BACKGROUND

Achilles tendon ruptures in humans might be treated more efficiently with the help of a growth factor. Cartilage-derived morphogenetic protein-2 has been shown to induce formation of tendon-like tissue.

OBJECTIVE

Cartilage-derived morphogenetic protein-2 has a positive effect on mechanical parameters for tendon healing in a rabbit model with Achilles tendon transection.

METHODS

Controlled laboratory study.

METHODS

The right Achilles tendon of 40 rabbits was transected without tendon suture. Cartilage-derived morphogenetic protein-2 (10 micro g) or vehicle control (acetate buffer) was injected locally 2 hours postoperatively. All tendons were tested biomechanically at 8 and 14 days, and treated tendons were histologically and radiographically evaluated at 56 days.

RESULTS

At 14 days, both failure load and stiffness of treated tendons were increased by 35%. The treated tendons had significantly larger callus size at 8 and 14 days. Histologic and radiographic examination showed no signs of ossification in the treated tendons after 56 days.

CONCLUSIONS

A single injection of cartilage-derived morphogenetic protein-2 led to a stronger and stiffer tendon callus than that in the controls without inducing bone formation.

CONCLUSIONS

Similar results from a larger animal model would suggest a possible future use of cartilage-derived morphogenetic protein-2 in the treatment of human Achilles tendon ruptures.

BACKGROUND

Fibroblast growth factor 2 (FGF2) is a growth factor that is immediately released after cartilage injury and plays a pivotal role in cartilage homeostasis. In human adult articular cartilage, FGF2 mediates anti-anabolic and potentially catabolic effects via the suppression of proteoglycan (PG) production along with the upregulation of matrix-degrading enzyme activity. The aim of the present study was to determine the biological effects of FGF2 in spine disc cells and to elucidate the complex biochemical pathways utilized by FGF2 in bovine intervertebral disc (IVD) cells in an attempt to further understand the pathophysiologic processes involved in disc degeneration.

METHODS

We studied the effect of FGF2 on IVD tissue homeostasis by assessing MMP-13 expression (potent matrix-degrading enzyme), PG accumulation, and PG synthesis in the bovine spine IVD, as well as evaluating whether FGF2 counteracts known anabolic factors such as BMP7. To understand the molecular mechanisms by which FGF2 antagonizes BMP7 activity, we also investigated the signaling pathways utilized by FGF2 in bovine disc tissue.

RESULTS

The primary receptor expressed in bovine nucleus pulposus cartilage is FGFR1, and this receptor is upregulated in degenerative human IVD tissue compared with normal IVD tissue. Stimulation of bovine nucleus pulposus cells cultured in monolayer with FGF2 augmented the production of MMP-13 at the transcriptional and translational level in a dose-dependent manner. Stimulation of bovine nucleus pulposus cells cultured in alginate beads for 21 days with FGF2 resulted in a dose-dependent decrease in PG accumulation, due at least in part to the inhibition of PG synthesis. Further studies demonstrate that FGF2 (<em>10</em> ng/ml) antagonizes BMP7-mediated acceleration of PG production in bovine nucleus pulposus cells via the upregulation of noggin, an inhibitor of the transforming growth factor beta/<em>bone</em> <em>morphogenetic</em> <em>protein</em> signaling pathway. Chemical inhibitor studies showed that FGF2 utilizes the mitogen-activated <em>protein</em> kinase and NF-kappaB pathways to upregulate noggin, serving as one potential mechanism for its anti-anabolic effects.

CONCLUSIONS

FGF2 is anti-anabolic in bovine spine disc cells, revealing the potential of FGF2 antagonists as unique biologic treatments for both prevention and reversal of IVD degeneration.

Calcium phosphate <em>bone</em> cements (CPBCs) are osteotransductive, i.e. after implantation in <em>bone</em> they are transformed into new <em>bone</em> tissue. Furthermore, due to the fact that they are mouldable, their osteointegration is immediate. Their chemistry has been established previously. Some CPBCs contain amorphous calcium phosphate (ACP) and set by a sol-gel transition. The others are crystalline and can give as the reaction product dicalcium phosphate dihydrate (DCPD), calcium-deficient hydroxyapatite (CDHA), carbonated apatite (CA) or hydroxyapatite (HA). Mixed-type gypsum-DCPD cements are also described. In vivo rates of osteotransduction vary as follows: gypsum-DCPD>> DCPD>> CDHA approximately CA>> HA. The osteotransduction of CDHA-type cements may be increased by adding dicalcium phosphate anhydrous (DCP) and/or CaCO3 to the cement powder. CPBCs can be used for healing of <em>bone</em> defects, <em>bone</em> augmentation and <em>bone</em> reconstruction. Incorporation of drugs like antibiotics and <em>bone</em> <em>morphogenetic</em> <em>protein</em> is envisaged. Load-bearing applications are allowed for CHDA-type, CA-type and HA-type CPBCs as they have a higher compressive strength than human trabecular <em>bone</em> (<em>10</em> MPa).

