This study describes a novel bioactive xerogel glass as a carrier for <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) and the value of this carrier in terms of stimulating osteogenic activity of rat stromal marrow cells in vitro. These cells were seeded onto the surface of xerogel glass disks with BMP either incorporated in the glass, adsorbed to the surface of the glass, or added to the culture media and then compared to cells on glass with no added BMP or to cells on tissue culture plastic (TCP) with and without BMP. Cells were cultured for 6 and <em>10</em> days and examined for total DNA, alkaline phosphatase activity, and osteocalcin and total <em>protein</em> production. Stromal cell differentiation, as measured by alkaline phosphatase activity and osteocalcin synthesis was most increased when the BMP was incorporated or adsorbed onto the xerogel glass. Cells on xerogel glass without BMP were more differentiated than cells grown on plastic with BMP, thereby demonstrating the additive effect of a bioactive substrate and BMP on osteoblastic cell differentiation. These data indicate that xerogel glass effects differentiation of cells with osteogenic potential and that it can serve as a delivery vehicle for BMP.

Fibrodysplasia ossificans progressiva (FOP) is a skeletal disorder with progressive heterotopic ossification in skeletal muscle. A mutation causing constitutive activation in a <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) type 1 receptor [ALK2(R206H)] is found in most patients with FOP. However, the details in the heterotopic ossification of muscle in FOP and the role of matrix metallo<em>protein</em>ase-<em>10</em> (MMP-<em>10</em>) in <em>bone</em> remain to be fully elucidated. In the present study, we investigated the role of MMP-<em>10</em> in the differentiation of mouse myoblastic C2C12 cells into osteoblasts. MMP-<em>10</em> was extracted as a factor, whose expression was most extensively enhanced by ALK2 (R206H) transfection in C2C12 cells. MMP-<em>10</em> significantly augmented the levels of Osterix, type 1 collagen, alkaline phosphatase (ALP) and osteocalcin mRNA as well as ALP activity enhanced by BMP-2 in C2C12 cells. Moreover, a reduction in endogenous MMP-<em>10</em> levels by siRNA significantly decreased the levels of Runx2, Osterix, type 1 collagen, ALP and osteocalcin mRNA enhanced by BMP-2 in these cells. In addition, MMP-<em>10</em> increased the phosphorylation of Smad1/5/8 as well as enhanced the levels of Smad6 and Smad7 mRNA induced by BMP-2. In conclusion, the present study first demonstrated that MMP-<em>10</em> promotes the differentiation of myoblasts into osteoblasts by interacting with the BMP signaling pathway. MMP-<em>10</em> may play some important role in the heterotopic ossification of muscle in FOP.

Controlled delivery systems play a critical role in the success of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (i.e., BMP2 and BMP7) for challenged <em>bone</em> repair. Instead of single-drug release that is currently and commonly prevalent, dual-drug delivery strategies are highly desired to achieve effective <em>bone</em> regeneration because natural <em>bone</em> repair process is driven by multiple factors. Particularly, angiogenesis is essential for osteogenesis and requires more than just one factor (e.g., Vascular Endothelial Growth Factor, VEGF). Therefore, we developed a novel mesoporous silicate nanoparticles (MSNs) incorporated-3D nanofibrous gelatin (GF) scaffold for dual-delivery of BMP2 and deferoxamine (DFO). DFO is a hypoxia-mimetic drug that can activate hypoxia-inducible factor-1 alpha (HIF-1α), and trigger subsequent angiogenesis. Sustained BMP2 release system was achieved through encapsulation into large-pored MSNs, while the relative short-term release of DFO was engineered through covalent conjugation with chitosan to reduce its cytotoxicity and elongate its half-life. Both MSNs and DFO were incorporated onto a porous 3D GF scaffold to serve as a biomimetic osteogenic microenvironment. Our data indicated that DFO and BMP2 were released from a scaffold at different release rates (<em>10</em> vs 28 days) yet maintained their angiogenic and osteogenic ability, respectively. Importantly, our data indicated that the released DFO significantly improved BMP2-induced osteogenic differentiation where the dose/duration was important for its effects in both mouse and human stem cell models. Thus, we developed a novel and tunable MSNs/GF 3D scaffold-mediated dual-drug delivery system and studied the potential application of the both FDA-approved DFO and BMP2 for <em>bone</em> tissue engineering.

