 16, 18, and 20 days of gestation and 2, 5, <em>10</em>, 15, 20, 30, 50, and 80 days after birth (n = 1<em>10</em>). Posterior frontal and coronal (patent, control) sutures were analyzed for IGF-I and IGF-II mRNA expression by in situ hybridization by using 35S-labeled IGF-I and IGF-II antisense riboprobes. Levels of IGF-I and IGF-II mRNA were quantified by counting the number of autoradiograph signals per cell. IGF-I and osteocalcin immunoreactivity were identified by avidin-biotin peroxidase immunohistochemistry. IGF-I and IGF-II mRNA were expressed in dural cells beneath fusing sutures, and the relative mRNA abundance increased between 2 and <em>10</em> days before initiation of fusion. Subsequently, IGF-I and IGF-II mRNA were detected in the suture connective tissue cells at 15 and 20 days during the time of active fusion. In contrast, within large osteoblasts of the osteogenic front, the expression of IGF-I and IGF-II mRNA was minimal. However, IGF-I peptide and osteocalcin <em>protein</em> were intensely immunoreactive within these osteoblasts at 15 days (during the period of suture fusion). These data suggest that the dura-suture interaction may be signaled in a paracrine fashion by dura-derived growth factors, such as IGF-I and IGF-II. These peptides, in turn, stimulate nearby osteoblasts to produce <em>bone</em>-promoting growth factors, such as osteocalcin.

BACKGROUND

Hepatocellular carcinoma (HCC) is a preventable disease rather than a curable one, since there is no well-documented effective treatment modality until now, making the molecular study of this disease mandatory.

RESULTS

We studied gene expression profile of 17 Egyptian HCC patients associated with HCV genotype-4 infection by c-DNA microarray. Out of the 15,660 studied genes, 446 were differentially expressed; 180 of them were up regulated and 134 were down regulated. Seventeen genes out of the 180 up-regulated genes are involved in 28 different pathways. <em>Protein</em> phosphatase 3 (PPP3R1) is involved in <em>10</em> different pathways followed by fibroblast growth factor receptor 1 (FGFR1), Cas-Br-M ecotropic retroviral transforming sequence b (CBLB), spleen tyrosine kinase (SYK) involved in three pathways; <em>bone</em> <em>morphogenetic</em> <em>protein</em> 8a (BMP8A), laminin alpha 3 (LAMA3), cell division cycle 23 (CDC23) involved in 2 pathways and NOTCH4 which regulate Notch signaling pathway. On the other hand, 25 out of the 134 down-regulated genes are involved in 20 different pathways. Integrin alpha V alpha polypeptide antigen CD51 (ITGVA) is involved in 4 pathways followed by lymphotoxin alpha (TNF superfamily, member 1) (LTA) involved in 3 pathways and alpha-2-macroglobulin (A2M), phosphorylase kinase alpha 2-liver (PHKA2) and MAGI1 membrane associated guanylate kinase 1 (MAGI1) involved in 2 pathways. In addition, 22 genes showed significantly differential expression between HCC cases with cirrhosis and without cirrhosis. Confirmation analysis was performed on subsets of these genes by RT-PCR, including some up-regulated genes such as CDK4, Bax, NOTCH4 and some down-regulated genes such as ISGF3G, TNF, and VISA.

CONCLUSIONS

This is the first preliminary study on gene expression profile in Egyptian HCC patients associated with HCV-Genotype-4 using the cDNA microarray. The identified genes could provide a new gate for prognostic and diagnostic markers for HCC associated with HCV. They could also be used to identify candidate genes for molecular target therapy.

METHODS

Posterolateral lumbar spinal fusion with use of recombinant human bone morphogenetic protein 2 (rhBMP-2) was tested in rabbits by implanting composites of rhBMP-2 and collagen carrier.

OBJECTIVE

To examine the bone-formation-inducing activity of rhBMP-2 and find the optimal amount of rhBMP to add to a collagen carrier to constitute bone-formation-inducing implants to be substituted for bone graft in posterolateral spinal fusion in rabbits.

BACKGROUND

In animal models, rhBMP-2--impregnated collagen has been successfully used for posterolateral spinal fusion, indicating that it is a potential substitute for the autogenous corticocancellous bone graft currently used most routinely in posterolateral lumbar spinal fusion.