In order to develop a successful gene therapy system for the healing of <em>bone</em> defects, we developed a murine leukemia virus (MLV)-based retroviral system expressing the human <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) 4 transgene with high transduction efficiency. The <em>bone</em> formation potential of BMP4 transduced cells was tested by embedding 2.5 x <em>10</em>(6) transduced stromal cells in a gelatin matrix that was then placed in a critical size defect in calvariae of syngenic rats. Gelatin matrix without cells or with untransduced stromal cells were the two control groups. The defect area was completely filled with new <em>bone</em> in experimental rats after 4 weeks, while limited <em>bone</em> formation occurred in either control group. <em>Bone</em> mineral density (BMD) of the defect in the gene therapy group was 67.8 +/- 5.7 mg/cm(2) (mean +/- s.d., n = 4), which was 119 +/- <em>10</em>% of the control BMD of <em>bone</em> surrounding the defect (57.2 +/- 1.5 mg/cm(2)). In contrast, BMD of rats implanted with untransduced stromal cells was five-fold lower (13.8 +/- 7.4 mg/cm(2), P < 0.001). Time course studies revealed that there was a linear increase in BMD between 2-4 weeks after inoculation of the critical size defect with 2.5 x <em>10</em>(6) implanted BMP4 cells. In conclusion, the retroviral-based BMP4 gene therapy system that we have developed has the potential for regeneration of large skeletal defects.

We previously reported that <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) and their endogenous antagonist noggin are expressed in the brain weeks after an ischemic insult. Here, to define their roles in ischemic brain tissue repair and remodeling, we infused recombinant BMP7 or noggin into the ipsilateral ventricle of mice for 2weeks starting 2weeks after transient middle cerebral artery occlusion (MCAO). Four weeks after MCAO, we measured ischemic brain volume, functional recovery, and molecules related to neurogenesis and angiogenesis such as synaptophysin, GAP-43, and VEGF. Noggin-treated mice but not BMP7-treated mice showed preserved ipsilateral brain volume and reduced neurological deficits compared with artificial cerebrospinal fluids (aCSF)-treated mice. Noggin treatment also decreased glial scar thickness, increased levels of GAP-43 and VEGF <em>protein</em>, and increased the number of Iba1-positive activated microglia in the ipsilateral brain. Furthermore, noggin treatment decreased M1 markers (IL-1β, TNF-α, IL-12, CCL2 and CD86) and increased M2 markers (IL-1ra, IL-<em>10</em>, arginase 1, CD206 and Ym1) of activated microglia, suggesting a shift from M1 to M2 phenotypes. These results suggest that noggin improves functional recovery from ischemic stroke and enhances alternatively activated microglia, thereby promoting tissue repair and remodeling.

During embryonic development, cartilage formation involves the condensation of mesenchymal stem cells and a series of maturation steps that ultimately results in the mineralized hypertrophic chondrocyte. The embryonic, murine, mesenchymal stem cell line, C3H/<em>10</em>T1/2, is pluripotent; exposure to azacytidine or to <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 or -4 results in low rates of differentiation to three mesengenic lineages. In contrast to previous studies, we report conditions for <em>10</em>T1/2 differentiation specifically to the cartilage lineage and at high yields. These conditions include high cell density micromass cultures, a purified mixture of osteoinductive-<em>proteins</em> (BP; Intermedics Orthopedics, Denver, CO), a serum substitute, 50 micrograms/ml ascorbic acid, and <em>10</em> mM beta-glycerophosphate. The cartilagenous fate was confirmed by 1) histological detection of sulfated proteoglycans, 2) electron microscopic detection of proteoglycan and rounded cells separated by extracellular matrix containing short, disorganized collagen fibrils, 3) morphological detection of chondrocytes surrounded by a territorial matrix and encompassed within a distinct perichondrium, and 4) immunocytochemical detection of type II collagen and link <em>protein</em>. After 4 weeks in culture, mature although unmineralized cartilage was observed, as indicated by hypertrophic morphology, immunocytochemical detection of osteocalcin, and histological detection of lacunae. These conditions promote overt chondrogenesis for most of the treated cells and preclude lineage determination to the fat, muscle, and <em>bone</em> lineage, as assayed by electron microscopy and histomorphology. The faithful recapitulation of cartilage differentiation that we have established in vitro provides a versatile alternative to the use of chondrocyte and limb bud explant cultures. We propose this as a model system to study the factors that regulate commitment to the chondrogenic lineage, exclusion to related mesengenic pathways, and maturation during chondrogenesis.