Collagen sponge disks (6 mm diameter, 1 mm thickness) were impregnated with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) (5 microg/disk) and implanted onto the back muscles of mice. Ten or 20 mg/kg per day of Rolipram, a selective inhibitory agent to phosphodiesterase type 4 (PDE-4), or vehicle, was injected subcutaneously into the host mice for 3 weeks. After treatment, rhBMP-2-induced ectopic ossicles were harvested and examined by radiographic and histologic methods to determine the size, <em>bone</em> quality, and mineral content of the ossicles. The ossicles from a group treated with 20 mg/kg per day Rolipram were significantly larger in size and higher in <em>bone</em> mineral density (BMD) and <em>bone</em> mineral content (BMC) than the control samples. No significant differences were noted in mice treated with <em>10</em> mg/kg per day of Rolipram. Histologically, ossicles from the high-dose (20 mg/kg per day) Rolipram-treated group showed densely packed, thicker trabeculae when compared with those from the control group. These experimental results indicate that the PDE-4 inhibitor, Rolipram, may enhance the <em>bone</em>-inducing capacity of BMP-2 in mesenchymal cells. This in turn may result in increased responsiveness to BMP-2 and point to a potential use of PDE-4 inhibitors for the promotion of rhBMP-dependent <em>bone</em> repair.

METHODS

Consecutive case series.

OBJECTIVE

The purpose of this study was to analyze the pseudarthrosis rate in a large series of recombinant human bone morphogenetic protein-2 (rhBMP-2) augmented multilevel >> or =3 levels) anterior cervical fusions.

BACKGROUND

The reported pseudarthrosis rate following anterior cervical fusion varies from 0% to 20% for single-level and up to 50% for multilevel fusions. It has been postulated that the use of rhBMP-2 may decrease the pseudarthrosis rate.

METHODS

A consecutive series of patients with cervical spondylosis and/or disc herniation who underwent anterior cervical fusion with rhBMP-2, structural allograft, and plate fixation with a minimum 2-year follow-up were analyzed by experienced, independent spine surgeons.

RESULTS

A total of 127 patients (54 men and 73 women with mean age of 54 +/- 10 years [range, 32-79]) were examined. Seventy-five (59.1%) patients underwent a 3-level fusion, 34 (26.7%) underwent a 4-level fusion, and 18 (14.2%) underwent a 5-level fusion. Of the 451 fusion segments, 14 segments (3.1%) in 13 of 127 patients (10.2%) had evidence of pseudarthrosis at 6 months following surgery. Of the 13 patients with a pseudarthrosis, 3 had a 3-level fusion (3/75 patients [4.0%]), 6 had a 4-level fusion (6/34 patients [17.4%]), and 4 had a 5-level fusion (4/18 patients [22.2%]). Five patients were asymptomatic and were not revised, but the remaining 8 patients required additional surgery. In 12 of 13 patients with a pseudarthrosis, the nonunion occurred at the lowest fusion level and at the cervicothoracic junction. The only statistically significant risk factor for developing a pseudarthrosis was the number of fusion levels.

CONCLUSIONS

In a large series of rhBMP-2 augmented multilevel fusions, the pseudarthrosis rate was 10.2% at 6 months following surgery. Since the risk of pseudarthrosis increases with the number of fusion levels, a long fusion lever arm may biomechanically overwhelm the biologic advantage of rhBMP-2. While rhBMP-2 is known to enhance fusion rates, it does not guarantee fusion in all situations.

<em>Bone</em> overgrowth is a known phenomenon occurring after fracture of growing long <em>bones</em> with possible long-term physical consequences for affected children. Here, the physeal expression of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) was investigated in a fracture-animal model to test the hypothesis that a diaphyseal fracture stimulates the physeal expression of these known key regulators of <em>bone</em> formation, thus stimulating <em>bone</em> overgrowth. Sprague-Dawley rats (male, 4 weeks old), were subjected to a unilateral mid-diaphyseal tibial fracture. Kinetic expression of physeal BMP-2, -4, -6, -7, and BMP receptor-1a (BMPR-1a) was analyzed in a monthly period by quantitative real time-polymerase chain reaction and immunohistochemistry. On Days 1, 3, <em>10</em>, and 14 post-fracture, no changes in physeal BMPs gene-expression were detected. Twenty-nine days post-fracture, when the fracture was consolidated, physeal expression of BMP-6 and BMPR-1a was significantly upregulated in the growth plate of the fractured and contra-lateral intact <em>bone</em> compared to control (p<0.005). This study demonstrates a late role of BMP-6 and BMPR-1a in fracture-induced physeal growth alterations and furthermore, may have discovered the existence of a regulatory "cross-talk" mechanism between the lower limbs whose function could be to limit leg-length-discrepancies following the breakage of growing <em>bones</em>.

