Estrogen deficiency causes postmenopausal osteoporosis. The relationship between estrogen deficiency and the high failure rate after osteoporotic fracture treatment is unclear, as is the effect of possible interventions, either with anti-resorptive agents or with anabolic agents such as <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs). To investigate the influence of estrogen deficiency as well as the effect of early intervention, forty female wistar rats underwent ovarectomy (OVX) followed by low calcium diet. Ten rats underwent sham operations, followed by normal diet. After 6 weeks, a closed midshaft femoral fracture was induced. Ten animals received a systemic bisphosphonate injection, <em>10</em> injection of BMP-7 in the fracture, and <em>10</em> a combination. All then received a normal diet. After 2 weeks healing was evaluated using radiographs, CT, biomechanical testing, and histology. Radiography showed significant increase of bridging in groups treated with BMP-7. Callus volume was higher in these groups. Bending stiffness and strength were similar between OVX and sham, and not influenced by bisphosphonates. Significant increase was seen in groups treated with BMP-7. Histology was in accordance with other endpoints. Early fracture healing was not affected by estrogen deficiency. While no beneficiary effect of bisphosphonate treatment was found, injection of BMP-7 stimulated healing in ovarectomized rats.

In women, the window of implantation is limited to a brief 2- to 3-day period characterized by optimal levels of circulating ovarian hormones and a receptive endometrium. Although the window of implantation is assumed to occur 8 to <em>10</em> days after ovulation in women, molecular markers of endometrial receptivity are necessary to determine optimal timing prior to embryo transfer. Previous studies showed that members of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) family are expressed in the uterus necessary for female fertility; however, the role of BMP7 during implantation and in late gestation is not known. To determine the contribution of BMP7 to female fertility, we generated Bmp7flox/flox-Pgr-cre+/- [BMP7 conditional knockout (cKO)] mice. We found that absence of BMP7 in the female reproductive tract resulted in subfertility due to uterine defects. At the time of implantation, BMP7 cKO females displayed a nonreceptive endometrium with elevated estrogen-dependent signaling. These implantation-related defects also affected decidualization and resulted in decreased expression of decidual cell markers such as Wnt4, Cox2, Ereg, and Bmp2. We also observed placental abnormalities in pregnant Bmp7 cKO mice, including excessive parietal trophoblast giant cells and absence of a mature placenta at <em>10</em>.5 days post coitum. To establish possible redundant roles of BMP5 and BMP7 during pregnancy, we generated double BMP5 knockout/BMP7 cKO [BMP5/7 double knockout (DKO)] mice; however, we found that the combined deletion had no additive disruptive effect on fertility. Our studies indicate that BMP7 is an important factor during the process of implantation that contributes to healthy embryonic development.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are crucial factors in follicular growth and development. Among the BMP ligands, BMP-7 which use ActRII as their type II receptor, strongly bind to ALK-2 as their type I receptor. However, whether their receptors are expressed and the regulatory mechanisms controlling their expression during the process of bovine follicle development are still unknown. The aim of the present study was to clarify the involvement of the receptor system for BMP-7 in follicular selection by examining the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the regulation of ActRII and ALK-2 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression, follicles were obtained from heifers and GCs were classified into two groups: pre-selection follicles (PRF; follicles with an average diameter of 7 mm and low E2) and post-selection follicles (POF; follicles with an average diameter of 15 mm and high E2). The theca cell (TC) layer and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7 mm diameter and cultured in Dulbecco's modified Eagle's/F12 (DMEM/F-12) medium with <em>10</em>% fetal calf serum for 24h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, <em>10</em>,<em>10</em>0 ng/ml), FSH (1, 5, <em>10</em> ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses (1, 5, <em>10</em> ng/ml). Total RNA was extracted from GCs and the mRNA expression of ActRII and ALK-2 was estimated by the quantitative PCR method using LightCycler. The expression of BMP-7 mRNA in TCs did not differ between the PRF and POF. ActRII and ALK-2 expression was detected in GCs from bovine antral follicles and was higher in the GCs of POF than in those of PRF, while the expression of the ActRII and ALK-2 genes in the TCs was not different between PRF and POF. Treatment of GCs with E2 (<em>10</em> ng/ml) alone increased the expression of both ActRII and ALK-2 mRNAs, whereas FSH alone had no effect. However, ActRII and ALK-2 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). The results of the present study provide the first evidence that FSH and E2 regulate the expression of the ActRII and ALK-2 genes in bovine GCs. Thus, our data suggest that the BMP7/ActRII/ALK-2 system may be critically involved in the process of selection of bovine follicles.

