OBJECTIVE

The aim of the present investigation was to histologically assess the effect of laser photobiomodulation (LBPM) on the repair of autologous bone grafts in a rodent model.

BACKGROUND

A major problem in modern dentistry is the recovery of bone defects caused by trauma, surgical procedures, or pathologies. Several types of biomaterials have been used to improve the repair of these defects. These materials are often associated with procedures of guided bone regeneration (GBR).

METHODS

Twenty four animals were divided into four groups: group I (control); group II (LPBM of the bone graft); group III (bone morphogenetic proteins [BMPs] + bone graft); and group IV (LPBM of the bed and the bone graft + BMPs). When appropriate the bed was filled with lyophilized bovine bone and BMPs used with or without GBR. The animals in the irradiated groups received 10 J/cm(2) per session divided over four points around the defect (4 J/cm(2)), with the first irradiation immediately after surgery, and then repeated seven times every other day. The animals were humanely killed after 40 d.

RESULTS

The results showed that in all treatment groups, new bone formation was greater and qualitatively better than the untreated subjects. Control specimens showed a less advanced repair after 40 d, and this was characterized by the presence of medullary tissue, a small amount of bone trabeculi, and some cortical repair.

CONCLUSIONS

We conclude that LPBM has a positive biomodulatory effect on the healing of bone defects, and that this effect was more evident when LPBM was performed on the surgical bed intraoperatively, prior to the placement of the autologous bone graft.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are members of the TGFbeta superfamily of cytokines that are involved in development, differentiation, and disease. In an analysis of normal ovarian surface epithelium (OSE) and ovarian cancer (OC) cells, we observed BMP4 mRNA expression and found that primary OC cells produce mature BMP4. In addition, each member of the downstream signaling pathway was expressed in primary OSE and OC cells. Smad1 was phosphorylated and underwent nuclear translocation in normal OSE and OC cells upon treatment with BMP4. Interestingly, the BMP target genes ID1 and ID3 were up-regulated <em>10</em>- to 15-fold in primary OC cells, compared with a 2- to 3-fold increase in normal OSE. The growth of several primary OC cells was relatively unaltered by BMP4 treatment; however, long-term BMP4 treatment of primary OC cells resulted in decreased cell density as well as increased cell spreading and adherence. These data demonstrate the existence and putative function of BMP signaling in normal OSE and OC cells, and thus the continued examination of BMP4 signaling in the regulation of these two processes will be critical to further our current understanding of the role of BMP biology in OC pathogenesis.

The objective of our study was to elucidate the potential of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP7) to initiate distinct mesenchymal lineage development of human adult mesenchymal stem cells (MSC) in three-dimensional micro-mass culture. Expanded MSC were cultured in high-density micro-masses under serum-free conditions that favor chondrogenic differentiation and were stimulated with 50-200 ng/ml BMP7 or <em>10</em> ng/ml transforming growth factor-beta3 (TGFbeta3) as control. Histological staining of proteoglycan with alcian blue, mineralized matrix according to von Kossa, and lipids with Oil Red O, immunostaining of type II collagen as well as real-time gene expression analysis of typical chondrogenic, adipogenic, and osteogenic marker genes showed that BMP7 promoted adipogenic differentiation of MSC. Micro-masses stimulated with BMP7 developed adipocytic cells filled with lipid droplets and showed an enhanced expression of the adipocyte marker genes fatty acid binding <em>protein</em> 4 (FABP4) and the adipose most abundant transcript 1 (apM1). Development along the chondrogenic lineage or stimulation of osteogenic differentiation were not evident upon stimulation with BMP7 in different concentrations. In contrast, TGFbeta3 directed MSC to form a cartilaginous matrix that is rich in proteoglycan and type II collagen. Gene expression analysis of typical chondrocyte marker genes like cartilage oligomeric matrix <em>protein</em> (COMP), link <em>protein</em>, aggrecan, and types IIalpha1 and IXalpha3 collagen confirmed chondrogenic differentiation of MSC treated with TGFbeta3. These results suggest that BMP7 promotes the adipogenic and not the osteogenic or chondrogenic lineage development of human stem cells when assembled three-dimensionally in micro-masses.