The aims of this study were to fabricate poly(ethylene glycol) (PEG) hydrogel micropatterns on a biomaterial surface to guide osteoblast behaviour and to study how incorporating vascular endothelial growth factor (VEGF) within the adhered hydrogel influenced cell morphology. Standard photolithographic procedures or photopolymerization through a poly(dimethyl siloxane) (PDMS) mould were used to fabricate patterned PEG hydrogels on the surface of silanized silicon wafers. Hydrogel patterns were evaluated by light microscopy and surface profilometry. Rat osteoblasts were cultured on these surfaces and cell morphology investigated by fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Release of <em>protein</em> trapped in the polymerized PEG was evaluated and VEGF-PEG surfaces were characterized for their ability to support cell growth. These studies show that photopolymerized PEG can be used to create anti-adhesive structures on the surface of silicon that completely control where cell interaction with the substrate takes place. Using conventional lithography, structures down to 50 microm were routinely fabricated with the boundaries exhibiting sloping sides. Using the PDMS mould approach, structures were fabricated as small as <em>10</em> microm and boundaries were very sharp and vertical. Osteoblasts exhibiting typical morphology only grew on the silicon wafer surface that was not coated with PEG. Adding BSA to the monomer solution showed that <em>protein</em> could be released from the hydrogel for up to 7 days in vitro. Incorporating VEGF in the hydrogel produced micropatterns that dramatically altered osteoblast behaviour. At boundaries with the VEGF-PEG hydrogel, there was striking formation of cellular processes and membrane ruffling indicative of a change in cell morphology. This study has explored the <em>morphogenetic</em> properties of VEGF and the applications of nano/microfabrication techniques for guided tissue (<em>bone</em>) regeneration in dental and orthopaedic applications using osteoinductive PEG hydrogel micropatterns.

OBJECTIVE

For human use, it is necessary to sterilize bone morphogenetic proteins (BMPs), in order to reduce the risk of infections and associated complications. We compared the effects of ethylene oxide and gamma irradiation in the sterilization of native reindeer BMP extract with regard to bone induction in the Balb/C mouse thigh muscle pouch model.

METHODS

BMP extract, sterilized with ethylene oxide gas (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h, ethylene oxide concentration 860 mg/l), or gamma irradiation at doses of 3.15 MRad was administered in implants containing 5 or 10 mg of BMP extract with collagen carrier. Non-sterilized collagen implants served as controls. New bone formation was evaluated based on the incorporation of Ca45 and radiographically three weeks after implantation.

RESULTS

The collagen was not able to induce new bone visible in radiographs. The mean Ca45 incorporation in the gamma sterilized group containing 5 mg of BMP extract was 30% (p = 0.04) and that containing 10 mg of BMP extract was 60% (p = 0.02) higher than seen in the corresponding ethylene oxide sterilized groups. The mean new bone areas were 45% higher in the gamma sterilized groups than in the corresponding ethylene oxide sterilized groups, but the differences were not significant. The mean optical density of new bone in the gamma sterilized group containing 5 mg of BMP extract was 75% (p = 0.00) and in that containing 10 mg of BMP extract was 70% (p = 0.00) higher than seen in the corresponding ethylene oxide sterilized groups.

CONCLUSIONS

Native reindeer BMP extract is more sensitive to the effects of ethylene oxide gas sterilization than gamma irradiation. These results suggest that gamma irradiation is recommendable for the sterilization of BMP extracts.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) heterodimers can trigger and sustain osteoblastic <em>bone</em> regeneration in significantly lower dosages than BMP homodimers. However, their effects on osteoclastic activity-a paramount coupling process with ostoblastic activity-remain undocumented. In this study, we delineated the functional characteristics of BMP2/7 heterodimer in inducing the in vitro osteoclastogenesis. We compared the dose-dependent effects of BMP2/7 heterodimer on the osteoclastogenesis of a preosteoclast cell line (RAW264.7) with those of BMP2 and BMP7 homodimers under the stimulation of 50 ng/mL receptor activator of nuclear factor-κB ligand. We quantitatively monitored the following parameters: cell proliferation, osteoclastic genes expression, morphological characteristics of osteoclasts, and calcium phosphate (CaP) resorption. BMP2/7 heterodimer could dose dependently modulate each osteoclastogenic event with different concentration patterns from the BMP homodimers. All BMPs of <em>10</em>-150 ng/mL could increase the numbers of osteoclasts. Not BMP7 but 50-200 ng/mL BMP2 homodimer and <em>10</em>0-200 ng/mL BMP2/7 heterodimer could significantly enlarge the average surface-area of an osteoclast. BMP2/7 of 5-150 ng/mL could significantly enhance the osteoclastic CaP resorption to a similar level as the two homodimers. BMP2/7 heterodimer affects every osteoclastogenic event in a complicated dose-dependent manner. Low-concentration BMP2/7 heterodimer may favor a rapid and spontaneous remodeling of its induced <em>bone</em> and, thus, bear a promising potential in cytokine-based tissue engineering.