Normal human <em>bone</em> marrow stroma cells include stem cells for both haemopoietic and osteochondrogenic lineages and express both <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) type I and type II receptors. As a member of the TGF-beta super-family, BMP-6 binds to both BMP type I and type II receptors and is involved in the developmental processes of renal and hepatic systems as well as of human foetal intestine. Also, BMP-6 induces osteoblastic differentiation of pluripotent mesenchymal cells and is an autocrine stimulator of chondrocyte differentiation. The present study was carried out to investigate the effect of BMP-6 on human cobblestone-area-forming cells (CAFC), that represent the functional primitive repopulating haemopoietic stem cell in long-term <em>bone</em> marrow culture. Also, the effect of BMP-6 on marrow stroma production of interleukin-6, -11 and their common receptor gp130 that is expressed in haemopoietic stem cells and is indispensable for their proliferation and tri-lineage differentiation was examined. Moreover, the effect of BMP-6 on marrow stroma release of soluble adhesion molecule VCAM-1 mediating the primitive haemopoietic stem cell adhesion to marrow stroma was examined. The number of CAFC was significantly reduced after BMP-6 treatment from 88+/-<em>10</em> per <em>10</em>(5)cells in control cultures in a dose dependent manner to only 48+/-3 per <em>10</em>(5)cells in 50 ng/ml BMP-6-treated cultures, P< 0.01. Quantitative ELISA measurement revealed 50 ng/ml BMP-6 was able to significantly reduce IL-6 and IL-11 production from marrow stroma, P< 0.01. Also, BMP-6 significantly increased soluble gp130 release by 7.4-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The profound rapid increase in this natural antagonist of human IL-6 cytokine family may reduce the gp130 signaling. Also, the soluble VCAM-1 released increased by two-fold in 50 ng/ml BMP-6-treated marrow stroma cultures. The marked increase in the soluble form may exert an antagonist effect on the function of VCAM-1 (ligand for VLA4). Recently, blocking the VLA4/VCAM-1 adhesion pathway was shown to mobilise haemopoietic CD34 positive cells in normal individuals. Also, we previously observed a significantly lower expression of VLA4 (CD49d) on G-CSF-mobilised blood CD34 positive cells than on <em>bone</em> marrow CD34 positive cells before mobilisation in the same normal donors. Since BMP are currently being used in clinical trials for <em>bone</em> repair and fracture healing, the present results suggest a possible role for BMP-6 in mobilising CD34 positive cells for transplantation. Further in vitro tests are required to evaluate this potential mobilising role of BMP-6 in human long-term <em>bone</em> marrow culture.

Delayed healing and non-union remain common problems in the treatment of open tibial shaft fractures. Additional surgical treatments may be required to facilitate healing. The efficacy of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2), as an adjunct to the standard of care, has been investigated in the BMP-2 Evaluation in Surgery for Tibial Trauma (BESTT) study. This prospective, randomised, multicentre, controlled study included 450 patients with acute, diaphyseal, open tibial fractures. A significant reduction in the risk of secondary intervention was observed in the 1.50 mg/mL rhBMP-2 group compared with the standard of care alone group (p = 0.0005). In a subgroup analysis of 131 patients with Gustilo-Anderson grade IIIA or IIIB open tibial fractures, using data combined from the BESTT study and a study conducted at <em>10</em> level I US trauma centres, a significant reduction in the incidence of secondary autologous <em>bone</em> graft procedures was observed with 1.50 mg/mL rhBMP-2 compared with the standard of care (p = 0.0005). The influence of fracture gap on the re-operation rate has also been examined in the BESTT study. In the 1.50 mg/mL rhBMP-2 group, patients with a 0 mm fracture gap had significantly less re-operations compared with those patients with a greater than 2 mm gap (p = 0.048).

<em>Bone</em> <em>Morphogenetic</em> <em>Proteins</em> (BMPs) induce the differentiation of Serum-free Mouse Embryo (SFME) cells into astrocytes (D'Alessandro et al., 1994) as demonstrated by change in morphology, increase in Glial Fibrillary Acidic <em>Protein</em> (GFAP) content and classification as both type 1 and 2 astrocytes. Further analyses showed that in the presence of BMP, cells which had differentiated into astrocytes were inhibited from proliferation. Moreover, removal of BMP resulted in a resumption of proliferation accompanied by a loss of GFAP expression over time, indicating that under these in vitro conditions the differentiation was reversible. Since EGF is absolutely required for the survival of SFME cells, we examined the effect of its removal in the presence of BMP. Cell survival was>> 80% in the presence of BMP-2, 7 or 2/7 and < <em>10</em>% in the presence of TGF-beta 1. These data demonstrate that BMPs have effects on the proliferation, differentiation and survival of cells in the astrocyte lineage.

MicroRNAs (miRNAs) regulate myriad biological processes; however, their role in cell fate choice is relatively unexplored. Pluripotent NT2/D1 embryonal carcinoma cells differentiate into an epithelial/smooth muscle phenotype when treated with <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2). To identify miRNAs involved in epithelial cell development, we performed miRNA profiling of NT2/D1 cells treated with BMP-2 at 6, 12, and 24 h, and on days 6 and <em>10</em>. Integration of the miRNA profiling data with previously obtained gene expression profiling (GEP) data of NT2/D1 cells treated with BMP-2 at the same time points identified miR-18b and miR-518b as the top two miRNAs with the highest number of up-regulated predicted targets with known functions in epithelial lineage development. Silencing of miR-18b and miR-518b in NT2/D1 cells revealed several up-regulated TFs with functions in epithelial lineage development; among these, target prediction programs identified FOXN1 as the only direct target of both miRNAs. FOXN1 has previously been shown to play an important role in keratinocyte differentiation and epithelial cell proliferation. NT2/D1 and H9 human embryonic stem cells with silenced miR-18b and miR-518b showed up-regulation of FOXN1 and the epithelial markers CDH1, EPCAM, KRT19, and KRT7. A 3'UTR luciferase assay confirmed FOXN1 to be a target of the two miRNAs, and up-regulation of FOXN1 in NT2/D1 cells led to the expression of epithelial markers. Overexpression of the two miRNAs in BMP-2-treated NT2/D1 cells led to down-regulation of FOXN1 and epithelial lineage markers. These results show that miR-18b and miR-518b are upstream controllers of FOXN1-directed epithelial lineage development.