The ability of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) to induce <em>bone</em> formation has led to an increasing interest in the potential for their use in fusion surgery. The purpose of this multi-center clinical pilot study was to evaluate the safety of one such BMP-osteogenic <em>protein</em> 1, in the form of OP-1 putty-combined with autograft for intertransverse process fusion of the lumbar spine in patients with symptomatic spinal stenosis and degenerative spondylolisthesis following spinal decompression. Twelve patients with spinal stenosis and degenerative lumbar spondylolisthesis underwent laminectomy and partial or complete medial facetectomy as required for decompression of the neural elements followed by intertransverse process fusion by placing iliac crest autograft and OP-1 putty between the decorticated transverse processes. No instrumentation was used. Patients were followed clinically using the Oswestry scale and radiographically using static and dynamic radiographs to assess their fusion status. Independent and blinded radiologists assessed the films for the presence of bridging <em>bone</em> between the transverse processes and measured translation and angulation on dynamic films using digital calipers. In addition to bridging <em>bone</em>, less than or equal to 5 degrees of angular motion and less than or equal to 2 mm of translation were required to classify the patients as successfully fused, as per the definition of successful fusion provided by the FDA for use in clinical trials involving investigational devices to attain spinal fusion. Radiographic outcome was compared to a historical control (autograft alone fusion without instrumentation for the treatment of degenerative spondylolisthesis). All adverse events were recorded prospectively. The results showed 9 of the 12 patients (75%) obtained at least a 20% improvement in their preoperative Oswestry score, while 6 of 11 patients (55%) with radiographic follow-up achieved a solid fusion by the criteria used in this study. Bridging <em>bone</em> on the anteroposterior film was observed in <em>10</em> of the 11 patients (91%). No systemic toxicity, ectopic <em>bone</em> formation, recurrent stenosis or other adverse events related to the OP-1 putty implant were observed. A successful fusion was observed in slightly over half the patients in this study, using stringent criteria without adjunctive spinal instrumentation. This study did not demonstrate the superiority of OP-1 combined with autograft over an autograft alone historical control, in which the fusion rate was approximately 45%. The lack of adverse events related to the OP-1 putty implant in this study is in agreement with other studies supporting the safety of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> in spinal surgery.

The major proteoglycan in macrophages and platelets is the chondroitin sulphate proteoglycan serglycin. To study the biological role of serglycin, its binding to secreted and cell-associated <em>proteins</em> from macrophages and blood platelets was examined. Affinity chromatography with serglycin-Sepharose and chondroitin sulphate-Sepharose was used to isolate proteoglycin-binding <em>proteins</em> from macrophages and platelets. Antibodies against human macrophage inflammatory <em>protein</em>-1 alpha (MIP-1 alpha) precipitated a 14-kDa 35S-methionine-labeled <em>protein</em> among the chondroitin sulfate binding <em>proteins</em> secreted from the macrophage-like U937 cells after stimulation. Two <em>proteins</em> from murine macrophage J774 cells with molecular masses of approximately <em>10</em> and 14 kDa were precipitated by an antiserum against the murine MIP-1 alpha. <em>Protein</em> sequencing of fragments obtained by trypsin digestion of a 14-kDa chondroitin sulfate-binding <em>protein</em> from cell extracts of stimulated U937 cells revealed <em>10</em>0% homology with lysozyme, a bacteriolytic enzyme. Fragment of one other <em>protein</em> with approximate molecular mass of 8 kDa showed high homology with <em>bone</em> <em>morphogenetic</em> <em>protein</em>. Inhibition studies showed that chondroitin 6-sulfate inhibited the bacteriolytic activity of lysozyme in a competitive manner more efficiently than heparin and chondroitin 4-sulphate. Amino-terminal sequencing of two <em>proteins</em> from platelet extracts that bound to serglycin-Sepharose revealed that they corresponded to multimeric forms of human platelet factor 4 (PE4). Chondroitin sulfate-Sepharose was shown to be equally efficient in retaining PF4 from platelet extracts as serglycin-Sepharose indicating that the glycosaminoglycan chains mediate the binding to PF4 in the intact proteoglycan molecule. Competition experiments showed that serglycin was as efficient as heparin sulfate in blocking the binding of [3H] chondrotin sulfate to PF4, whereas heparin was one order of magnitude more efficient. Affinity measurements using fluoresceinamine-labeled glycosaminoglycans showed that the affinity of heparin for PF4 is on the order of 30 nM, whereas chondroitin sulfate has an affinity of 260 nM. Both PF4, MIP-1 alpha, and lysozyme play important role in different types of inflammatory reactions. The interaction with serglycin may indicate that this proteoglycan is involved in the regulation of the inflammatory response.