BACKGROUND

Mechanical forces play critical roles in the development and remodelling process of bone. As an alternative cell source for bone engineering, adipose-derived stem cells (ASCs) should be fully investigated for their responses to mechanical stress and the mechanisms responsible for osteogenic induction in response to mechanical signals.

METHODS

We hypothesized that appropriate application of uniaxial cyclic tensile strain to ASCs could increase bone morphogenetic protein-2 (BMP-2) expression and improve osteogenesis of ASCs. To test our hypothesis, ASCs from the same flask of the same donor were subjected to tensile strain with different patterns in order to eliminate the difference of donor site and passage. After surface markers investigation, the osteo-induced ASCs were subjected to uniaxial cyclic tensile stretch with the following two loading patterns: long duration continuous pattern (6 h, 1 HZ, 2000 µɛ) and short duration consecutive pattern (17 min every day for 10 consecutive days, 1 HZ, 2000 µɛ). Then osteogenic related genes were analysed by real-time PCR.

RESULTS

The ASCs were positive for the markers STRO-1, CD90 and CD44 and negative for CD34. Cyclic tensile strain of 6 continuous h' duration significantly increased gene expressions of BMP-2 and Runx2, and depressed OCN mRNA expression. In contrast, mechanical loading of 17 min every day did not significantly affect gene expression of BMP-2, Runx2, OCN or ALP.

CONCLUSIONS

We indicate that ASCs may sense mechanical loading in a duration-dependent manner and cyclic tensile stretch may modulate the osteogenic differentiation of ASCs via the BMP-2 signalling pathway.

OBJECTIVE

To identify genetic variants underlying six anthropometric traits: body height, body weight, body mass index, brachial circumference, waist circumference, and hip circumference, using a genome-wide association study.

METHODS

The study was carried out in the isolated population of the island of Korcula, Croatia, with 898 adult examinees who participated in the larger DNA-based genetic epidemiological study in 2007. Anthropometric measurements followed standard internationally accepted procedures. Examinees were genotyped using HumanHap 370CNV chip by Illumina, with a genome-wide scan containing 316730 single nucleotide polymorphisms (SNP).

RESULTS

A total of 11 SNPs were associated with the investigated traits at the level of P(<em>10</em>(-5), with one SNP (rs7792939 in gene zinc finger <em>protein</em> 498, ZNF498) associated with body weight, hip circumference, and brachial circumference (P=3.59-5.73 x <em>10</em>(-6)), and another one (rs157350 in gene delta-sarcoglycan, SGCD) with both brachial and hip circumference (P=3.70-6.08 x <em>10</em>(-6). Variants in CRIM1, a gene regulating delivery of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> to the cell surface, and ITGA1, involved in the regulation of mesenchymal stem cell proliferation and cartilage production, were also associated with brachial circumference (P=7.82 and 9.68 x <em>10</em>(-6), respectively) and represent interesting functional candidates. Other associations involved those between genes SEZ6L2 and MAX and waist circumference, XTP6 and brachial circumference, and AMPA1/GRIA1 and height.

CONCLUSIONS

Although the study was underpowered for the reported associations to reach formal threshold of genome-wide significance under the assumption of independent multiple testing, the consistency of association between the 2 variants and a set of anthropometric traits makes CRIM1 and ITGA1 highly interesting for further replication and functional follow-up. Increased linkage disequilibrium between the used markers in an isolated population makes the formal significance threshold overly stringent, and changed allele frequencies in isolate population may contribute to identifying variants that would not be easily identified in large outbred populations.

METHODS

Randomized clinical trial.

OBJECTIVE

To perform a cost-utility analysis using actual cost data from a randomized clinical trial of patients over 60 years old who underwent posterolateral fusion using either rhBMP-2/ACS or iliac crest bone graft (ICBG).

BACKGROUND

Bone morphogenetic protein has been shown to be an effective bone graft substitute for spine fusion. However, a clinical trial-based economic analysis of rhBMP-2/ACS compared with iliac crest bone graft has not been done.

METHODS

Patients over 60 years old requiring decompression and posterolateral fusion were randomized to rhBMP-2/ACS (n = 50) or ICBG (n = 52). A dedicated hospital coder and research nurse tracked each patient to determine direct costs of inpatient care and all postoperative healthcare encounters up to 2 years after surgery. Preoperative and 2-year-postoperative SF-6D utility scores for each patient were determined. A decision tree was created, which included the probability of complications, need for additional treatments and revision surgery; and the costs associated with initial surgery and treatment for complications and additional treatment for continued spine symptoms; and utility scores.