OBJECTIVE

To determine the degree of bone resorption and stability of 3-cm, full-thickness canine mandibular defects reconstructed with recombinant human bone morphogenetic protein 2 (rhBMP-2) and a bioerodible particle carrier followed for 30 months after reconstruction.

METHODS

Nine dogs, divided into three groups, underwent reconstruction of surgically created 3-cm, full-thickness defects of the body of the mandible.

METHODS

Mandibular reconstruction was performed via a combined intraoral and extraoral approach. Using standard plating techniques, a unilateral full-thickness, 3-cm defect was created in the body of the mandible. After stabilizing the defects with titanium reconstruction plates, test implants composed of rhBMP-2 and poly(lactide-co-glycolide) bioerodible particles were placed in the mandibular defects of six animals. Reconstruction plates were removed from test animals at 10 weeks. Three short-term test animals were sacrificed 3 months after reconstruction. Three long-term test animals were sacrificed 30 months after reconstruction to determine the degree of resorption and long-term stability of the rhBMP-2-induced bone. Control implants (carrier without rhBMP-2) were used in three animals and were sacrificed at 3 months. At 9 months, long-term animals were advanced to a solid diet. Masticatory function and body weight were monitored periodically to assess diet tolerance. Roentgenographic photodensitometry was performed on serial dental roentgenograms of the reconstructed segments to determine bone density and the degree of bone resorption over 30 months. After sacrifice, reconstructed segments were harvested and embedded in plastic for histological analysis and histomorphometry to determine the percentage of the defect replaced by mineralized bone (area density) and degree of resorption from 3 to 30 months after reconstruction. The main outcome measures were bone density and bone height determined from serial roentgenograms and percentage of the reconstructed segment replaced by mineralized bone (area density) determined from histomorphometry.

RESULTS

Control animals without rhBMP-2 showed no evidence of bone formation across the defect. Histological examination revealed good bone formation in two of three of the short-term test animals with a mean area density of 41.0%. The long-term test animals treated with rhBMP-2 demonstrated good bone formation that was comparable to that of normal host bone by 3 months. The roentgenographic photodensity measurements stabilized at 5 months without evidence of persistent bone resorption. The height of the reconstructed segment (rhBMP-2-induced bone) initially decreased, then stabilized by 11 months after reconstruction with no indication of resorption or failure. Histological examination of the long-term test animals revealed good bone formation across the mandibular defects. However, there were localized areas of thinning of the cortical bone as compared with the short-term test animals sacrificed at 3 months. Histological examination verified the loss of height of the bone in the reconstructed segments. The area density (mean) of the long-term test animals was 56.5%. Despite the decrease in height of the induced bone, there was an increase in area density of the bone over time.

CONCLUSIONS

This study demonstrated that rhBMP-2 in a bioerodible particle carrier induced new host bone formation across critical-size mandibular defects. The newly formed bone successfully integrated with existing host bone creating a stable union capable of withstanding the forces of masticatory function in a canine. There was some evidence of early bone resorption (thinning of the cortical bone and decrease in height) in the rhBMP-2-induced bone. The rhBMP-2-induced bone stabilized by 11 months after reconstruction and no further resorption was noted. The percentage of area of the defect replaced by rhBMP-2-induced bone (area density) increased over 30 months. (ABSTRACT TRUNCATED)

We examined the expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) and proliferating cell nuclear antigen (PCNA) during distraction osteogenesis in the mandible in rabbits. Twenty-four rabbits each had an osteotomy of the left mandibular body, and distraction devices were fixed. The <em>bone</em> was distracted at a rate of 1mm/day for <em>10</em> days. Four rabbits were killed at each of 1, 3, 7, 14, and 28 days after completion of distraction, and the mandibles examined radiographically, histologically, and immunohistochemically. Four rabbits that had not been operated on served as controls. Immunohistochemical analysis showed that BMP-2 and PCNA both appeared initially at the edge of the osteogenesis, but tended to disappear after 14 days. After 1, 3, 7, and 14 days after distraction, the ratio of stained cells was significantly higher than in the control group (p<0.05), during the period that active <em>bone</em> formation was shown radiographically and histologically. These results suggest that BMP-2 plays an important part in the induction of <em>bone</em> formation during distraction osteogenesis.

BACKGROUND

Recombinant human bone morphogenetic protein-2 (rhBMP-2) has had limited success in stimulating osteogenesis at the site of posterolateral lumbar spine arthrodesis when used at the currently approved human dose for anterior lumbar interbody arthrodesis. The objective of the present study was to investigate the effect of co-administration of fresh harvested autologous bone marrow aspirate and platelet-rich plasma on rhBMP-2-mediated in vivo murine posterolateral lumbar spine arthrodesis.