BACKGROUND

Approximately 5% to 20% of fractures have delayed or impaired healing. Therefore, it is desirable to develop new therapies to enhance fracture-healing that can be used in conjunction with traditional treatment methods. The purpose of this study was to evaluate the ability of a single application of recombinant human bone morphogenetic protein-2 to accelerate fracture-healing in a rabbit ulnar osteotomy that heals spontaneously.

METHODS

Bilateral mid-ulnar osteotomies (approximately 0.5 to 1.0 mm wide) were created in seventy-two skeletally mature male rabbits. The limbs were assigned to one of three groups: those treated with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2, those treated with an absorbable collagen sponge containing buffer, and those left untreated. In the first two groups, an 8 20-mm strip of absorbable collagen sponge containing either 40 g of recombinant human bone morphogenetic protein-2 or buffer only was wrapped around the osteotomy site. The rabbits were killed at two, three, four, or six weeks after surgery. In addition, twenty-four age-matched rabbits were used to provide data on the properties of intact limbs. The retention of recombinant human bone morphogenetic protein-2 at the osteotomy site was determined with scintigraphic imaging of (125)I-labeled recombinant human bone morphogenetic protein-2. After the rabbits were killed, the limbs were scanned with peripheral quantitative computed tomography to assess the area and mineral content of the mineralized callus. The limbs were then tested to failure in torsion, and undecalcified specimens were evaluated histologically.

RESULTS

Gamma scintigraphy of (125)I-recombinant human bone morphogenetic protein-2 showed that 73% +/- 6% (mean and standard deviation) of the administered dose was initially retained at the fracture site. Approximately 37% +/- 10% of the initial dose remained at the site one week after surgery, and 8% +/- 7% remained after two weeks. The mineralized callus area was similar in all groups at two weeks, but it was 20% to 60% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 than in either the ulnae treated with buffer or the untreated ulnae at three, four, and six weeks (p < 0.05). Biomechanical properties were similar in all groups at two weeks, but they were at least 80% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 at three and four weeks than in either the ulnae treated with buffer (p < 0.005) or the untreated ulnae (p < 0.01). By four weeks, the biomechanical properties of the ulnae treated with recombinant human bone morphogenetic protein-2 were equivalent to those of the intact ulnae, whereas the biomechanical properties of both the ulnae treated with buffer and the untreated ulnae had reached only approximately 45% of those of the intact ulnae. At six weeks, the biomechanical properties were similar in all groups and were equivalent to those of the intact ulnae. The callus geometry and biomechanical properties of the ulnae treated with buffer were equivalent to those of the untreated ulnae at all time-points.

CONCLUSIONS

These findings indicate that treatment with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2 enhances healing of a long-bone osteotomy that heals spontaneously. Specifically, osteotomies treated with recombinant human bone morphogenetic protein-2 healed 33% faster than osteotomies left untreated. The results of this study provide a rationale for testing the ability of recombinant human bone morphogenetic protein-2 to accelerate healing in patients with fractures requiring open surgical management.

BACKGROUND

Adipose-derived adult stem (ADAS) cells are multipotent cells capable of differentiating into osteoblasts, adipocytes and chondrocytes. The aim of this study was to determine whether BMP-7-expressing ADAS cells would elicit bone formation invitro and in vivo.

METHODS

ADAS cells were harvested from Lewis rats and transduced with adenovirus carrying the recombinant human bone morphogenetic protein-7 (Ad-BMP-7) gene. Untransduced cells and cells transduced with adenovirus carrying the enhanced green fluorescence protein (Ad-EGFP) gene served as controls. BMP-7 expression was assessed by RT-PCR, immunofluorescence on day 1, and Western blot on days 4, 8 and 12. Alkaline phosphatase (ALP) activity was assayed on days 2, 4, 6, 8, 10 and 12. Osteocalcin production and bone nodule formation were detected by immunohistochemistry and von Kossa stain on day 12. A total of 1 x 10(6) cells mixed with type I collagen were implanted into the subcutaneous pocket in Lewis rat and subjected to histologic analysis 1, 2 and 4 weeks post-implantation.

RESULTS

The Ad-BMP-7-transduced ADAS cells expressed BMP-7 at both mRNA and protein levels. ALP activity was detected in Ad-BMP-7-transduced cells from day 2 to day 12, peaking on day 8. Osteocalcin production and matrix mineralization further confirmed that these cells differentiated into osteoblasts and induced bone formation in vitro. Histologic examination revealed that implantation of BMP-7-expressing ADAS cells could induce new bone formation in vivo.