RESULTS

The mean total 2-year cost for care (excluding complication and additional spine treatment costs) was $34,235 in the ICBG group and $36,530 in the rhBMP-2/ACS group. For the entire group, the mean cost to treat a major complication was $10,888, the cost of revision surgery for nonunion was $46,852, and additional treatment for spine-related events was $5892. In the ICBG group, 8 patients had complications; 20 had additional interventions, 5 of whom required revision for nonunion. In the rhBMP-2/ACS group, 6 patients had complications, 10 had additional interventions, and 1 required revision for nonunion. The cost of using rhBMP-2/ACS was $39,967 with a 0.11 mean improvement in SF-6D; and for ICBG the cost was $42,286 with a mean improvement of 0.10 in SF-6D.

CONCLUSIONS

There are more complications, increased need for additional treatment and revision surgery in patients over 60 years old receiving ICBG compared with rhBMP-2/ACS. This may account for higher costs and lower improvements in utility seen in patients receiving ICBG compared with rhBMP-2/ACS in this study population.

Osteointegration of metal implants into aged organisms can be severely compromised due to reduced healing capacity of <em>bone</em>, lack of precursor cells for new <em>bone</em> formation, or osteoporosis. Here, we report on successful implant healing in a novel model of aged sheep in the presence of nonglycosylated <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2). Ewes of 8 to 12 years with significant radiologic and histologic signs of osteoporosis and adipocytic <em>bone</em> marrow received a cylindrical hydroxyapatite-titanium implant of 12 x <em>10</em> mm. BMP-2 has been produced as a bacterial recombinant fusion <em>protein</em> with maltose-binding <em>protein</em> and in vitro generation of mature BMP-2 by renaturation and proteolytic cleavage. A BMP-2 inhibition ELISA was developed to measure the in vitro release kinetics of bioactive human BMP-2 from immersed solid implant materials by using Escherichia coli expressed and biotinylated recombinant human BMP-2 receptor IA extracellular domain (ALK-3 ECD). The implants were placed laterally below both tibial plateaus, with the left leg implant carrying 380 microg BMP-2. Both implant types became integrated within the following 20 weeks. The control implant only integrated at the cortical <em>bone</em>, and little new <em>bone</em> formation was found within the pre-existing trabecular <em>bone</em> or the marrow cavity. Marrow fat tissue was partially replaced by unspecific connective tissue. In contrast, BMP-2-coated implants initiated significant new <em>bone</em> formation, initially in trabecular arrangements to be replaced by cortical-like <em>bone</em> after 20 weeks. The new <em>bone</em> was oriented towards the cylinder. Highly viable <em>bone</em> marrow appeared and filled the lacunar structures of the new <em>bone</em>. In mechanical tests, the BMP-2-coated implants displayed in average 50% higher stability. This animal model provided first evidence that application of nonglycosylated BMP-2 coated on solid implants may foster <em>bone</em> healing and regeneration even in aged-compromised individuals.

BACKGROUND

Bone morphogenetic proteins (BMPs) are being evaluated for periodontal and bone regenerative therapy. The objective of this study was to evaluate the effect of recombinant human bone morphogenetic protein-4 (rhBMP-4) dose on local bone formation in a rat calvaria defect model.

METHODS

Calvarial, 8 mm diameter, critical-size osteotomy defects were created in 140 male Sprague-Dawley rats. Seven groups of 20 animals each received either 1) rhBMP-4 (2.5 microg) in an absorbable collagen sponge (ACS) carrier, 2) rhBMP-4 (5 microg)/ACS, 3) rhBMP-4 (2.5 microg) in a beta-tricalcium phosphate (beta-TCP) carrier, 4) rhBMP-4 (5 microg)/beta-TCP, 5) ACS or 6) beta-TCP carrier controls, or 7) a sham-surgery control, and were evaluated by histologic and histometric parameters following a 2- or 8-week healing interval (10 animals/group/healing interval).

RESULTS

Surgical implantation of rhBMP-4/ACS and rhBMP-4/beta-TCP resulted in enhanced local bone formation at both 2 and 8 weeks. Within the dose range examined, rhBMP-4 did not exhibit an appreciable dose-dependent response. Defect closure was not significantly different between the rhBMP-4/ACS and rhBMP-4/beta-TCP groups. New bone area of the rhBMP-4/ beta-TCP group was significantly greater than that of the rhBMP-4/ ACS group; however, bone density in the rhBMP-4/ACS group was significantly greater than that in the rhBMP-4/beta-TCP group at 8 weeks (P < 0.05).

CONCLUSIONS

rhBMP-4 combined with ACS or beta-TCP has a significant potential to induce bone formation in the rat calvaria defect model. Within the selected rhBMP-4 dose range and observation interval, there appeared to be no meaningful differences in bone formation.