METHODS

Forty adult male mice underwent posterolateral intertransverse process arthrodesis from L4 to L6. In three experimental groups, a collagen sponge was placed on each side, overlaying the decorticated transverse processes. Each collagen sponge was presoaked for fifteen minutes with 31 microg of rhBMP-2 in a 100-microL solution containing either saline solution (n = 10), platelet-rich plasma (n = 10), or donor bone-marrow cells (n = 10). Control mice underwent decortication alone (n = 10). The lumbar spine was harvested four weeks after surgery, and spinal fusion was evaluated on the basis of radiographs, computed tomography, and histological analysis.

RESULTS

Control mice showed no evidence of spinal fusion. The rate of fusion was radiographically and histologically similar in all three experimental groups. The area, volume, and density of the fusion mass were significantly greater (p < 0.05) for the group treated with rhBMP-2 and bone marrow as compared with the group treated with rhBMP-2 alone. The group treated with rhBMP-2 and platelet-rich plasma had intermediate fusion area and density. Histologically, the spines treated with rhBMP-2 alone consistently showed the presence of cortical bone between the two transverse processes but fewer trabeculae within the fusion mass; bone marrow co-augmentation resulted in more trabeculae within the fusion mass and a thicker cortical perimeter.

CONCLUSIONS

The present study quantitatively confirmed a synergistic effect of bone marrow cells when added to rhBMP-2 in an in vivo mouse posterolateral lumbar spine fusion model. The volume, area, and density of the fusion mass were significantly increased by augmentation with bone marrow cells.

High-dose estrogen administration is known to induce new <em>bone</em> formation in mouse long <em>bones</em>. To study the role of regulatory <em>proteins</em> in this response, we examined associated changes in femoral messenger RNA (mRNA) for candidate factors. 17beta-estradiol (E2) 0.5 mg was administered to intact female mice by weekly injection, and Northern blot analysis was performed 1, 2, 4, 8, 12, and 16 days after the first injection. In contrast to other factors, an increase was observed in mRNA for <em>bone</em> <em>morphogenetic</em> <em>protein</em>-6 (BMP-6), which reached significance at day 8 and subsequent time-points. Estrogen-induced changes in BMP-6 <em>protein</em> expression were assessed by immunocytochemistry in longitudinal femoral sections. In untreated animals, BMP-6 was expressed by a significant proportion of growth plate chondrocytes and a subpopulation of <em>bone</em> marrow cells. In contrast, osteoblasts were consistently BMP-6 negative. From as early as 4 days after starting estrogen, clusters of slightly elongated BMP-6-positive cells were observed within the marrow cavity; the majority were close to active <em>bone</em> formation surfaces. Double immunolabeling studies revealed that only approximately <em>10</em>% of BMP-6-positive <em>bone</em> marrow cells co-expressed the osteoblast transcription factor Cbfa1 suggesting that they are largely distinct from the osteoblast precursor population generated concurrently. BMP-6-positive cells expressed neither leukocyte nor erythroid markers (CD45 and TER-119, respectively), consistent with a stromal origin. We conclude that estrogen-induced osteogenesis in female mice is associated with increased levels of BMP-6 mRNA in mouse femurs, which seems to reflect the emergence of clusters of BMP-6 positive stromal cells adjacent to active <em>bone</em> formation surfaces. These findings raise the possibility that BMP-6 serves as a paracrine mediator of estrogen's osteogenic action in mice.

OBJECTIVE

In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells enter the vitreous and proliferate. They become fibroblast-like and participate in the formation of contractile membranes, which can lead to retinal detachment. Vitreous treatment of RPE cells in vitro results in similar morphologic changes. This study was conducted to examine vitreous-induced modulation of gene expression in RPE cells.

METHODS

Low-passage human RPE cell lines derived from three donors were each treated for 6, 12, 24, or 48 hours with complete medium or complete medium containing 25% vitreous. Changes in mRNA levels were examined by using microarrays. Real-time quantitative PCR (qPCR) was used to measure mRNA expression of a subset of genes in cells from three additional donors. Immunohistochemistry and immunoblot analysis were used to examine protein expression.

RESULTS

Vitreous treatment caused a progressive reprogramming of gene expression. qPCR confirmed vitreous modulation of mRNA levels of 10 of 10 genes. Changes consistent with a transition from an epithelial to a mesenchymal phenotype were observed. Downregulated genes included genes associated with differentiated RPE cells. Upregulated genes included genes associated with stress and inflammation. Pathway analysis indicated that the transforming growth factor-beta/bone morphogenetic protein (BMP) pathway and the focal adhesion pathway may play a role in this process. BMP-2 protein and mRNA were increased.

CONCLUSIONS

Despite the biological variation in vitreous and RPE donors, vitreous reproducibly modulated a limited number of mRNAs. Many of these changes were consistent with the more fibroblast-like appearance of vitreous-treated cells and with the pathobiology of PVR. TGF-beta and BMP-2 may be important modulators of vitreous-induced changes in gene expression.