METHODS

Nine rabbits were divided into three equal groups. The bilateral L4-L5 transverse processes were exposed, and collagen strips impregnated with rhBMP-2 (10, 50, or 200 micrograms) were placed on the left transverse processes, and collagen strips alone were inserted on the right. All rabbits were killed 24 weeks after surgery. The implanted sites were assessed for new bone formation and bony fusion by radiography and histologic examination.

RESULTS

New bone formation was noted in intertransverse spaces on the left side of all rabbits except one (10 micrograms rhBMP-2). Twelve weeks after implantation, no new bone formation was seen on the right side of all animals. The newly formed bone masses were significantly larger in the 50-microgram and 200-microgram rhBMP-2 groups than in the 10-microgram rhBMP-2 group (P < 0.01), but there was no significant difference between bone formation in the 50-microgram and 200-microgram groups (P = 0.647).

CONCLUSIONS

The rhBMP-2/collagen composite implant was an effective bone graft substitute for achieving posterolateral spinal fusion. When combined with a collagen carrier, the optimal rhBMP-2 dose for achieving posterolateral spinal fusion seemed to be approximately 50 micrograms per segment in rabbits.

OBJECTIVE

Antiangiogenic therapy, mostly targeting VEGF, has been applied in cancer patients for the last decade. However, resistance to anti-VEGF therapy and/or no significant benefit as monotherapeutic agent is often observed. Therefore, new antiangiogenic strategies are needed. In the current study, we investigated the therapeutic effect of interfering with the bone morphogenetic protein (BMP)9/activin receptor-like kinase (ALK)1 signaling pathway by using an ALK1-Fc ligand trap.

METHODS

We analyzed the potential antiangiogenic and antitumor effects of ALK1-Fc protein as monotherapy and in combination with chemotherapy in vivo in mouse models of melanoma, head and neck cancer, and invasive lobular breast carcinomas. ALK1-Fc sequesters BMP9 and 10 and prevents binding of these ligands to endothelial ALK1, which regulates angiogenesis.

RESULTS

Treatment of mice with ALK1-Fc strongly decreased the tumors' microvascular density in the three different mouse cancer models. However, this effect was not accompanied by a reduction in tumor volume. An immunohistochemical analysis of the tumor samples revealed that ALK1-Fc treatment increased the pericyte coverage of the remaining tumor vessels and decreased the hypoxia within the tumor. Next, we observed that combining ALK1-Fc with cisplatin inhibited tumor growth in the breast and head and neck cancer models more efficiently than chemotherapy alone.

CONCLUSIONS

The addition of ALK1-Fc to the cisplatin treatment was able to enhance the cytotoxic effect of the chemotherapy. Our results provide strong rationale to explore combined targeting of ALK1 with chemotherapy in a clinical setting, especially in the ongoing phase II clinical trials with ALK1-Fc.

Renal ischemia/reperfusion injury (IRI) is a major cause of acute renal failure. The aim of this study was to investigate whether propofol pretreatment in a rat model protects kidney tissue against IRI. Thirty-two Wistar rats were equally divided into four groups: a sham-operated group, untreated renal IRI group, and low-dose (5 mg/kg) and high-dose (<em>10</em> mg/kg) propofol-treated groups which were treated with propofol prior to the induction of IRI. The rats were subjected to renal ischemia by bilateral clamping of the pedicles for 50 min, followed by reperfusion. The low-dose and high-dose propofol treatment groups were pretreated via femoral vein injection with a propofol suspension prior to the induction of ischemia/reperfusion. The untreated IRI group showed significantly higher serum creatinine (SCr), blood urea nitrogen (BUN), interleukin 6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and malondialdehyde (MDA) levels compared with the sham-operated rats. Superoxide dismutase (SOD) levels were significantly reduced following IRI; however, they significantly increased following propofol administration. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) levels were significantly increased in the propofol-treated groups compared with the untreated IRI group. These results suggest that propofol reduces renal oxidative injury and facilitates repair following IRI. Propofol may play a protective role by regulating BMP2 expression in renal IRI.