CONCLUSIONS

ADAS cells would be a promising source of adult autologous stem cells for BMP gene therapy and tissue engineering.

The <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) play an inductive role in the generation of cerebellar granule cells embryonically. Therefore, we chose to look at their effects on cerebellar granule cell survival and differentiation postnatally. The cells express mRNA for both BMP-6 and BMP-7, as well as for the receptors BMPRIA and BMPRII, demonstrating that the postnatal cells have the ability to form the heterodimer receptors needed to respond to BMPs. BMP-7 promotes cell survival, with a maximal effect at <em>10</em> ng/ml, whereas tenfold more BMP-6 is needed: Both were active over the course of 8 days in culture. In addition, both BMPs were able to protect the neurons against death from induced apoptosis (exposure to serum-free, low-potassium medium) or exposure to glutamate. However, only BMP-6 could stimulate neurite outgrowth, measured with a neurofilament ELISA, an effect that was seen over the first 6 days in culture. These results, taken together with others in the literature, suggest that the BMPs have strong neurotrophic effects that are both neuron specific and BMP specific.

BACKGROUND

The transforming growth factor-β (TGF-β) signaling pathway is involved in many aspects of tumorigenesis, including angiogenesis and metastasis. The authors evaluated this pathway in association with survival after a diagnosis of colon or rectal cancer.

METHODS

The study included 1553 patients with colon cancer and 754 patients with rectal cancer who had incident first primary disease and were followed for a minimum of 7 years after diagnosis. Genetic variations were evaluated in the genes TGF-β1 (2 single nucleotide polymorphisms [SNPs]), TGF-β receptor 1 (TGF-βR1) (3 SNPs), smooth muscle actin/mothers against decapentaplegic homolog 1 (Smad1) (5 SNPs), Smad2 (4 SNPs), Smad3 (37 SNPs), Smad4 (2 SNPs), Smad7 (11 SNPs), <em>bone</em> <em>morphogenetic</em> <em>protein</em> 1 (BMP1) (11 SNPs), BMP2 (5 SNPs), BMP4 (3 SNPs), <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor 1A (BMPR1A) (9 SNPs), BMPR1B (21 SNPs), BMPR2 (11 SNPs), growth differentiation factor <em>10</em> (GDF<em>10</em>) (7 SNPs), Runt-related transcription factor 1 (RUNX1) (40 SNPs), RUNX2 (19 SNPs), RUNX3 (9 SNPs), eukaryotic translation initiation factor 4E (eiF4E) (3 SNPs), eukaryotic translation initiation factor 4E-binding <em>protein</em> 3 (eiF4EBP2) (2 SNPs), eiF4EBP3 (2 SNPs), and mitogen-activated <em>protein</em> kinase 1 (MAPK1) (6 SNPs).

RESULTS

After adjusting for American Joint Committee on Cancer stage and tumor molecular phenotype, 12 genes and 18 SNPs were associated with survival in patients with colon cancer, and 7 genes and 15 tagSNPs were associated with survival after a diagnosis of rectal cancer. A summary score based on "at-risk" genotypes revealed a hazard rate ratio of 5.<em>10</em> (95% confidence interval, 2.56-<em>10</em>.15) for the group with the greatest number of "at-risk" genotypes; for rectal cancer, the hazard rate ratio was 6.03 (95% confidence interval, 2.83-12.75).

CONCLUSIONS

The current findings suggest that the presence of several higher risk alleles in the TGF-β signaling pathway increase the likelihood of dying after a diagnosis of colon or rectal cancer.