The present study investigates the differential effect of two vitamin D receptor agonists, calcitriol and paricalcitol, on human aortic smooth muscle cells calcification in vitro. Human vascular smooth muscle cells were incubated in a high phosphate (HP) medium alone or supplemented with either calcitriol <em>10</em>(-8)M (HP + CTR) or paricalcitol 3·<em>10</em>(-8) M (HP + PC). HP medium induced calcification, which was associated with the upregulation of mRNA expression of osteogenic factors such as <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2), Runx2/Cbfa1, Msx2, and osteocalcin. In these cells, activation of Wnt/β-catenin signaling was evidenced by the translocation of β-catenin into the nucleus and the increase in the expression of direct target genes as cyclin D1, axin 2, and VCAN/versican. Addition of calcitriol to HP medium (HP + CTR) further increased calcification and also enhanced the expression of osteogenic factors together with a significant elevation of nuclear β-catenin levels and the expression of cyclin D1, axin 2, and VCAN. By contrast, the addition of paricalcitol (HP + PC) not only reduced calcification but also downregulated the expression of BMP2 and other osteoblastic phenotype markers as well as the levels of nuclear β-catenin and the expression of its target genes. The role of Wnt/β-catenin on phosphate- and calcitriol-induced calcification was further demonstrated by the inhibition of calcification after addition of Dickkopf-related <em>protein</em> 1 (DKK-1), a specific natural antagonist of the Wnt/β-catenin signaling pathway. In conclusion, the differential effect of calcitriol and paricalcitol on vascular calcification appears to be mediated by a distinct regulation of the BMP and Wnt/β-catenin signaling pathways.

Providing fully mature and functional osteoblasts is challenging for <em>bone</em> tissue engineering and regenerative medicine. Such cells could be obtained from multipotent <em>bone</em> marrow mesenchymal stem cells (MSCs) after induction by different osteogenic factors. However, there are some discrepancies in results, notably due to the use of sera and to the type of osteogenic factor. In this study, we compared the osteogenic differentiation of <em>bone</em> marrow MSCs induced by dexamethasone (Dex) or <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) by assessing phenotypes in vitro and functional osteoblasts in vivo. Reducing the content of fetal calf serum from <em>10</em>% to 2% significantly increased the mineral deposition and expression of osteoblastic markers during osteogenesis. In comparison to Dex condition, the addition of BMP4 greatly improved the differentiation of MSCs into fully mature osteoblasts as seen by high expression of Osterix. These results were confirmed in different supportive matrixes, plastic flasks, or biphasic calcium phosphate biomaterials. In contrast to Dex-derived osteoblasts, BMP4-derived osteoblasts from MSCs were significantly able to produce new <em>bone</em> in subcutis of nude mice in accordance with in vitro results. In conclusion, we describe a convenient ex vivo method to produce consistently mature functional osteoblasts from human MSCs with use of BMP4 and low serum.

The <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) family is a class of transforming growth factor (TGF-beta) superfamily molecules that have been implicated in neuronal differentiation. We studied the effects of BMP2 and glial cell line-derived neurotrophic factor (GDNF) on inducing differentiation of enteric neurons and the signal transduction pathways involved. Studies were performed using a novel murine fetal enteric neuronal cell line (IM-FEN) and primary enteric neurons. Enteric neurons were cultured in the presence of vehicle, GDNF (<em>10</em>0 ng/ml), BMP2 (<em>10</em> ng/ml), or both (GDNF + BMP2), and differentiation was assessed by neurite length, markers of neuronal differentiation (neurofilament medium polypeptide and beta-III-tubulin), and neurotransmitter expression [neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and Substance P]. BMP2 increased the differentiation of enteric neurons compared with vehicle and GDNF-treated neurons (P < 0.001). BMP2 increased the expression of the mature neuronal markers (P < 0.05). BMP2 promoted differentiation of NPY-, nNOS-, and TH-expressing neurons (P < 0.001) but had no effect on the expression of cholinergic neurons (ChAT, Substance P). Neurons cultured in the presence of BMP2 have higher numbers of TH-expressing neurons after exposure to 1-methyl 4-phenylpyridinium (MPP(+)) compared with those cultured with MPP(+) alone (P < 0.01). The Smad signal transduction pathway has been implicated in TGF-beta signaling. BMP2 induced phosphorylation of Smad1, and the effects of BMP2 on differentiation of enteric neurons were significantly reduced in the presence of Smad1 siRNA, implicating the role of Smad1 in BMP2-induced differentiation. The effects of BMP2 on catecholaminergic neurons may have therapeutic implications in gastrointestinal motility disturbances.