Cellular behavior is dependent on a variety of physical cues required for normal tissue function. In order to mimic native tissue environments, human alveolar <em>bone</em>-derived mesenchymal stem cells (hABMSCs) were exposed to orbital shear stress (OSS) in a low-speed orbital shaker. The synergistic effects of OSS on proliferation and differentiation of hABMSCs were investigated. In particular, we induced the osteoblastic differentiation of hABMSCs cultured in the absence of OM by exposing hABMSCs to OSS (0.86-1.51 dyne/cm(2)). Activation of Cx43 was associated with exposure of hABMSCs to OSS. The viability of cells stimulated for <em>10</em>, 30, 60, 120, and 180 min/day increased by approximately <em>10</em>% compared with that of control. The OSS groups with stimulation of <em>10</em>, 30, and 60 min/day had more intense mineralized nodules compared with the control group. In quantification of vascular endothelial growth factor (VEGF) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) <em>protein</em>, VEGF <em>protein</em> levels under stimulation for <em>10</em>, 60, and 180 min/day and BMP-2 levels under stimulation for 60, 120, and 180 min/day were significantly different compared with those of the control. In conclusion, the results indicated that exposing hABMSCs to OSS enhanced their differentiation and maturation.

Angiogenesis is a hallmark of cancer and is now a validated therapeutic target in the clinical setting. Despite the initial success, anti-angiogenic compounds impinging on the vascular endothelial growth factor (VEGF) pathway display limited survival benefits in patients and resistance often develops due to activation of alternative pathways. Thus, finding and validating new targets is highly warranted. Activin receptor-like kinase (ALK)1 is a transforming growth factor beta (TGF-β) type I receptor predominantly expressed in actively proliferating endothelial cells (ECs). ALK1 has been shown to play a pivotal role in regulating angiogenesis by binding to <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)9 and <em>10</em>. Two main pharmacological inhibitors, an ALK1-Fc fusion <em>protein</em> (Dalantercept/ACE-041) and a fully human antibody against the extracellular domain of ALK1 (PF-03446962) are currently under clinical development. Herein, we briefly recapitulate the role of ALK1 in blood vessel formation and the current status of the preclinical and clinical studies on inhibition of ALK1 signalling as an anti-angiogenic strategy. Future directions in terms of new combination regimens will also be presented.

Human mesenchymal stem cells (hMSCs) typically range in size from <em>10</em> to 50 μm and <em>proteins</em> that mediate hMSC adhesion and differentiation usually have a size of a few nanometers. Nanomaterials with a feature size smaller than <em>10</em>0 nm have demonstrated the unique capability of promoting osteoblast (<em>bone</em> forming cell) adhesion and long-term functions, leading to more effective <em>bone</em> tissue regeneration. For new <em>bone</em> deposition, MSCs have to be recruited to the injury or disease sites and then differentiate into osteoblasts. Therefore, designing novel nanomaterials that are capable of attracting MSCs and directing their differentiation is of great interest to many clinical applications. This in vitro study investigated the effects of nanophase hydroxyapatite (nano-HA), nano-HA/poly(lactide-co-glycolide) (PLGA) composites and a <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP-7) derived short peptide on osteogenic differentiation of hMSCs. The short peptide was loaded by physical adsorption to nano-HA or by dispersion in nanocomposites and in PLGA to determine their effects on hMSC adhesion and differentiation. The results showed that the nano-HA/PLGA composites promoted hMSC adhesion as compared to the PLGA controls. Moreover, nano-HA/PLGA composites promoted osteogenic differentiation of hMSCs to a similar extent with or without the presence of osteogenic factors in the media. In the MSC growth media without the osteogenic factors, the nanocomposites supported greater calcium-containing <em>bone</em> mineral deposition by hMSC than the BMP-derived short peptide alone. The nanocomposites provided promising alternatives in controlling the adhesion and differentiation of hMSCs without osteogenic factors from the culture media, and, thus, should be further studied for clinical translation and the development of novel nanocomposite-guided stem cell therapies.

BACKGROUND

Recombinant human bone morphogenetic protein (rhBMP)-2 has been shown to stimulate significant regeneration of alveolar bone and cementum in periodontal defects clinically. The aim of this study was to evaluate the osteopromotive effect of oligopeptide domain-coated bovine bone mineral granules in a rabbit calvarial defect model.

METHODS

The peptide-coated bovine bone was fabricated by incubating the graft material in a solution of oligopeptide domain. The cell attachment and proliferation were measured in vitro. Peptide-coated (test group) or uncoated (control group) bone minerals were implanted into 10-mm calvarial defects in rabbits, and the animals were sacrificed at 1, 2, or 4 weeks post-surgery.

RESULTS

The cells grown with peptide-coated bone showed significantly higher proliferation activity at all times compared to cells grown with peptide-uncoated bone mineral. At 2 weeks post-surgery, the control wounds showed a limited amount of osteoid formation in a centripetal pattern around the grafted bone, but the oligopeptide domain-coated grafted bone had formed new bone around the grafted area. At 4 weeks post-surgery, the control sites showed increased new bone formation, but they still showed a significant difference from the peptide-coated bone-implanted sites.