Growth differentiation factor 9 (GDF9), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 15 (BMP15), and anti-Müllerian hormone (AMH) play an important role in the primary to secondary follicle transition and follicle activation in vivo. In organ culture of neonatal mouse ovaries, it was observed that significantly fewer primary follicles develop to the secondary stage. The objectives of this study were: (1) to compare ovarian follicular populations between organ-cultured neonatal mouse ovaries and freshly isolated age-matched control ovaries; (2) to quantify RNA levels of Gdf9, Bmp15, and Amh in cultured primary follicles; and (3) to immunolocalize GDF9 and AMH in cultured ovaries. Ovaries from 3-day-old (PND 3) mice were cultured for 7 or <em>10</em> days in the absence or presence of FSH. Follicular populations were counted in freshly isolated 13-day-old (PND 13) ovaries and organ-cultured ovaries. Transcripts were quantified in isolated primary follicles using real-time RT-PCR, and <em>protein</em> expressions were localized using immunohistochemistry. The number of secondary follicles in organ-cultured ovaries was significantly lower than in vivo controls. Gdf9 and Bmp15 mRNA expression levels were similar as in controls. Amh mRNA levels were significantly (P<0.05) lower after day <em>10</em> of culture in the absence of FSH. GDF9 and AMH <em>proteins</em> were respectively detected in the oocytes and the granulosa cells (GC) beginning at the primary and primordial stages onward. GDF9 and BMP15 production in cultured primary follicles are not different from in vivo controls; hence abnormal early follicular growth was not related to a deficient transcription of these factors.

Adipose stem cell preparations, either obtained as a freshly isolated so-called stromal vascular fraction (SVF) or as cells cultured to homogeneity and then referred to as adipose stem cells (ASCs), have found widespread use in a broad variety of studies on tissue engineering and regenerative medicine applications, including <em>bone</em> repair.For newcomers within the field, but also for established research laboratories having up to <em>10</em> years of expertise in this research area, it may be convenient to strive for, and use consensus protocols (1) for studying the osteogenic differentiation potential of ASC preparations in vitro, and (2) for osteogenic induction regimes for in vivo implementation. To assist in achieving this goal, this chapter describes various step-by-step osteogenic differentiation protocols for adipose-derived stem cell populations (SVF as well as ASCs) currently applied within our laboratory, with particular emphasis on protocols aimed at intra-operative use. The protocols describe the use of inducing compounds, including the <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), 1,25-dihydroxyvitamin-D3, and polyamines, as well as methods and parameters for evaluating the level of differentiation achieved.We would appreciate receiving feedback on the protocols described; this will facilitate the development of consensus protocols, which in turn will allow better comparison of data sets generated by different research groups. This continuing standardization, which might be reported on at international meetings like those of IFATS ( http://www.IFATS.org ), might be of benefit for the whole ASC research community.

BACKGROUND

Cell-based therapy using mesenchymal stem cells (MSCs) seems promising to obtain regeneration of dental tissues. A comparison of tissue sources, including periodontal ligament (PDL) versus pulp (P), could provide critical information to select an appropriate MSC population for designing predictable regenerative therapies. The purpose of this study is to compare the proliferation and stemness and the MSC-specific and mineralized tissue-specific gene expression of P-MSCs and PDL-MSCs.

METHODS

MSCs were obtained from PDL and P tissue of premolars (n = 3) extracted for orthodontic reasons. MSC proliferation was evaluated using a real-time cell analyzer for 160 hours. Telomerase activity was evaluated by a telomeric repeat amplification protocol assay based on enzyme-linked immunosorbent assay. Total RNA was isolated from the MSCs on day 3. A polymerase chain reaction (PCR) array was used to compare the expression of MSC-specific genes. The expression of mineralized tissue-associated genes, including Type I collagen (COL I), runt-related transcription factor 2 (RunX2), bone sialoprotein (BSP), and osteocalcin (OCN) messenger RNA (mRNA), was evaluated using quantitative real-time PCR.

RESULTS

Higher proliferation potential and telomerase activity were observed in the P-MSCs compared to PDL-MSCs of premolar teeth. Fourteen of 84 genes related to MSCs were expressed differently in the PDL-MSCs versus the P-MSCs. The expressions of bone morphogenetic protein 2 (BMP2) and BMP6; sex-determining region Y-box 9 (SOX9); integrin, alpha 6 (ITGA6); melanoma cell adhesion molecule (MCAM); phosphatidylinositol glycan anchor biosynthesis, class S (PIGS); prominin 1 (PROM1); ribosomal protein L13A (RPL13A); and microphthalmia-associated transcription factor (MITF) were higher in the P-MSCs compared to the PDL-MSCs, and higher expression of matrix metalloproteinase 2 (MMP2), interleukin (IL)-6, insulin (INS), alanyl (membrane) aminopeptidase (ANPEP), and IL-10 were observed in the PDL-MSCs. However, there was no statistically significant difference in the expression of mineralized tissue-associated genes, including BSP and RunX2, between the P-MSCs and the PDL-MSCs. Higher expression of COL I and lower expression of OCN mRNA transcripts were noted in the PDL-MSCs compared to the P-MSCs.