Osteochondral tissue-engineered grafts are proposed to hold greater potential to repair/regenerate damaged cartilage through enhanced biochemical and mechanical interactions with underlying subchondral <em>bone</em> as compared to simple engineered cartilage. Additionally, biomechanical stimulation of articular chondrocytes (ACs) or osteoblasts (OBs) was shown to induce greater morphogenesis of the engineered tissues composed of these cells. In this report, to define the advantages of biomechanical stimulation to osteochondral grafts for tissue engineering, we examined whether (1) ACs and OBs in three-dimensional (3D) osteochondral constructs support functional development of each other at the molecular level, and (2) biomechanical stimulation of osteochondral constructs further promotes the regenerative potential of such grafts. Various configurations of cell/scaffold assemblies, including chondral, osseous, and osteochondral constructs, were engineered with mechano-responsive electrospun poly(ɛ-caprolactone) scaffolds. These constructs were subjected to either static or dynamic (<em>10</em>% cyclic compressive strain at 1 Hz for 3 h/day) culture conditions for 2 weeks. The expression of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) was examined to assess the regenerative potential of each treatment on the cells. Biomechanical stimulation augmented a marked upregulation of Bmp2, Bmp6, and Bmp7 as well as downregulation of BMP antagonist, Bmp3, in a time-specific manner in the ACs and OBs of 3D osteochondral constructs. More importantly, the presence of biomechanically stimulated OBs was especially crucial for the induction of Bmp6 in ACs, a BMP required for chondrocytic growth and differentiation. Biomechanical stimulation led to enhanced tissue morphogenesis possibly through this BMP regulation, evident by the improved effective compressive modulus of the osteochondral constructs (7<em>10</em> kPa of dynamic culture vs. 280 kPa of static culture). Similar BMP regulation was observed in the femoral cartilages of the rats subjected to gentle exercise, demonstrating the physiological relevance of in vitro biomechanical stimulation of osteochondral constructs. Overall, our findings show that biomechanical stimulation may be critical for cross signaling between ACs and OBs to support chondrocytic growth in 3D osteochondral tissues.

Asymmetrically porous polycaprolactone (PCL)/Pluronic F127 guided <em>bone</em> regeneration (GBR) membranes were fabricated. The top surface of the membrane had nanosize pores (∼<em>10</em> nm) which can effectively prevent invasion by fibrous connective tissue but permeate nutrients, whereas the bottom surface had microsize pores (∼200 μm) which can enhance the adhesiveness with <em>bone</em> tissue. Ultrasound was applied to a <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP-2)-immobilized PCL/F127 GBR membrane to investigate the feasibility of using dual biological (BMP-2) and physical (ultrasound) stimulation for enhancing <em>bone</em> regeneration through the membrane. In an animal study using SD rats (cranial defect model), the <em>bone</em> regeneration behavior that occurred when using BMP-2-loaded GBR membranes with ultrasound treatment (GBR/BMP-2/US) was much faster than when the same GBR membrane was used without the ultrasound treatment (GBR/BMP-2), as well as when GBR membranes were used without stimulations (GBR). The enhanced <em>bone</em> regeneration of the GBR/BMP-2/US group can be interpreted as resulting from the synergistic or additive effect of the asymmetrically porous PCL/F127 membrane with unique properties (selective permeability, hydrophilicity, and osteoconductivity) and the stimulatory effects of BMP-2 and ultrasound (osteoinductivity). The asymmetrically porous GBR membrane with dual BMP-2 and ultrasound stimulation may be promising for the clinical treatment of delayed and insufficient <em>bone</em> healing.

OBJECTIVE

To study the effect of the recombinant human bone morphogenetic protein 2 (rhBMP-2) on sinus volumetric and histometric changes after sinus floor augmentation compared to a conventional approach of non-biologic bone grafting materials.

METHODS

An electronic search of 4 databases (January 1990-February 2015), including PubMed/MEDLINE, EMBASE, Web of Science and Cochrane Central, and a hand search of peer-reviewed journals for relevant articles were performed. Human clinical trials with data on comparison of sinus volumetric and/or histometric outcomes with and without the use of rhBMP-2 in sinus grafting procedures, with ≥10 augmentation sites in each study group, and with a follow-up period of at least 6 months, were included. Random-effects meta-analyses were performed to analyze weighted mean difference (WMD) and confidence interval (CI) for the recorded variables according to PRISMA guidelines.

RESULTS

Six randomized controlled trials (RCTs) were included. The results of the meta-analyses showed that the WMD of vertical bone height gain was -0.14 mm (95% CI = -1.91 to 1.62 mm, P = 0.87), the WMD of bone density was -142.42 mg/cm3 (95% CI = -310.62-25.78 mg/cm3 , P = 0.10), the WMD of the percentage of vital bone was -4.59% (95% CI = -11.73-2.56%, P = 0.21), and the WMD of the percentage of residual bone grafting materials was -9.90% (95% CI = -26.38-6.58%, P = 0.21). The comparison of implant survival rate presented an overall risk ratio of 1.00 (95% CI = 0.94-1.07). The two approaches (conventional bone grafting compared to BMPs) demonstrated comparable effectiveness for both clinical and histomorphometric measures.

CONCLUSIONS

This systematic review revealed that the use of rhBMP-2 in maxillary sinus floor augmentation achieved similar clinical and histometric outcomes when compared to conventional sinus grafting procedures after a healing period of 6-9 months. However, previous studies showed the morbidity and other patient-reported outcomes were improved in rhBMP-2 approaches as compared to bone autograft procedures (both intraoral and extraoral bone harvesting because no donor site is required). Long-term studies are required to determine the cost-benefit of sinus floor augmentation procedures for patients requiring implant reconstruction.