CONCLUSIONS

At 2 and 4 weeks, accelerated new bone formation was observed within the experimental sites compared to control groups. The use of deproteinized bovine bone combined with a synthetic oligopeptide seems to be a more beneficial material for bone regeneration in the early healing period.

OBJECTIVE

Previously, we reported that (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, increased the osteogenic differentiation of murine bone marrow mesenchymal stem cells by increasing the messenger RNA expression of osteogenesis-related genes, alkaline phosphatase activity, and, eventually, mineralization. The present study further investigated the effects of EGCG on bone microstructure change and possible mechanisms in ovariectomy (OVX)-induced osteopenic rats.

METHODS

Rats subjected to OVX were administered EGCG systemically for 12 weeks. Proximal tibial bone mineral densities before and after treatment were compared between groups. Changes in the microarchitecture of both the proximal tibia and the third lumbar spine were compared between EGCG-treated and nontreated groups using micro-CT (μCT). Bone histology and immunohistochemistry in the proximal tibia were evaluated.

RESULTS

Results showed that EGCG 3.4 mg/kg/day (estimated peak serum concentration, 10 μmol/L) hampered the decrease in bone mineral density (from 7.97% to 3.96%) and improved the parameters of μCT measurements, including bone volume (from 18% to 27%), trabecular thickness (from 0.17 to 0.22 mm), trabecular number (from 1.13 to 1.37 mm(-1)), and trabecular separation (from 0.91 to 0.69 mm), compared with nontreated ovariectomized rats. Similar improvements in bone volume (from 30% to 49%) and trabecular thickness (from 0.14 to 0.26 mm) were also found in the third lumbar spine. Bone volume in the tibial cortex also increased after EGCG treatment (from 9% to 28%). A higher trabecular number and greater trabecular volume were also seen in histology, further confirming the results of μCT. The immunolocalized bone morphogenetic protein 2 brown-stained area increased from 31% in the OVX group to 53% in the OVX + 10 EGCG group (P < 0.01). Serial biochemistry data revealed no significant systemic toxic effect of EGCG.

CONCLUSIONS

Intraperitoneal treatment with EGCG 3.4 mg/kg/day for 3 months can mitigate bone loss and improve bone microarchitecture in ovariectomized rats, and increased expression of bone morphogenetic protein 2 may contribute to this effect.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7) regulates cartilage metabolism and promotes matrix synthesis. However, the effect of BMP-7 on inflammatory arthritis remains unknown. We investigated the effect and mechanism of exogenous BMP-7 on cartilage and synovium in vivo in rat zymosan-induced arthritis. Zymosan was injected into the left knees of Wistar rats. Phosphate-buffered saline or BMP-7 at <em>10</em>, <em>10</em>0, or <em>10</em>00 ng per joint was injected into the left knee every 2 days. Normal joints acted as normal controls. The knee joints were analyzed histologically and immunohistologically at 14 days. Joint swelling was evaluated by measuring the transverse diameter of the knee joints. Synovial lysates were collected, and the concentrations of interleukin-1β (IL-1β), IL-6, and IL-<em>10</em> were measured by enzyme-linked immunosorbent assay. Intra-articular injection of zymosan resulted in acute inflammation and was followed by cartilage degeneration. Local administrations of BMP-7 inhibited this loss of cartilage matrix in a dose-dependent manner. Immunohistochemical analysis demonstrated enhanced type II collagen levels in cartilage and enhanced BMP-7 levels in cartilage and synovium after exogenous BMP-7 treatment. Joint swelling and cell infiltration into synovium were significantly reduced by BMP-7 injections. Administration of BMP-7 decreased IL-1β production significantly and increased IL-<em>10</em> production in the synovium. Thus, intra-articular injections of BMP-7 had a protective effect on cartilage degeneration in the inflammatory arthritis model by enhancing levels of BMP-7 in cartilage and suppressing the production of IL-1β in synovium.

Intestinal ischemia/reperfusion (I/R) injury has been shown to cause intestinal mucosal injury and adversely affect function. Ischemic preconditioning (IPC) has been shown to protect against intestinal I/R injury by reducing polymorphonuclear leukocyte infiltration, intestinal mucosal injury, and liver injury, and preserve intestinal transit. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP-7) has been shown to protect against I/R injury in the kidney and brain. Recently, microarray analysis has been used to examine the possible IPC candidate pathways. This work revealed that IPC may work through upregulation of BMP-7. The purpose of this study was to examine if pretreatment with BMP-7 would replicate the effects seen with IPC in the intestine and liver after intestinal I/R. Rats were randomized to six groups: sham, I/R (30 min of superior mesenteric artery occlusion and 6 h of R), IPC+R (three cycles of superior mesenteric artery occlusion for 4 min and R for <em>10</em> min), IPC+I/R, BMP-7+R (<em>10</em>0 microm/kg recombinant human BMP-7), or BMP-7+I/R. A duodenal catheter was placed, and 30 min before sacrifice, fluorescein isothiocyanate-Dextran was injected. At sacrifice, dye concentrations were measured to determine intestinal transit. Ileal mucosal injury was determined by histology and myeloperoxidase activity was used as a marker of polymorphonuclear leukocyte infiltration. Serum levels of aspartate aminotransferase were measured at sacrifice to determine liver injury. Pretreatment with BMP-7 significantly improved intestinal transit and significantly decreased intestinal mucosal injury and serum aspartate aminotransferase levels, comparable to animals undergoing IPC. In conclusion, BMP-7 protected against intestinal I/R-induced intestinal and liver injury. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 7 may be a more logical surrogate to IPC in the prevention of injury in the setting of intestinal I/R.