CONCLUSIONS

The results of this study suggest that MSCs isolated from P and PDL tissues show different cellular behavior. To increase the predictability of MSC-based regenerative treatment, differences in dental tissue-derived MSCs and favorable aspects of cell sources should be further clarified.

Bone morphogenetic protein 9 (BMP9) has previously been characterized as one of the most osteogenic growth factors of the BMP family. To the best of the authors' knowledge, previous experiments have only used adenovirus transfection (gene therapy). With the recent development of recombinant human BMP9 (rhBMP9), the present study investigates the osteopromotive potential of BMP9 versus rhBMP2 when loaded onto collagen membranes.

ST2 stromal bone marrow cells were seeded onto: 1) control; 2) low-dose rhBMP2 (10 ng/mL); 3) high-dose rhBMP2 (100 ng/mL); 4) low-dose rhBMP9 (10 ng/mL); and 5) high-dose rhBMP9 (100 ng/mL) porcine collagen membranes. The following parameters were compared among groups: 1) cell adhesion (at 8 hours); 2) cell proliferation (at 1, 3, and 5 days); 3) real-time polymerase chain reaction for genes encoding runt-related transcription factor 2; 4) alkaline phosphatase (ALP); 5) bone sialoprotein ([BSP] at 3 and 14 days); and 6) alizarin red staining (at 14 days).

rhBMP2 and rhBMP9 demonstrated little effect on cell attachment and proliferation; however, pronounced increases were observed in osteoblast differentiation. All groups significantly induced ALP messenger RNA (mRNA) levels at 3 days and BSP levels at 14 days; however, high-dose rhBMP9 showed significantly higher values compared with all other groups for ALP levels (five-fold increase at 3 days and two-fold increase at 14 days). Alizarin red staining further revealed both concentrations of rhBMP9 induced up to three-fold more staining compared with rhBMP2.

Results indicate that the combination of collagen membranes with rhBMP9 induced significantly higher ALP mRNA expression and alizarin red staining compared with rhBMP2. These findings suggest that rhBMP9 may be a suitable growth factor for future regenerative procedures in bone biology.

The percentage of delayed or non-unions after fractures of long <em>bones</em> depends on the individual risk profile at approximately <em>10</em> %. The current definition states that a non-union is a fracture that will not consolidate without any further intervention - independent from the treatment time. At the early stage of a non-union a conservative treatment is possible in case of an adequately stable situation. The operative treatment depends on the type of the non-union. There are one-step or two-step procedures, all according to the principles of the "diamond concept". This means improvement of the mechanical situation - in most cases by means of a reosteosynthesis - and vascularization, local application of osteoconductive carriers e.g. tricalciumphosphate, vital cells from autologous <em>bone</em> and osteoinductive substances like <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP-2 or BMP-7). Hypertrophic and atrophic non-unions without large defect gaps or signs of infection can be treated with a one-step procedure. For treating infected non-unions or non-unions with large defect gaps the Masquelet technique is recommended.