TAT (a 13-mer derived from the HIV-1 Tat <em>protein</em>)-linked cell-permeable peptides deliver plasma membrane impermeable cargos into the cell. We investigated the effect of a TAT-linked intracellular third loop of the endothelin-1 type B receptor on endothelin-1 activation of ERK. The effect of this peptide on ERK activation was determined in ETB receptor cDNA-transfected Chinese hamster ovary cells and in ETA- and ETB-expressing human pulmonary artery smooth muscle cells obtained from a normal and a <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 receptor, exon 1-8 deletion subject, with pulmonary hypertension. In the Chinese hamster ovary cells the peptide, at optimum <em>10</em> μm concentration, suppressed endothelin-1 activation. In the normal human pulmonary artery smooth muscle cells, the peptide marginally enhanced endothelin-1 activation of ERK. However, it markedly enhanced the endothelin-1 activation of ERK in the <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 receptor human pulmonary artery smooth muscle cells. While the effective concentration for endothelin-1 activation of ERK remained unchanged in the <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 receptor human pulmonary artery smooth muscle cells, the number of ETB receptors declined by 2/3. These data point to the intracellular third loop peptide as having variable receptor interactive effects with both signal repressive and enhancing capabilities. Peptides that can alter endothelin-1 signal capabilities are potentially important in the study and treatment of pulmonary hypertension.

Aim: Effective treatment of premature infants with bronchopulmonary dysplasia (BPD) is lacking. We hypothesize that <em>bone</em> <em>morphogenetic</em> <em>protein</em> 9 (BMP9), a ligand of the TGF-β family that binds to the activin receptor-like kinase 1 (ALK1)-BMP receptor type 2 (BMPR2) receptor complex, may be a novel therapeutic option for BPD. Therefore, we investigated the cardiopulmonary effects of BMP9 in neonatal Wistar rats with hyperoxia-induced BPD. Methods: Directly after birth Wistar rat pups were exposed to <em>10</em>0% oxygen for <em>10</em> days. From day 2 rat pups received BMP9 (2.5 μg/kg, twice a day) or 0.9% NaCl by subcutaneous injection. Beneficial effects of BMP9 on aberrant alveolar development, lung inflammation and fibrosis, and right ventricular hypertrophy (RVH) were investigated by morphometric analysis and cytokine production. In addition, differential mRNA expression of BMP9 and its receptor complex: ALK1, BMPR2, and Endoglin, and of the ALK1 downstream target transmembrane <em>protein</em> <em>10</em>0 (TMEM<em>10</em>0) were studied during the development of experimental BPD. Expression of the BMP9 receptor complex and TMEM<em>10</em>0 was studied in human endothelial and epithelial cell cultures and the effect of BMP9 on inflammatory cytokine production and TMEM<em>10</em>0 expression was studied in endothelial cell cultures. Results:ALK1, ALK2, BMPRII, TMEM<em>10</em>0, and Endoglin were differentially expressed in experimental BPD, suggesting a role for BMP9-dependent signaling in the development of (experimental) BPD. TMEM<em>10</em>0 was expressed in the wall of blood vessels, showing an elastin-like expression pattern in arterioles. Expression of TMEM<em>10</em>0 mRNA and <em>protein</em> was decreased after exposure to hyperoxia. BMP9 treatment of rat pups with hyperoxia-induced experimental BPD reduced alveolar enlargement, lung septal thickness and fibrosis, and prevented inflammation, but did not attenuate vascular remodeling and RVH. The anti-inflammatory effect of BMP9 was confirmed in vitro. Highest expression of ALK1, BMPR2, and TMEM<em>10</em>0 was observed in human endothelial cell cultures. Stimulation of human endothelial cell cultures with BMP9 reduced their pro-inflammatory cytokine response and induced TMEM<em>10</em>0 expression in pulmonary arterial endothelial cells. Conclusion: BMP9 protects against neonatal hyperoxia-induced BPD by improving aberrant alveolar development, inflammation and fibrosis, demonstrating its therapeutic potential for premature infants with severe BPD.

A new method was developed in this study for testing excised sheep mandibles as a cantilever. The method was used to determine the strength and stiffness of sheep hemi-mandibles including a 35 mm defect bridged by regenerated <em>bone</em>. Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (rhBMP-7) in a bovine collagen type-I carrier was used for the <em>bone</em> regeneration. Initial tests on ten intact sheep mandibles confirmed that the strength, stiffness and area beneath the load-deformation curves of the right and left hemi-mandibles were not significantly different, confirming the validity of using the contra-lateral hemi-mandible as a control side. Complete <em>bone</em> regeneration occurred in six hemi-mandibles treated with rhBMP, but the quality and mechanical properties of the <em>bone</em> were very variable. The new <em>bone</em> in three samples contained fibrous tissue and was weaker and less stiff than the contra-lateral side (strength, <em>10</em>-20 per cent; stiffness, 6-15 per cent). The other half had better-quality <em>bone</em> and was significantly stiffer and stronger (p < 0.05), with strength 45-63 per cent and stiffness 35-46 per cent of the contra-lateral side. Hemi-mandibles treated with collagen alone had no regenerated <em>bone</em> bridge suggesting that 35 mm is a critical-size <em>bone</em> defect.