In the hen ovary, selection of a follicle into the preovulatory hierarchy occurs from a small cohort of prehierarchal (6-8 mm) follicles. Prior to follicle selection the granulosa layer remains in an undifferentiated state despite elevated follicle-stimulating hormone receptor (FSHR) expression. The present studies describe a role for <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) in supporting FSHR mRNA expression in granulosa cells from prehierarchal follicles and promoting differentiation at follicle selection. Culture of undifferentiated granulosa cells in culture medium alone resulted in a significant decline in levels of FSHR mRNA (by ~80% compared to freshly collected cells). By comparison, granulosa cultured with BMP4 (<em>10</em>-<em>10</em>0 ng/ml) maintained FSHR and expression at approximately in vivo levels. Because both granulosa and theca tissues from prehierarchal follicles express BMP4, it is suggested that BMP4 acts in a paracrine and/or autocrine fashion to support elevated FSHR expression prior to follicle selection. Granulosa cells cultured with BMP4 for 24 h also initiated FSH-induced cAMP production and indirectly initiated anti-Mullerian hormone (AMH), CYP11A, and STAR expression plus progesterone production. However, pretreatment with the BMP antagonist NOGGIN or the mitogen-activated <em>protein</em> kinase (MAPK) agonist transforming growth factor alpha attenuated or blocked each action promoted by BMP4. We conclude that prior to and immediately after selection, BMP4 serves to support FSHR expression within the granulosa layer, yet prior to selection, multiple factors (including inhibitory MAPK signaling, AMH, and BMP antagonists) can modulate FSHR expression and suppress FSH-mediated cell signaling to prevent granulosa cell differentiation prior to follicle selection.

BACKGROUND

Mesenchymal stem cell (MSC)-based cell therapy and gene transfer have converged and show great potential for accelerating bone healing. Gene therapy can provide more sustained expression of osteogenic factors such as bone morphogenetic protein-2 (BMP-2). We previously demonstrated that low-dose BMP-2 enhanced spinal posterolateral fusion by MSCs in a rabbit model. Herein, we genetically modified rabbit MSCs with a recombinant baculovirus encoding BMP-2 (Bac-CB) and vascular endothelial growth factor (Bac-VEGF) seeded into porous scaffolds to enhance spinal fusion.

OBJECTIVE

This study evaluates the success rate of the MSC-based cell therapy and gene transfer approach for single-level posterolateral spine fusion. We hypothesize that combining three-dimensional tricalcium phosphate (TCP) scaffolds and genetically modified allogeneic MSCs with baculovirus-mediated growth factor expression would increase the success rate of spinal fusion.

METHODS

The study design was based on an animal model (approved by the Institutional Animal Care and Use Committee) using 18 adult male New Zealand rabbits.

METHODS

This study included 18 male New Zealand rabbits, weighing 3.5 to 4 kg. Allogeneic bone marrow-derived MSCs were isolated and genetically modified with Bac-CB and Bac-CV seeded onto TCP scaffolds (MSC/Bac/TCP). The animals were divided into three groups according to the material implanted into the bilateral L4-L5 intertransverse space: TCP scaffold (n=6), MSC/TCP (n=6), and MSC/Bac/TCP (n=6). After 12 weeks, the rabbits were euthanized for radiographic examination, manual palpation, and histologic study.

RESULTS

Bilateral fusion areas in each animal were evaluated independently. The radiographic fusion rates at 12 sites were 0 of 12 in the TCP scaffold group, 4 of 12 in the MSC/TCP group, and 10 of 12 in the MSC/Bac/TCP group. By manual palpation, there were zero solid fusions in the TCP scaffold group, two solid fusions in the MSC/TCP group, and five solid fusions in the MSC/Bac/TCP group. Fusion rates were significantly greater in the MSC/Bac/TCP group.

CONCLUSIONS

The results indicate the potential of using baculovirus as a vector for gene/cell therapy approaches to improve bone healing and support the feasibility of using allogeneic MSCs for inducing bone formation and intertransverse process fusion.