Block-type biphasic calcium phosphate (BCP) carriers are more effective at delivering recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) in various clinical situations than are particle-type carriers, due to their potential for highly successful three-dimensional <em>bone</em> regeneration. The aim of this study was to confirm the <em>bone</em>-regenerative capabilities of three-dimensional BCP blocks with a low hydroxyapatite/β-tricalcium phosphate ratio (20/80) combined with collagen (<em>10</em>% wt) as an rhBMP-2 delivery system in a craniofacial vertical <em>bone</em> augmentation model. BCP blocks and BCP-collagen blocks (with average macropore sizes of 296 and 390 μm, respectively) with or without rhBMP-2 were fixed with osteosynthesis screws to the calvarial surface of rabbits. After 8 weeks, histologic and histomorphometric analyses were performed to evaluate the resulting new <em>bone</em> area, augmented area, <em>bone</em> density, and degree of integration. The area of new <em>bone</em> was significantly greater in specimens containing rhBMP-2 than in the control group (p < 0.05). Moreover, the area fractions of newly formed <em>bone</em> within the augmented area and a degree of integration between the regenerative <em>bone</em> and the calvarium were both significantly greater in the BCP-collagen/rhBMP-2 group than in the BCP/rhBMP-2 group (p < 0.05), whereas the two carrier systems exhibited similar rhBMP-2 release profiles, with sustained and linear release. The BCP and BCP/rhBMP-2 blocks exhibited excellent structural integrity, with large fragments of residual ceramic. In conclusion, the BCP-collagen composite block exhibited enhanced osteoinductive potential and could be a good candidate as a carrier of rhBMP-2 due to its characteristics of favorable volumetric stability, ease of handling, and excellent remodeling properties.

Inflammation plays a fundamental role in the inception and development of atherosclerosis (ATH). Mechanisms of inflammation include the infiltration of monocytes into the injured area and subsequent differentiation into either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. We have previously published data suggesting <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) enhances M2 macrophage differentiation and anti-inflammatory cytokine secretion in vitro. In this regard, we hypothesized BMP-7 would inhibit plaque formation in an animal model of ATH through monocytic plasticity mediation. ATH was generated in male and female Apo E(-/-) mice via partial left carotid artery (PLCA) ligation and mice were divided into 3 groups: Sham, PLCA, and PLCA+BMP-7 (200 ug/kg; i.v.). Our data suggest that BMP-7 inhibits plaque formation and increases arterial systolic velocity. Furthermore, we report inhibition of monocyte infiltration and a decrease in associated pro-inflammatory cytokines (MCP-1, TNF-α, and IL-6) in the PLCA+BMP-7 mice. In contrast, our data suggest a significant (p<0.05) increase in M2 macrophage populations with consequential enhanced anti-inflammatory cytokine (IL-1RA, IL-<em>10</em>, and Arginase 1) expression following BMP-7 treatment. We have also observed that mechanisms promoting monocyte into M2 macrophage differentiation by BMP-7 involve the upregulation and activation of the BMP-7 receptor (BMP-7RII). In conclusion, we report that BMP-7 has the potential to mediate cellular plasticity and mitigate the inflammatory immune response, which results in decreased plaque formation and improved blood velocity.

OBJECTIVE

Use of recombinant human bone morphogenetic protein-2 has risen steadily since its approval by the FDA for use in anterior lumbar interbody fusion in 2002. The FDA has not approved the use of bone morphogenetic protein (BMP) in children. Age less than 18 years or lack of evidence of epiphyseal closure are considered by the manufacturer to be contraindications to BMP use. In light of this, the authors performed a query of the database of one of the nation's largest health insurance companies to determine the rate of BMP use and complications in pediatric patients undergoing spinal fusion.

METHODS

The authors used the PearlDiver Technologies private payer database containing all records from United Health-Care from 2005 to 2011 to query all cases of pediatric spinal fusion with or without BMP use. A review of the literature was also performed to examine the complications associated with BMP use in pediatric spinal fusion.

RESULTS

A total of 4658 patients underwent spinal fusion. The majority was female (65.4%), and the vast majority was age 10-19 years (94.98%) and underwent thoracolumbar fusion (93.13%). Bone morphogenetic protein was used in 1752 spinal fusions (37.61%). There was no difference in the rate of BMP use when comparing male and female patients or age 10 years or older versus less than 10 years. Anterior cervical fusions were significantly less likely to use BMP (7.3%). Complications occurred in 9.82% of patients treated with versus 9.88% of patients treated without BMP. The complication rate was nearly identical in male versus female patients and in patients older versus younger than 10 years. Comparison of systemic, wound-related, CNS, and other complications showed no difference between groups treated with and without BMP. The reoperation rate was also nearly identical.

CONCLUSIONS

Bone morphogenetic protein is used in a higher than expected percentage of pediatric spinal fusions. The rate of acute complications in these operations does not appear to be different in patients treated with versus those treated without BMP. Caution must be exercised in interpreting these data due to the many limitations of the administrative database as a data source, including the short length of follow-up.