Fracture healing involves multiple stages of repair and coordinated actions of multiple cell types. Consequently, it may be possible to enhance healing through treatment strategies that target more than one repair process or cell type. The goal of this study was to determine the combined effects of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (rhBMP-7) and parathyroid hormone (PTH(1-34)) on metaphyseal <em>bone</em> healing. A wedge-shaped defect was created in the lateral aspect of the distal tibia in female New Zealand white rabbits (n=64) and was filled with tricalcium phosphate (TCP). Animals were assigned to four groups: 1) BMP-7 and PTH; 2) BMP-7; 3) PTH; and 4) control (TCP alone). In groups 1 and 2, 200 microg rhBMP-7 was incorporated into the TCP. Animals received daily subcutaneous injections of <em>10</em> microg/kg PTH(1-34) (groups 1 and 3) or saline (groups 2 and 4). Healing at 4 weeks was assessed using micro-computed tomography, histology, immunohistochemistry, and mechanical testing. Combined treatment with rhBMP-7 and PTH resulted in increased callus total volume (TV), mineralized volume (BV), average cross-sectional area, and <em>bone</em> mineral content (BMC) as compared to the control group (p<0.02). BV and BMC were also higher in the combined treatment group as compared to the BMP-7 group (p<0.02); however, tissue mineral density was highest in the BMP-7 group (p=0.002). New <em>bone</em> formation in the BMP-7 group was largely restricted to the defect site, while PTH promoted <em>bone</em> formation throughout the defect and surrounding regions. Combined treatment led to greater quantities of woven trabecular <em>bone</em>, increased trabecular thickness, decreased trabecular separation (p<0.04), and a trend towards increased numbers of osteoclasts (p=0.09). Combined treatment also resulted in increased torsional rigidity and compressive strength as compared to the control and BMP-7 groups (p<0.001). These results suggest that the improvements in mechanical function obtained with the combined treatment resulted from differing biological activities of rhBMP-7 and PTH. While the activities of rhBMP-7 appeared to be strictly anabolic, those of PTH appeared to work in the context of coupled remodeling. The combination of both agents led to greater <em>bone</em> volume as well as better microstructural organization and integration of this <em>bone</em> with the surrounding tissues.

In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing <em>10</em>% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P < 0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P < 0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P < 0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P>> 0.<em>10</em>). Treatment with 600 ng/ml GDF-9 increased (P < 0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P < 0.05) progesterone production (by 28%), increased (P < 0.05) cell numbers (by 60%), and had no effect (P>> 0.<em>10</em>) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P < 0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.

BACKGROUND

Segmental bone loss remains a challenging clinical problem. A frequent mitigating factor is inadequate blood supply. Small molecules that activate the hypoxia-inducible factor pathway can be used to stimulate angiogenesis. We investigated an approach to promote healing using angiogenic and osteogenic compounds in combination with a biodegradable, weightbearing scaffold.

METHODS

Adult rats underwent removal of a 5-mm segment of femur stabilized by a cylindrical biodegradable implant and intramedullary fixation. Treatment groups included 1) saline (negative control); 2) desferrioxamine (DFO, a hypoxia-inducible factor activator; 3) low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 μg); 4) DFO and low-dose rhBMP-2 (5 μg); or 5) rh-BMP-2 (10 μg). Angiography was used to evaluate vascularity. Bone healing was assessed by radiographs, microcomputed tomography, histology, and biomechanical testing.

RESULTS

Increased vascularity was seen at 6 weeks in the DFO treatment group. There appeared to be increased bone bridging as assessed by radiographic scores and microcomputed tomography in the BMP groups, although the quantification of bone volume did not show statistically significant differences. Biomechanical testing revealed improved stiffness in the treatment groups.

CONCLUSIONS

DFO improved angiogenesis and stiffness of bone healing in segmental defects. BMP improved radiographic scores and stiffness. Use of angiogenic compounds in segmental bone loss is promising.

CONCLUSIONS

Activation of the hypoxia-inducible factor pathway may prove useful for bone defects, particularly where impaired blood supply exists.The low-cost approach could be useful in segmental bone defects clinically.

Activin receptor-like kinase 1 (ALK1) is an endothelial serine-threonine kinase receptor for <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) 9 and <em>10</em>. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/<em>10</em> ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/<em>10</em> ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2.