Ethanol inhibits cell-cell adhesion mediated by the L1 cell adhesion molecule. 1-Octanol potently antagonizes this cellular action of ethanol and also prevents ethanol-induced dysmorphology and cell death in mouse whole embryo culture. NAPVSIPQ (NAP) and SALLRSIPA (SAL) are active peptide fragments of two neuroprotective <em>proteins</em>: activity-dependent neuroprotective <em>protein</em> and activity-dependent neurotrophic factor. NAP and SAL are neuroprotective at femtomolar concentrations against a variety of neurotoxins and also prevent ethanol teratogenesis in mice. To explore the cellular basis for this action, we asked whether NAP and SAL antagonize ethanol inhibition of L1 adhesion. Aggregation assays were carried out in ethanol-sensitive, human L1-transfected NIH/3T3 cells in the absence and presence of NAP and SAL. Neither NAP nor SAL altered L1 adhesion or L1 expression; however, both peptides potently and completely antagonized the inhibition of L1 adhesion by <em>10</em>0 mM ethanol (EC(50): NAP, 6 x <em>10</em>(-14) M; SAL, 4 x <em>10</em>(-11) M). NAP also antagonized ethanol inhibition of cell-cell adhesion in <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7-treated NG<em>10</em>8-15 cells. In L1-expressing NIH/3T3 cells, SAL antagonism was reversible and could be overcome by increasing concentrations of ethanol. In contrast, NAP antagonism was irreversible and could not be overcome by increasing agonist concentration. Two scrambled NAP peptides (ASPNQPIV and PNIQVASP) were not antagonists at concentrations as high as <em>10</em>(-7) M. Thus, two structurally unrelated classes of compounds, alcohols and small polypeptides, share two common actions: antagonism of ethanol inhibition of L1-mediated cell adhesion and prevention of ethanol teratogenesis. These findings support the hypothesis that ethanol inhibition of L1 adhesion contributes to ethanol teratogenesis.

There is mounting evidence that innate and adaptive immunity are critical for periodontal disease-mediated <em>bone</em> resorption. These studies examined the role of B and CD4 T cells in adaptive immunity of rats infected with Aggregatibacter actinomycetemcomitans (Aa). Sprague-Dawley male rats were fed Aa-containing mash or control-mash for 2 weeks. B and CD4 T cells were obtained from draining lymph nodes at 2, 4 and 12 weeks, postinoculation. Quantitative polymerase chain reaction-based messenger RNA expression was conducted for 89 cytokine family genes. Disease-relevance of the differentially expressed genes was assessed using a biological interaction pathway analysis software. B and CD4 T cells of Aa-infected rats increased and were activated, resulting in enhanced isotype-switched serum immunoglobulin G by 2 weeks postinoculation. <em>Bone</em> resorption was evident 12 weeks after Aa-feeding. In B cells, interleukin-2 (IL-2), macrophage-inhibiting factor, IL-19, IL-21, tumor necrosis factor (TNF), CD40 ligand (CD40L), CD70, <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2), BMP3, and BMP<em>10</em> were upregulated early; while IL-7, Fas ligand (FasL), small inducible cytokine subfamily E1, and growth differentiation factor 11 (GDF11; BMP11) were upregulated late (12 weeks). BMP<em>10</em> was sustained throughout. In CD4 T cells, IL-<em>10</em>, IL-16, TNF, lymphotoxin-beta (LTbeta), APRIL, CD40L, FasL, RANKL and osteoprotegerin were upregulated early, whereas IL-1beta, IL-1RN, IL-1F8, IL-24, interferon-alpha1, GDF11 (BMP11), and GDF15 were upregulated late (12 weeks). Adaptive immunity appears crucial for <em>bone</em> resorption. Several of the deregulated genes are, for the first time, shown to be associated with <em>bone</em> resorption, and the results indicate that activated B cells produce BMP<em>10</em>. The study provides a rationale for a link between periodontal disease and other systemic diseases.

OBJECTIVE

Mechanical stress has been considered one of the important factors in ossification of the spinal ligaments. According to previous clinical and in vitro studies, the accumulation of tensile stress to these ligaments may be responsible for ligament ossification. To elucidate the relationship between such mechanical stress and the development of ossification of the spinal ligaments, the authors established an animal experimental model in which the in vivo response of the spinal ligaments to direct repetitive tensile loading could be observed.

METHODS

The caudal vertebrae of adult Wistar rats were studied. After creating a novel stimulating apparatus, cyclic tensile force was loaded to rat caudal spinal ligaments at <em>10</em> N in 600 to 1800 cycles per day for up to 2 weeks. The morphological responses were then evaluated histologically and immunohistochemically. After the loadings, ectopic cartilaginous formations surrounded by proliferating round cells were observed near the insertion of the spinal ligaments. Several areas of the cartilaginous tissue were accompanied by woven <em>bone</em>. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-2 expression was clearly observed in the cytoplasm of the proliferating round cells. The histological features of the rat spinal ligaments induced by the tensile loadings resembled those of spinal ligament ossification observed in humans.