The present work assesses the potential of three-dimensional (3D) porous titanium (pore size of 188-390 μm and porosity of 70%) fabricated by vacuum diffusion bonding of titanium meshes for applications in <em>bone</em> engineering. Rat <em>bone</em> marrow mesenchymal stem cells were used to investigate the proliferation and differentiation of cells on titanium scaffolds with different pore sizes at day 7, day 14 and day 21 based on DNA contents, alkaline phosphatase (ALP) activity, collagen (COL) secretion and osteogenic gene expressions including ALP, COL-1, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2), osteopontin (OPN), runt-related transcription factor 2 (RUNX2), using smooth solid titanium plate as reference material. The rabbit models with distal femoral condyles defect were used to investigate the <em>bone</em> ingrowth into the porous titanium. All samples were subjected to Micro-CT and histological analysis after 4 and 12 weeks of healing. A one-way ANOVA followed by Tukey post hoc tests was used to analyze the data. It was found that the differentiation stage of cells on the porous titanium delayed compared with the smooth solid titanium plate and Ti 188 was more inclined to promote cell differentiation at the initial stage (day 14) while cell proliferation (day 1, 4, 7, <em>10</em>, 14 and 21) and <em>bone</em> ingrowth (4 and 12 weeks) were biased to Ti 313 and Ti 390. The study indicates that the hybrid porous implant design which combines the advantages of different pore sizes may be meaningful and promising for <em>bone</em> defect restoration.

UNASSIGNED

One of the significant challenges in bone defect restoration is the integration of biomaterials and surrounding bone tissue. Porous titanium may be a promising choice for bone ingrowth and mineralization with appropriate mechanical and biological properties. In this study, based on porous titanium fabricated by vacuum diffusion bonding of titanium meshes, we have evaluated the influence of various pore sizes on rat bone marrow mesenchymal stem cells (rBMMSCs) penetration in vitro and bone ingrowth in vivo. It was interesting that we found the proliferation and differentiation abilities of rBMMSCs, as well as bone ingrowth were related to different pore sizes of such porous scaffolds. The results may provide guidance for porous titanium design for bone defect restoration.

Adipocytes control <em>bone</em> mass, but the mechanism is unclear. To explore the effect of postnatal adipocyte elimination on <em>bone</em> cells, we mated mice expressing an inducible primate diphtheria toxin receptor (DTR) to those bearing adiponectin (ADQ)-Cre. DTR activation eliminates peripheral and marrow adipocytes in these DTR^ADQ mice. Within 4 days of DTR activation, the systemic <em>bone</em> mass of DTR^ADQ mice began to increase due to stimulated osteogenesis, with a 1,000% expansion by <em>10</em>-14 days post-DTR treatment. This adipocyte ablation-mediated enhancement of skeletal mass reflected <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) receptor activation following the elimination of its inhibitors, associated with simultaneous epidermal growth factor (EGF) receptor signaling. DTR^ADQ-induced osteosclerosis is not due to ablation of peripheral adipocytes but likely reflects the elimination of marrow ADQ-expressing cells. Thus, anabolic drugs targeting BMP receptor inhibitors with short-term EGF receptor activation may be a means of profoundly increasing skeletal mass to prevent or reverse pathological <em>bone</em> loss.

Keywords: BMPR activation; adipocyte; bone formation; heparin-binding epidermal like growth factor.

To clarify the effect of recombinant human basic fibroblast growth factor (FGF-2) on the osteoinductive activity of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) in vivo, different amounts of FGF-2 (0, 16, 80 and 400 ng, and 2, <em>10</em> and 50 micro g: n=<em>10</em> in each group), BMP-2 (2 micro g) and type I collagen as a carrier were mixed and implanted into rat calf muscles. Three weeks after implantation, compared with the controls, the radiopaque shadows of the implants were increased in the 16, 80 and 400 ng FGF-2-treated groups, but decreased in the 2, <em>10</em> and 50 micro g FGF-2-treated groups. In addition, alkaline phosphatase activity was increased in the 16, 80 and 400 ng FGF-2-treated groups but decreased in the 50 micro g FGF-2-treated group. Histological examination revealed increased <em>bone</em> formation in the 16, 80 and 400 ng FGF-2-treated groups. These results show that combined treatment with FGF-2 and BMP-2 has a biphasic effect on osteoinductive activity, i.e. it increases with low doses of FGF-2 and decreases with high doses of FGF-2.