Cell-cell communication between multiple-cell populations is believed to be important for the activation of many cellular functions. Previously, we showed that, in the rat hepatoma cell line Fao, grown in a layered co-culture system with a human umbilical vein endothelial cell (HUVEC) sheet, the expression of albumin and apolipo<em>protein</em> A-I increased time-dependently for <em>10</em> days and was maintained at a significantly higher level than Fao without the HUVEC sheet. Because the gene-expression profile of hepatocytes and HUVECs under double-layered co-culture has not previously been elucidated, in the present study, we examined the difference in messenger ribonucleic acid expression between Fao or HUVEC monolayer cells and double-layered co-cultured Fao cells/HUVECs using suppression subtractive hybridization. More than 200 transcripts were differentially screened to ensure unique expression. The expression levels of genes in the co-cultured and monolayer cells were determined using SYBR Green I real-time reverse transcription polymerase chain reaction with species-specific primers. We found 23 genes that showed at least a 2-fold difference in expression level. Five hepatocyte-specific genes--alpha1-acidglyco<em>protein</em>, alpha2-microglobulin, hepcidin, transferrin, and transthyretin--were identified from the Fao cells. Two cell-surface <em>protein</em> genes--<em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor type II and CD82--which may be related to cell-cell communication, showed greater expression in the HUVECs co-cultured with Fao cells. These results indicate that many hepatocyte and endothelial cell functions increase in intensity upon layered co-culture.

Craniofacial injuries can result from trauma, tumor ablation, or infection and may require multiple surgical revisions. To address the challenges associated with treating craniofacial <em>bone</em> defects, an ideal material should have the ability to fit complex defects (i.e. be conformable), provide temporary protection to the brain until the <em>bone</em> heals, and enhance tissue regeneration with the delivery of biologics. In this study, we evaluated the ability of injectable lysine-derived polyurethane (PUR)/allograft biocomposites to promote <em>bone</em> healing in critical-size rabbit calvarial defects. The biocomposites exhibited favorable injectability, characterized by a low yield stress to initiate flow of the material and a high initial viscosity to minimize the adverse phenomena of extravasation and filter pressing. After injection, the materials cured within <em>10</em>-12 min to form a tough, elastomeric solid that maintained mechanical integrity during the healing process. When injected into a critical-size calvarial defect in rabbits, the biocomposites supported ingrowth of new <em>bone</em>. The addition of 80 µg mL(-1) recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) enhanced new <em>bone</em> formation in the interior of the defect, as well as bridging of the defect with new <em>bone</em>. These observations suggest that injectable reactive PUR/allograft biocomposites are a promising approach for healing calvarial defects by providing both mechanical stability as well as local delivery of rhBMP-2.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are multifunctional <em>proteins</em> that comprise the largest subfamily of the transforming growth factor-beta. These <em>proteins</em> bind to types I and II serine/threonine kinase receptors. Ligand-induced heteromeric dimerization of these receptors is the key event in initiation of biological responses. We report here large-scale expression and purification of extracellular domain of the type I receptor for BMP-2/4, using a silkworm expression system. This soluble form of BMP receptor (sBMPR) was in monomer form in solution and bound to BMP-4 but not to activin or transforming growth factor-beta1. Surface plasmon resonance studies showed that kinetic parameters of sBMPR for BMP-4 consisted of a relatively rapid association rate constant (ka = 3.81 +/- 0.19 x <em>10</em>(4) s-1 M-1) and an extremely slow dissociation rate constant (kd = 3.69 +/- 0.26 x <em>10</em>(-4) s-1). From these two kinetic parameters, affinity was determined to be similar to that of the intact membrane-associated receptor expressed on COS cells. sBMPR inhibited the alkaline phosphatase activity in BMP responsive cell lines such as mouse osteoblastic cell MC3T3-E1 and <em>bone</em> marrow stromal cell ST2. These data indicate that the extracellular domain of type I receptor for BMP-2 and BMP-4 is sufficient for high-affinity binding to its ligands and should prove useful in understanding the role of BMP-2/4 in vivo, because a suitable high-affinity anti-BMP antibody has yet to be developed.

The aim of the present study was to determine the relationships between <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs), BMP receptor type IA and carcinogenesis of oral epithelium. A retrospective study was performed on material obtained from oral mucosa, including nine cases of normal mucosa (NB), eight cases of nonspecific chronic inflammation (NCI), seven cases of hyperkeratosis (HK), five cases of squamous cell papilloma (SCP), 29 cases of squamous cell carcinoma (SCC) with various grades of differentiation and <em>10</em> cases of epithelium adjacent to carcinoma (EAC). Six cases of NB from hard palate (NHP) were chosen as a control group. The benign groups consisted of NCI, HK and SCP. The antibodies against BMP-2/4, -5, receptor BMPR-IA and purified bovine BMP (bBMP-McAb) were utilised using an immunocytochemical method. The results demonstrated that the immunostaining of BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb was weak and not consistent in normal and benign groups. The immunoreactivity level was independent of the clinical and pathological grading of SCC. All cases of SCC showed positive staining for BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb except for three cases and one case of SCC which negatively stained for BMP-2/4 and BMP-5, respectively. The staining intensity and proportion of the positively stained cells were markedly increased in SCC when compared with that of the normal and benign groups except for EAC. The metastatic carcinoma cells in lymph nodes were strongly and positively stained for BMP-2/4 and BMP-5 when compared with the primary lesions. Our results indicate that there was an overexpression of BMP-2/4, BMP-5, bBMP-McAb and BMPR-IA in the high-risk premalignant and malignant lesions of oral epithelium. Our findings suggest that BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis of oral carcinoma cells.