CONCLUSIONS

The findings obtained in the present study strongly suggest that repetitive tensile stress to the spinal ligaments is one of the important causes of ligament ossification in the spine.

OBJECTIVE

Bone morphogenetic proteins (BMPs) are involved in the growth and development of many tissues, but it is their role in skeletal development and their unique ability to induce ectopic and orthotopic osteogenesis that have attracted the greatest interest. Expression of the BMP-13 gene is predominantly localized to hypertrophic chondrocytes in regions of endochondral bone formation during development, as well as in mature articular cartilage in the adult. In addition, the application of BMP-13 on a collagen carrier induces neotendon/neoligament formation when delivered subcutaneously or intramuscularly in rodents. The aim of the present study was to determine the histological and ultrastructural changes that occur after the intramuscular injection of a first-generation BMP-13 adenoviral vector.

METHODS

Athymic nude rats were injected with 3.75 x 10(10) plaque-forming units of adenovirus (Ad)-BMP-13 or Ad-beta-galactosidase in the thigh musculature, and the region was examined using light and electron microscopy at various time points between 2 days and 100 days postinjection. As early as 2 days after injection of Ad-BMP-13, progenitor cells were observed infiltrating between the transduced muscle fibers. These cells subsequently proliferated, differentiated, and secreted large amounts of collagenous extracellular matrix. By 100 days postinjection, the treated tissue displayed the histological and ultrastructural appearance of neotendon/neoligament, which was clearly demarcated from the surrounding muscle. Small foci of bone and fibrocartilage were also seen within the treated tissue. A short-term bromodeoxyuridine study also demonstrated rapid mesenchymal cell proliferation at the Ad-BMP-13 injection site as early as 48 hours postinjection. At all time points, the control AD-beta-gal injection sites were found to contain only normal muscle, without evidence of inflammation or mesenchymal cell proliferation.

CONCLUSIONS

The results of this study indicate that in the future the use of the BMP-13 gene may have therapeutic utility for the healing of tendon and ligament tears and avulsion injuries.

To dissect the possible role of the transforming growth factor-beta (TGF-beta) family in the regulation of skeletal muscle growth during the early postnatal period, the <em>protein</em> abundances of the TGF-beta family and their correlation with <em>protein</em> synthesis were determined in skeletal muscle of neonatal rats. To obtain direct evidence of the role of these growth factors in the regulation of <em>protein</em> synthesis, the TGF-beta inhibitor, follistatin, was infused into <em>10</em>-d-old rats for 11 d and <em>protein</em> synthesis and phosphorylation of S6 kinase 1 (S6K1) and ribosomal <em>protein</em> (rpS6) were measured. TGF-beta2 abundance and <em>protein</em> synthesis in muscle decreased with development and were positively correlated. The abundances of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2), BMP-7, and myostatin increased with development and were negatively correlated with <em>protein</em> synthesis. The abundances of BMP-2 and BMP-7 were positively correlated with BMP receptor IA (BMP-RIA) abundance. Activin A abundance was positively correlated with follistatin abundance and activin receptor IIB (Act-RIIB) abundance. Infusion of follistatin increased muscle <em>protein</em> synthesis and S6K1 and rpS6 phosphorylation. The results provide indirect and direct evidence of TGF-beta family involvement in the regulation of muscle <em>protein</em> synthesis during the neonatal period.

Angiogenesis, a complex biologic process, is regulated by a large number of angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). Whether <em>Bone</em> <em>morphogenetic</em> <em>proteins</em>-2 (BMP-2), the osteoinductive factor, could significantly reinforce the effect of VEGF and FGF-2 on angiogenesis has not been studied in detail. To study the positive effects of multiple growth factors on angiogenesis, HUVECs were treated with BMP-2, VEGF, or FGF-2 singly and in binary and ternary combinations. This study further investigates the optimal timing of the ternary combination of BMP-2, VEGF and FGF-2 for angiogenesis in the chorioallantoic membrane (FGF-2 CAM). Results of single applications of BMP-2, VEGF, or FGF-2 suggested that HUVECs angiogenesis could be promoted in a dose-dependent manner and that the optimal concentration of BMP, VEGF and FGF-2 was <em>10</em>, 50 and 1 ng/mL, respectively. These results indicated that the angiogenic activity of VEGF and FGF-2 was amplified by combining with BMP-2. The ternary combination of BMP-2, VEGF and FGF-2 exhibited a positive and synergistic effect on HUVECs angiogenesis, with the lower concentrations of each factor (1 ng/mL of BMP-2, 25 ng/mL of VEGF and 0.1 ng/mL of FGF-2) being sufficient to show synergistic promotion. When VEGF and FGF-2 were added in the initial activation stage and BMP-2 was added in the maturation stage, both HUVECs angiogenesis in vitro and CAM angiogenesis in vivo could be enhanced more effectively. These results could provide a basis for the controlled release systems capable of delivering multiple factors sequentially to promote angiogenesis in tissue engineering.