Human <em>bone</em> marrow-derived mesenchymal stem cells (hBM-MSCs) are widely used in a number of cell therapies and have osteogenic differentiation capacity. Exposure to electromagnetic fields (EMFs) increases the osteogenic differentiation of hBM-MSCs. Nanomagnetic particles (MPs) also promote the differentiation potential of stem cells. In the present study, we investigated the effects of EMFs and MPs on the osteogenic differentiation of hBM-MSCs. hBM-MSCs were treated with 50 µg/ml of Fe3O4 MPs or exposed to a frequency of 45 Hz and an intensity of 1 mT EMF twice every 8 h per day for 7 days. MP incorporation, EMF exposure and MP incorporation with exposure to EMFs did not induce cytotoxic effects. A strong expression of osteogenic markers (osteocalcin, osteopontin and osteonectin) and von Kossa staining intensity was observed in the cells treated with MPs, the cells exposed to EMFs and in the cells treated with MPs and exposed to EMFs compared with the control group, as shown by immunohistochemical staining. Quantitative RT-PCR revealed that the mRNA expression levels of osteoblast markers [osteocalcin, osteopontin, osteonectin, collagen Ⅰ, collagen Ⅲ, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2), <em>bone</em> sialo<em>protein</em> (BSP) and runt-related transcription factor 2 (runx-2)] were markedly increased in the cells treated with MPs and exposed to EMFs. Furthermore, the mRNA expression of calcium channels (CACNA1C, CACNA1E, CACNA1G and CACNA1I) was activated during osteogenic differentiation. The expression levels of osteogenesis-related <em>proteins</em> (BSP, BMP-2, osteopontin and osteonectin) and phosphorylated extracellular signal-regulated kinase (p-ERK) were increased in the cells treated with MPs, those exposed to EMFs and in the cells treated with MPs and exposed to EMFs compared with the control group, as shown by western blot analysis. Fluorescence-activated cell sorting (FACS) analysis was performed for the hBM-MSC markers, CD73, CD90 and CD<em>10</em>5. The expression levels of hBM-MSC surface antigens were decreased in the cells treated with MPs, those exposed to EMFs and in the cells treated with MPs and exposed to EMFs compared with the control group. The cell numbers were determined to be approximately 3.4 x <em>10</em>(5) cells in the control group, 3.7 x <em>10</em>(5) cells in the MP-treated group, 3.1 x <em>10</em>(5) cells in the group exposed to EMFs and 3.9 x <em>10</em>(5) cells in the group treated with MPs and exposed to EMFs. The cell mitochondrial activity among the 4 experimental groups was similar. The hBM-MSCs treated with MPs and exposed to EMFs showed an increase in alkaline phosphatase (ALP) activity. Taken together, these results suggest that the treatment of hBM-MSCs with MPs or exposure to EMFs increases osteogenic differentiation, and that treatment with MPs in conjunction with EMF exposure is more effective in increasing osteogenic differentiation.

A porcine calvaria defect study was carried out to investigate the <em>bone</em> repair potential of three-dimensional (3D)-printed poly-ε-caprolactone (PCL) scaffolds embedded with nanoporous PCL. A microscopic grid network was created by rapid prototyping making a 3D-fused deposition model (FDM-PCL). Afterward, the FDM-PCL scaffolds were infused with a mixture of PCL, water, and 1,4-dioxane and underwent a thermal-induced phase separation (TIPS) followed by lyophilization. The TIPS process lead to a nanoporous structure shielded by the printed microstructure (NSP-PCL). Sixteen Landrace pigs were divided into two groups with 8 and 12 weeks follow-up, respectively. A total of six nonpenetrating holes were drilled in the calvaria of each animal. The size of the cylindrical defects was h <em>10</em> mm and Ø <em>10</em> mm. The defects were distributed randomly using following groups: (a) NSP-PCL scaffold, (b) FDM-PCL scaffold, (c) autograft, (d) empty defect, (a1) NSP-PCL scaffold + autologous mononuclear cells, and (a2) NSP-PCL scaffold + <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2. <em>Bone</em> volume to total volume was analyzed using microcomputed tomography (µCT) and histomorphometry. The µCT and histological data showed significantly less <em>bone</em> formation in the NSP-PCL scaffolds in all three variations after both 8 and 12 weeks compared to all other groups. The positive autograft control had significantly higher new <em>bone</em> formation compared to all other groups except the FDM-PCL when analyzed using histomorphometry. The NSP-PCL scaffolds were heavily infiltrated with foreign body giant cells suggesting an inflammatory response and perhaps active resorption of the scaffold material. The unmodified FDM-PCL scaffold showed good osteoconductivity and osseointegration after both 8 and 12 weeks.