Reduced estrogen levels occurring during menopause in women are accompanied by a variety of disorders, e.g. hot flushes, depressions, osteoporosis, increase in body weight and reduced movement drive. The phytoestrogen genistein (GEN) has been demonstrated to have a significant <em>bone</em>-protective potency. In order to study the ER subtype-specific effects of this phytoestrogen on <em>bone</em> in an animal model, ovariectomized (OVX) female Wistar rats were either treated with 17beta-estradiol (E(2)) (4 microg/kg/day), the ER alpha-specific agonist (ALPHA) 16 alpha-LE(2) (<em>10</em> microg/kg/day), the ER beta-specific agonist (BETA) 8 beta-VE(2) (<em>10</em>0 microg/kg/day) or GEN (<em>10</em> mg/kg/day) for 3 weeks. Vehicle-treated OVX animals served as controls. All animals had the opportunity of voluntary wheel running. Movement activity, changes of body weight and trabecular <em>bone</em> mineral density (BMD) in the tibia were analyzed. E(2) and ALPHA treatment, but not treatment with BETA, significantly increased the movement activity of OVX rats. Treatment with GEN resulted in a significant decrease of movement activity as compared to OVX animals. <em>Bone</em> mineral density in the trabecular area of the tibia and the expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) were significantly reduced in OVX- and BETA-treated rats as compared to rats substituted with E(2), ALPHA and GEN. The <em>bone</em>-protective effect of ALPHA was antagonized by co-treatment with the pure antiestrogen Faslodex (ICI). In order to distinguish hormone-dependent effects from those of exercise, we performed an additional experiment where the animals had no opportunity of wheel running. The results demonstrate that physically inactive rats have a stronger decrease of <em>bone</em> mineral density than physically active animals. Very surprisingly, our data demonstrate that GEN has no <em>bone</em>-protective activity in the absence of physical activity. In contrast, ALPHA and E(2) are <em>bone</em>-protective in the presence and absence of physical activity. In conclusion, our data provide evidence that the effects of E(2) on body weight, movement drive and protection of <em>bone</em> mineral density are mediated via ER alpha, whereas activation of ER beta has only a limited effect. Our data also indicate that the <em>bone</em>-protective effects of GEN may be mediated via ER alpha-dependent mechanisms and that physical activity has a strong impact on the <em>bone</em>-protective potency of this phytoestrogen.

OBJECTIVE

To understand the chemotherapy response program in ovarian cancer cells at deep transcript sequencing levels.

METHODS

Two next-generation sequencing technologies--MPSS (massively parallel signature sequencing) and SBS (sequencing by synthesis)--were used to sequence the transcripts of IGROV1 and IGROV1-CP cells, and to sequence the transcripts of a highly chemotherapy responsive and a highly chemotherapy resistant ovarian cancer tissue.

RESULTS

We identified 3422 signatures (2957 genes) that are significantly different between IGROV1 and IGROV1-CP cells (P<0.001). Gene Ontology (GO) term GO:0001837 (epithelial-to-mesenchymal transition) and GO:0034330 (cell junction assembly and maintenance) are enriched in genes that are over expressed in IGROV1-CP cells while apoptosis-related GO terms are enriched in genes over expressed in IGROV1 cells. We identified 1187 tags (corresponding to <em>10</em>40 genes) that are differentially expressed between the chemotherapy responsive and the persistently chemotherapy resistant ovarian cancer tissues. GO term GO:0050673 (epithelial cell proliferation) and GO:0050678 (regulation of epithelial cell proliferation) are enriched in the genes over expressed in the chemotherapy resistant tissue while the GO:0007229 (integrin-mediated signaling pathway) is enriched in the genes over expressed in the chemotherapy sensitive tissue. An integrative analysis identified 111 common differentially expressed genes including two <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP4 and BMP7), six solute carrier <em>proteins</em> (SLC<em>10</em>A3, SLC16A3, SLC25A1, SLC35B3, SLC7A5 and SLC7A7), transcription factor POU5F1 (POU class 5 homeobox 1), and KLK<em>10</em> (kallikrein-related peptidase <em>10</em>). A network analysis revealed a subnetwork with three genes BMP7, NR2F2 and AP2B1 that were consistently over expressed in the chemoresistant tissue or cells compared to the chemosensitive tissue or cells.

CONCLUSIONS

Our database offers the first comprehensive view of the digital transcriptomes of ovarian cancer cell lines and tissues with different chemotherapy response phenotypes.