The use of biochemical inducers of mesenchymal stem cell (MSC) differentiation into tenogenic lineage represents an investigated aspect of tendon disorder treatment. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 12 (BMP-12) is a widely studied factor, representing along with ascorbic acid (AA) and basic fibroblast growth factor (bFGF) one of the most promising stimulus in this context so far. Quantitative gene expression of specific tenogenic marker is commonly used to assess the efficacy of these supplements. Nevertheless, the reliability of these data is strongly associated with the choice of stable housekeeping genes. To date, no published studies have evaluated the stability of housekeeping genes in MSCs during tenogenic induction. Three candidate housekeeping genes (YWHAZ, RPL13A, and GAPDH) in human MSCs from <em>bone</em> marrow (BMSCs), adipose tissue (ASCs), and tendon cells (TCs) supplemented with BMP-12 or AA and bFGF in comparison with control untreated cells for 3 and <em>10</em> days were evaluated. GeNorm, NormFinder, and BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability and the overall ranking was determined by using by the RefFinder algorithm. In all culture conditions, YWHAZ was the most stable gene and RPL13A was the second choice. YWHAZ and RPL13A were the two most stable genes also for ASCs and BMSCs, regardless of the time point analyzed, and for TCs at <em>10</em> days of tenogenic induction. Only for TCs at 3 days of tenogenic induction were GAPDH and YWHAZ the best performers. In conclusion, our findings will be useful for the proper selection of housekeeping genes in studies involving MSCs cultured in the presence of tenogenic factors, to obtain accurate and high-quality data from quantitative gene expression analysis.

Hydrogels are based on hydrophilic polymers which are cross-linked to prevent dissolution in water. Because hydrogels can contain large amounts of water, they are interesting devices for the delivery of <em>protein</em> drugs. In this contribution, biodegradable dextran-co-gelatin hydrogel microspheres (DG-MPs) are described which are based on physical interactions and are particularly suitable for the controlled delivery of pharmaceutically active <em>proteins</em>. The unique feature of this preparation system is that the hydrogel microsphere formation takes place in an all-aqueous solution, by which the use of organic solvents is avoided. We investigated the preparation and biological activities of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP2)-loaded dextran-co-gelatin hydrogel microspheres (rhBMP2-DG-MPs), which aimed to keep rhBMP2's biological activity and to achieve a long-term sustained release of rhBMP2. The microspheres' average diameter was about 20-40 microm and rhBMP2 release in vitro could be maintained for>><em>10</em> days. Cytology studies showed that using rhBMP2-DG-MPs could promote the proliferation and osteoblastic differentiation of periodontal ligament cells better than using rhBMP2 aqueous solution. By a freeze-drying method, rhBMP2-DG-MPs could be adhered in chitosan membranes for guided tissue regeneration use, namely functionalized membranes. To evaluate <em>bone</em> regeneration induced by rhBMP2-DG-MPs, an animal experiment with canine class III furcation defects was adopted and the results indicated that using rhBMP2-DG-MPs incorporating scaffolds and functionalized membranes could gain more periodontal tissue regeneration than using scaffolds and general membranes soaked with concentrated rhBMP2 aqueous solution. Therefore, those studies demonstrate the potential of DG-MPs in the sustained delivery of low dosages of rhBMP2 to periodontal defects.

OBJECTIVE

To determine whether an intra-articular injection of recombinant human bone morphogenetic protein-2 (rhBMP-2) alleviates cartilage degradation in a rat model of osteoarthritis (OA) of the lumbar facet joint.

METHODS

The right-side facet joint OA model was created by an intra-articular injection of collagenase (type II) 2 weeks before treatment. The OA rats were divided into four groups: (1) no treatment, or intra-articular injection of either (2) saline, (3) rhBMP-2 10 ng, or (4) rhBMP-2 100 ng. The left-side facet joint served as the normal control. At 3 and 6 weeks after treatment, histological analyses were performed on the cartilage, synovium, subchondral bone and bone marrow. The cartilage and synovium were graded using a modified Mankin score and a synovium score system. Extracellular type II collagen was evaluated by immunohistochemistry.

RESULTS

Intra-articular injection of collagenase causes OA-like changes in the facet joint. OA rats treated with rhBMP-2 at both dosages tested showed reduced severity of their cartilage lesions compared with untreated and saline-treated groups. There was a statistically significant difference in the modified Mankin score compared to the untreated and saline-treated groups. However, some rhBMP-2-treated rats at the higher dose (100 ng) showed, as a side effect, joint space obliteration caused by cartilage overgrowth. Also OA rats treated with 100 ng of rhBMP-2 displayed a significant synovium reaction at 3 weeks compared with that in other groups. Immunohistochemical analysis showed that treatment with rhBMP-2 significantly increased the content of type II collagen.

CONCLUSIONS

This study demonstrates the potential efficacy of rhBMP-2 in the alleviation of arthritic changes in a rat model of OA of the lumbar facet joint. However, treatment with a high dosage of rhBMP-2 caused adverse side effects in some animals.