To facilitate <em>bone</em> healing in difficult circumstances, and to replace conventional therapeutic modalities, highly purified <em>bone</em> marrow-derived human mesenchymal stem cells (hMSCs) were investigated for induction of their osteogenic lineage upon provision of cytokine cues in vitro and in the cranial defect model in vivo. Alkaline phosphatase-expressing cells were most frequently observed when the hMSCs were treated with 2.5 ng/ml of basic fibroblast growth factor (bFGF) and 50 ng/ml of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2 for 4 days in culture after a 6-day incubation in osteogenic medium containing dexamethasone, ascorbic acid-2-phosphate, and beta-glycerophosphate. Four-millimeter full-thickness cranial defect wounds were made in male nude rats (F344/NJCl-rnu), whose deficit in the T cell compartment prevented T-cell-mediated cellular rejection. The animals were treated for 4 weeks with hMSCs and application of <em>10</em> microg each of bFGF and BMP-2 that had been soaked into a gelatin sponge carrier. Significant <em>bone</em> mineral density was observed by dual X-ray absorptiometry and this treatment also produced histologically mature osteocytes surrounded by both osteoblasts and osteoclasts expressing alkaline phosphatase and osteocalcin. The <em>bone</em> mineral densities and histological structures were matched at 8 weeks post-transplantation. Therefore, human <em>bone</em> marrow-derived mesenchymal stem cells are able to differentiate into an osteogenic lineage upon cytokine stimulation and accelerate healing in a nude rat cranial <em>bone</em> healing model.

Delayed fracture healing and nonunion occurs in up to 5-<em>10</em>% of all fractures, and can present a challenging clinical scenario for the treating physician. Methods for the enhancement of skeletal repair may benefit patients that are at risk of, or have experienced, delayed healing or nonunion. These methods can be categorized into either physical stimulation therapies or biological therapies. Physical stimulation therapies include electrical stimulation, low-intensity pulsed ultrasonography, or extracorporeal shock wave therapy. Biological therapies can be further classified into local or systemic therapy based on the method of delivery. Local methods include autologous <em>bone</em> marrow, autologous <em>bone</em> graft, fibroblast growth factor-2, platelet-rich plasma, platelet-derived growth factor, and <em>bone</em> <em>morphogenetic</em> <em>proteins</em>. Systemic therapies include parathyroid hormone and bisphosphonates. This article reviews the current applications and supporting evidence for the use of these therapies in the enhancement of fracture healing.

METHODS

Retrospective database analysis.

OBJECTIVE

To investigate national trends of cervical spine surgical procedures from 2002 to 2011.

BACKGROUND

There is a paucity of literature assessing the current practice trends and outcomes of cervical spine surgery following the 2008 Food and Drug Administration public health notifications regarding bone morphogenetic protein (BMP) utilization in cervical spine surgical procedures.

METHODS

The National Inpatient Sample database was accessed for each year across 2002 to 2011. Patients undergoing anterior cervical fusion, posterior cervical fusion, and posterior cervical decompression were identified. Patient and hospitalization parameters including demographics, BMP utilization, costs, early postoperative outcomes, and mortality were assessed for each surgical cohort. A Pearson correlation coefficient with a 95% confidence interval (P < 0.05) was used to analyze trends in patient and hospital outcome parameters during this 10-year period.

RESULTS

A total of 307,188 cervical spine procedures were performed from 2002 to 2011. Both the anterior cervical fusion and posterior cervical fusion cohort demonstrated a statistically significant increase in the number of procedures performed over time (r = +0.9, P < 0.001). A significant uptrend in patient age (r = +1.0, P < 0.001) and comorbidity burden (r = +0.9, P < 0.001) was demonstrated during the studied decade. Overall, BMP utilization (r = +0.7, P = 0.02) also demonstrated a significant increase during this time period, but demonstrated a decline after peaking in 2007. The posterior cervical fusion cohort demonstrated the greatest comorbidity, length of stay, costs, and mortality.