As an approach for improving the outcome and predictability of periodontal regenerative therapies, we have focused on determining the responses of cells within the local environment to putative regenerative factors. This study examined the effects of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) on murine cementoblasts in vitro. Northern blot analysis indicated that BMP-2 decreased mRNA levels of <em>bone</em> sialo<em>protein</em> and type I collagen dose-dependently (<em>10</em>-300 ng/mL). At low doses, up to <em>10</em>0 ng/mL, BMP-2 had no effect on transcripts for osteocalcin and osteopontin, whereas at 300 ng/mL, BMP-2 greatly increased expression of these two genes. BMP-2 also inhibited cementoblast-mediated mineral nodule formation in a dose-dependent manner (inhibition was noted at <em>10</em> ng/mL). Noggin reversed the effects of BMP-2 on gene expression and on mineralization. These findings reflect the diverse responses of periodontal cells to BMP-2 and highlight the need to consider the complexity of factors involved in designing predictable regenerative therapies.

Ischemia predisposes orthopedic trauma patients to delayed fracture healing or nonunion. The goal of this study was to test the ability of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (BMP7) to stimulate fracture repair in an ischemic environment. Ischemic fractures were generated in male adult mice by resecting the femoral artery prior to the creation of a nonstabilized tibia fracture. Recombinant human BMP7 (rhBMP7, 50 microg) was injected into the fracture site immediately after surgery. At 7 days after injury, more tissue vascularization was observed in rhBMP7 treated fractures. Histomorphometric analyses revealed that rhBMP7 induced more cartilage at day 7, more callus and <em>bone</em> at days 14 and 28, and more adipose tissue and fibrous tissue at days 7, 14, and 28 compared to controls (n=5/group/time). At day 28, all fractures treated with rhBMP7 (50 microg, n=5) healed, whereas only three of five control fractures exhibited slight bony bridging. In addition, we found that rhBMP7 (both <em>10</em> and 50 microg) significantly increased the amount of cartilage compared to controls in stabilized fractures, confirming its chondrogenic effect. Lastly, using <em>bone</em> marrow transplantation, we determined that no donor-derived osteocytes or chondrocytes were present in rhBMP7-treated fractures, suggesting rhBMP7 did not recruit mesenchymal stem cells from the <em>bone</em> marrow to the fracture site. In conclusion, our results indicate that rhBMP7 is a promising treatment for fractures with severely disrupted blood supply.

The involvement of osteoprotegerin (OPG) in <em>bone</em> metabolism has previously been established; however, whether OPG regulates chondrocytes directly and exerts precise cellular and molecular effects on chondrocytes remains to be determined. Thus, the present study aimed to investigate the direct effect of OPG on the viability, proliferation and functional consequences of chondrocytes. Primary chondrocytes were isolated from the knee of Sprague-Dawley rats. Passage 1 chondrocytes were identified by toluidine blue staining and used in the experiments. The cell proliferation induced by OPG at various concentrations was measured by a Cell Counting kit-8 (CCK-8) assay. Following pretreatment with mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126, extracellular signal-regulated kinase (ERK) inhibitor PD098059, and P38 mitogen-activated <em>protein</em> kinase (P38MAPK) inhibitor SB203580 for 30 min, chondrocytes were treated with OPG, and CCK-8 was performed. The cellular signals of MAPKs, including ERK, P38MAPK and c-Jun N-terminal <em>protein</em> kinase (JNK), were investigated by western blot analysis following treatment with OPG. The functional consequences following treatment with soluble OPG were analyzed by qPCR and western blot analysis. OPG increased chondrocyte proliferation with maximal effect at <em>10</em> ng/ml, and induced the phosphorylation of MEK and ERK but not P38MAPK or JNK. Suppression of ERK activity via PD098095 inhibited OPG-induced chondrocyte proliferation. Administration of OPG significantly downregulated ADAMTS‑5 and upregulated tissue inhibitor of metallo<em>protein</em>ase (TIMP)-4 production, but had no effect on the expression of TIMP-1, -2 and -3, insulin-like growth factor I, transforming growth factor-β, basic fibroblast growth factor, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2, collagen II, aggrecan and ADAMTS-4. Suppression of ERK activity via PD098095 inhibited the alteration of ADAMTS-5 and TIMP-4 expression induced by OPG. OPG therefore regulated the proliferation of chondrocytes via MEK/ERK signaling, and directly affected chondrocytes by influencing the expression profile of ADAMTS-5 and TIMP-4.

OBJECTIVE

Juvenile polyps are benign hamartomas with neoplastic potential that are the most frequent gastrointestinal polyp of childhood. Most information about juvenile polyps in childhood comes from small published series that lack detailed outcome data. We sought to identify a large cohort of children with one or more polyps and analyze clinical characteristics, including polyp recurrence, which might contribute to the development of management guidelines.