Hyaline cartilage repair is a significant challenge in orthopedics and current techniques result in formation of fibrocartilage. Human infrapatellar fat pad (hIPFP)-derived mesenchymal stem cells (MSCs) are capable of differentiation into multiple tissue lineages, including cartilage and <em>bone</em>. Chondrogenesis is a crucial part of normal skeletal development but the molecular mechanisms are yet to be completely defined. In this study we sourced hIPFP-derived MSCs utilizing chondrogenic growth factors, transforming growth factor beta-3, and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-6, to form hyaline-like cartilage in micromass cultures and we studied chondrogenic development of 7, 14, and 28 days. The purpose of this study was (1) to characterize chondrogenesis from MSCs derived from hIPFP tissue by conventional techniques and (2) to characterize temporal changes of key molecular components during chondrogenesis using microarray gene expression. Endpoints included histology, immunohistochemistry (IHC), gene expression profiles using a microarray technique, and changes in expression of specific genes using quantitative real-time polymerase chain reaction. Over 14-28 days, clusters of encapsulated chondrocytes formed surrounded by collagen type II and aggrecan in the extracellular matrix (ECM). Collagen type II and aggrecan production was confirmed using IHC and chondrogenic lineage markers were studied; SRY-related transcription factor (SOX9), collagen type II alpha 1 (COL2A1), and aggrecan gene expression increased significantly over the time course. Normalized microarray highlighted 608 differentially expressed genes; <em>10</em> chondrogenic genes were upregulated (2- to 87-fold), including COL2A1, COL<em>10</em>A1, COL9A1, COL11A1, COL9A2, COL11A2, COL1A1, COMP, SOX9, and COL3A1. We found that the upregulated genes (twofold or greater) represent significant level of expression (enrichment score) for the ECM structural constituent of the molecular functional at days 7, 14, and 28 during chondrogenesis. Therefore, we have successfully demonstrated in vitro production of hyaline-like cartilage from IPFP-derived MSCs in micromass culture. Microarray has provided information concerning genes involved in chondrogenesis of hIPFP-derived MSCs and our approach offers a viable strategy for generating clinically relevant cartilage for therapeutic use.

Fresh platelet-rich plasma (PRP) accelerates <em>bone</em> union in rat model. However, fresh PRP has a short half-life. We suggested freeze-dried PRP (FD-PRP) prepared in advance and investigated its efficacy in vivo. Spinal posterolateral fusion was performed on 8-week-old male Sprague-Dawley rats divided into six groups based on the graft materials (n = <em>10</em> per group): sham control, artificial <em>bone</em> (A hydroxyapatite-collagen composite) -alone, autologous <em>bone</em>, artificial <em>bone</em> + fresh-PRP, artificial <em>bone</em> + FD-PRP preserved 8 weeks, and artificial <em>bone</em> + human recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP) as a positive control. At 4 and 8 weeks after the surgery, we investigated their <em>bone</em> union-related characteristics including amount of <em>bone</em> formation, histological characteristics of trabecular <em>bone</em> at remodeling site, and biomechanical strength on 3-point bending. Comparable radiological <em>bone</em> union was confirmed at 4 weeks after surgery in 80% of the FD-PRP groups, which was earlier than in other groups (p < 0.05). Histologically, the trabecular <em>bone</em> had thinner and more branches in the FD-PRP. Moreover, the biomechanical strength was comparable to that of autologous <em>bone</em>. FD-PRP accelerated <em>bone</em> union at a rate comparable to that of fresh PRP and BMP by remodeling the <em>bone</em> with thinner, more tangled, and rigid trabecular <em>bone</em>.

BACKGROUND

Hyperpigmentation of the visceral peritoneum (HVP) has recently garnered much attention in the poultry industry because of the possible risk to the health of affected animals and the damage it causes to the appearance of commercial chicken carcasses. However, the heritable characters of HVP remain unclear. The objective of this study was to investigate the genetic parameters of HVP by genome-wide association study (GWAS) in chickens.

RESULTS

HVP was found to be influenced by genetic factors, with a heritability score of 0.33. HVP had positive genetic correlations with growth and carcass traits, such as leg muscle weight (rg = 0.34), but had negative genetic correlations with immune traits, such as the antibody response to Newcastle disease virus (rg = -0.42). The GWAS for HVP using 39,833 single nucleotide polymorphisms indicated the genetic factors associated with HVP displayed an additive effect rather than a dominance effect. In addition, we determined that three genomic regions, involving the 50.5-54.0 Mb region of chicken (Gallus gallus) chromosome 1 (GGA1), the 58.5-60.5 Mb region of GGA1, and the <em>10</em>.5-12.0 Mb region of GGA20, were strongly associated (P < 6.28 × <em>10</em>-7) with HVP in chickens. Variants in these regions explained >50% of additive genetic variance for HVP. This study also confirmed that expression of BMP7, which codes for a <em>bone</em> <em>morphogenetic</em> <em>protein</em> and is located in one of the candidate regions, was significantly higher in the visceral peritoneum of Huiyang Beard chickens with HVP than in that of chickens without pigmentation (P < 0.05).

CONCLUSIONS

HVP is a quantitative trait with moderate heritability. Genomic variants resulting in HVP were identified on GGA1 and GGA20, and expression of the BMP7 gene appears to be upregulated in HVP-affected chickens. Findings from this study should be used as a basis for further functional validation of candidate genes involved in HVP.