The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal <em>bone</em> tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage <em>10</em> (P<em>10</em>), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent <em>bone</em> phenotype from P<em>10</em> to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (<em>10</em> ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate <em>bone</em> development, cell/biomaterial interactions, and may be a useful screen for putative <em>bone</em> differentiating factors.

Novel anabolic drug targets are needed to treat osteoporosis. Having established a large national cohort with unexplained high <em>bone</em> mass (HBM), we aimed to identify a novel monogenic cause of HBM and provide insight into a regulatory pathway potentially amenable to therapeutic intervention. We investigated a pedigree with unexplained HBM in whom previous sequencing had excluded known causes of monogenic HBM. Whole exome sequencing identified a rare (minor allele frequency 0.0023), highly evolutionarily conserved, missense mutation in SMAD9 (c.65T>C, p.Leu22Pro) segregating with HBM in this autosomal dominant family. The same mutation was identified in another two unrelated individuals both with HBM. In-silico <em>protein</em> modelling predicts the mutation severely disrupts the MH1 DNA-binding domain of SMAD9. Affected individuals have <em>bone</em> mineral density [BMD] Z-Scores +3 to +5, mandible enlargement, a broad frame, torus palatinus/ mandibularis, pes planus, increased shoe size and a tendency to sink when swimming. Peripheral quantitative computer tomography (pQCT) measurement demonstrates increased trabecular volumetric BMD and increased cortical thickness conferring greater predicted <em>bone</em> strength; <em>bone</em> turnover markers are low/normal. Notably, fractures and nerve compression are not seen. Both genome-wide, and gene-based association testing involving estimated-BMD measured at the heel in 362,924 white British subjects from the UK Biobank Study showed strong associations with SMAD9 (P_GWAS = 6 x <em>10</em>^-16 ; P_GENE = 8 × <em>10</em>^-17 ). Furthermore, we found Smad9 to be highly expressed in both murine cortical <em>bone</em> derived osteocytes and skeletal elements of zebrafish larvae. Our findings support SMAD9 as a novel HBM gene, and a potential novel osteoanabolic target for osteoporosis therapeutics. SMAD9 is thought to inhibit <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) dependent target gene transcription to reduce osteoblast activity. Thus, we hypothesise SMAD9 c.65T>C is a loss-of-function mutation reducing BMP inhibition. Lowering SMAD9 as a potential novel anabolic mechanism for osteoporosis therapeutics warrants further investigation.

Several million people are exposed to dioxin and dioxin-like compounds, primarily through food consumption. Skin lesions historically called "chloracne" are the most specific sign of abnormal dioxin exposure and classically used as a key marker in humans. We followed for 5 years a man who had been exposed to the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a single oral dose of 5 million-fold more than the accepted daily exposure in the general population. We adopted a molecular medicine approach, aimed at identifying appropriate therapy. Skin lesions, which progressively covered up to 40% of the body surface, were found to be hamartomas, which developed parallel to a complete and sustained involution of sebaceous glands, with concurrent transcriptomic alterations pointing to the inhibition of lipid metabolism and the involvement of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> signaling. Hamartomas created a new compartment that concentrated TCDD up to <em>10</em>-fold compared with serum and strongly expressed the TCDD-metabolizing enzyme cytochrome P450 1A1, thus representing a potentially significant source of enzymatic activity, which may add to the xenobiotic metabolism potential of the classical organs such as the liver. This historical case provides a unique set of data on the human tissue response to dioxin for the identification of new markers of exposure in human populations. The herein discovered adaptive cutaneous response to TCDD also points to the potential role of the skin in the metabolism of food xenobiotics.

We evaluated whether dexamethasone augments the osteogenic capability of <em>bone</em> marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic <em>bone</em> formation induced by <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without <em>10</em>-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic <em>bone</em> formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic <em>bone</em> formation model, <em>bone</em> formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2.

CONCLUSIONS

1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.