METHODS

A retrospective chart review study of patients with juvenile polyps of the colon was performed. Cases were identified by searching a single hospital pathology database from 1990 to 2009 for the diagnosis of juvenile polyps. Recorded information included basic demographics, family history, genetic testing, and colonoscopy and pathology reports.

RESULTS

A total of 257 children (median age, 5.6 y; 61.5% male) with juvenile polyps were identified. Among 192 patients who underwent complete colonoscopy at initial diagnosis, 117 (60.9%) had a single polyp, 75 (39.1%) had multiple polyps, 8 (4.2%) had polyps restricted to the right colon, and a total of 1653 polyps were found during 350 colonoscopy examinations. Polyps recurred in 21 of 47 (44.7%) patients after initial eradication, including 3 (16.7%) of 18 presenting with a single polyp. Neoplasia was found in <em>10</em> of 257 (3.9%) patients (right colon in 7 patients). Germline DNA abnormalities in mothers against decapentaplegic Drosophila (SMAD4), <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 1A (BMPR1A), and phosphatase and tensin homolog (PTEN) were detected in <em>10</em> of 23 (43.5%) patients with multiple polyps.

CONCLUSIONS

Recurrent polyp formation is common in children with juvenile polyps and occurs in patients with multiple and solitary polyps. Standardized protocols for detecting polyp recurrence, associated gene mutations, and neoplasia should be developed for children with juvenile polyps.

BACKGROUND

Progressive cellular and extracellular matrix (ECM) changes related to age and disease severity have been demonstrated in rotator cuff tendon tears. Larger rotator cuff tears demonstrate structural abnormalities that potentially adversely influence healing potential. This study aimed to gain greater insight into the relationship of pathologic changes to tear size by analyzing gene expression profiles from normal rotator cuff tendons, small rotator cuff tears, and large rotator cuff tears.

METHODS

We analyzed gene expression profiles of 28 human rotator cuff tendons using microarrays representing the entire genome; 11 large and 5 small torn rotator cuff tendon specimens were obtained intraoperatively from tear edges, which we compared with 12 age-matched normal controls. We performed real-time polymerase chain reaction and immunohistochemistry for validation.

RESULTS

Torn rotator cuff tendons demonstrated upregulation of a number of key genes, such as matrix metallo<em>protein</em>ase 3, <em>10</em>, 12, 13, 15, 21, and 25; a disintegrin and metallo<em>protein</em>ase (ADAM) 12, 15, and 22; and aggrecan. Amyloid was downregulated in all tears. Small tears displayed upregulation of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 5. Chemokines and cytokines that may play a role in chemotaxis were altered; interleukins 3, <em>10</em>, 13, and 15 were upregulated in tears, whereas interleukins 1, 8, 11, 18, and 27 were downregulated.

CONCLUSIONS

The gene expression profiles of normal controls and small and large rotator cuff tear groups differ significantly. Extracellular matrix remodeling genes were found to contribute to rotator cuff tear pathogenesis. Rotator cuff tears displayed upregulation of a number of matrix metallo<em>protein</em>ase (3, <em>10</em>, 12, 13, 15, 21, and 25), a disintegrin and metallo<em>protein</em>ase (ADAM 12, 15, and 22) genes, and downregulation of some interleukins (1, 8, and 27), which play important roles in chemotaxis. These gene products may potentially have a role as biomarkers of failure of healing or therapeutic targets to improve tendon healing.

Abnormally elevated <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) expression and mediated signaling play a critical role in the pathogenesis of chronic hypoxia-induced pulmonary hypertension (CHPH). In this study, we investigated the expression level and functional significance of four reported naturally occurring BMP4 antagonists, noggin, follistatin, gremlin1, and matrix gla <em>protein</em> (MGP), in the lung and distal pulmonary arterial smooth muscle cell (PASMC). A 21-day chronic hypoxic (<em>10</em>% O2) exposure rat model was utilized, which has been previously shown to successfully establish experimental CHPH. Among the four antagonists, noggin, but not the other three, was selectively downregulated by hypoxic exposure in both the lung tissue and PASMC, in correlation with markedly elevated BMP4 expression, suggesting that the loss of noggin might account for the hypoxia-triggered BMP4 signaling transduction. Then, by using treatment of extrogenous recombinant noggin <em>protein</em>, we further found that noggin significantly normalized 1) BMP4-induced phosphorylation of cellular p38 and ERK1/2; 2) BMP4-induced phosphorylation of cellular JAK2 and STAT3; 3) hypoxia-induced PASMC proliferation; 4) hypoxia-induced store-operated calcium entry (SOCE), and 5) hypoxia-increased expression of transient receptor potential cation channels (TRPC1 and TRPC6) in PASMC. In combination, these data strongly indicated that the hypoxia-suppressed noggin accounts, at least partially, for hypoxia-induced excessive PASMC proliferation, while restoration of noggin may be an effective way to inhibit cell proliferation by suppressing SOCE and TRPC expression.