CONCLUSIONS

This study demonstrates that the number of cervical spine procedures has increased between 2002 and 2011, irrespective of the change in BMP utilization after the 2008 Food and Drug Administration warning. Despite an older patient population with greater comorbidities undergoing cervical spine surgeries, hospital length of stay and mortality has not significantly changed. However, we did note a significant increase in costs during this time period. These findings may be related to advances in surgical technology and instrumentation that may be associated with rising hospital costs.

Frizzled <em>proteins</em> are the principal receptors for the Wnt family of ligands. They mediate canonical Wnt signaling together with Lrp5 and Lrp6 coreceptors. In conjunction with Celsr, Vangl, and a small number of additional membrane and membrane-associated <em>proteins</em>, they also play a central role in tissue polarity/planar cell polarity (PCP) signaling. Targeted mutations in 9 of the <em>10</em> mammalian Frizzled genes have revealed their roles in an extraordinarily diverse set of developmental and homeostatic processes, including <em>morphogenetic</em> movements responsible for palate, ventricular septum, ocular furrow, and neural tube closure; survival of thalamic neurons; <em>bone</em> formation; central nervous system (CNS) angiogenesis and blood-brain barrier formation and maintenance; and a wide variety of processes that orient subcellular, cellular, and multicellular structures relative to the body axes. The last group likely reflects the mammalian equivalent of tissue polarity/PCP signaling, as defined in Drosophila, and it includes CNS axon guidance, hair follicle and tongue papilla orientation, and inner ear sensory hair bundle orientation. Frizzled receptors are ubiquitous among multicellular animals and, with other signaling molecules, they very likely evolved to permit the development of the complex tissue architectures that provide multicellular animals with their enormous selective advantage.

Mechanical loading, a potent stimulator of <em>bone</em> formation, is governed by osteocyte regulation of osteoblasts. We developed a three-dimensional (3D) in vitro co-culture system to investigate the effect of loading on osteocyte-osteoblast interactions. MLO-Y4 cells were embedded in type I collagen gels and MC3T3-E1(14) or MG63 cells layered on top. Ethidium homodimer staining of 3D co-cultures showed <em>10</em>0% osteoblasts and 86% osteocytes were viable after 7 days. Microscopy revealed osteoblasts and osteocytes maintain their respective ovoid/pyriform and dendritic morphologies in 3D co-cultures. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) of messenger ribonucleic acid (mRNA) extracted separately from osteoblasts and osteocytes, showed that podoplanin (E11), osteocalcin, and runt-related transcription factor 2 mRNAs were expressed in both cell types. Type I collagen (Col1a1) mRNA expression was higher in osteoblasts (P < 0.001), whereas, alkaline phosphatase mRNA was higher in osteocytes (P = 0.001). Immunohistochemistry revealed osteoblasts and osteocytes express E11, type I pro-collagen, and connexin 43 <em>proteins</em>. In preliminary experiments to assess osteogenic responses, co-cultures were treated with human recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) or mechanical loading using a custom built loading device. BMP-2 treatment significantly increased osteoblast Col1a1 mRNA synthesis (P = 0.031) in MLO-Y4/MG63 co-cultures after 5 days treatment. A 16-well silicone plate, loaded (5 min, <em>10</em> Hz, 2.5 N) to induce 4000-4500 με cyclic compression within gels increased prostaglandin E2 (PGE2) release 0.5 h post-load in MLO-Y4 cells pre-cultured in 3D collagen gels for 48, 72 h, or 7 days. Mechanical loading of 3D co-cultures increased type I pro-collagen release 1 and 5 days later. These methods reveal a new osteocyte-osteoblast co-culture model that may be useful for investigating mechanically induced osteocyte control of osteoblast <em>bone</em> formation.

OBJECTIVE

Previously we demonstrated that both hypoxia inducible factor-1 (HIF-1) and bone morphogenetic protein-4 (BMP4) up-regulate transient receptor potential canonical (TRPC) 1 and TRPC6, resulting in increased basal intracellular Ca(2+) concentration ([Ca(2+)]i) in pulmonary arterial smooth muscle cells (PASMCs), driving development of chronic hypoxia (CH)-induced pulmonary hypertension (CHPH). This study aims to determine whether HIF-1 regulates BMP4, and whether BMP4 mediates TRPC and basal [Ca(2+)]i increases in hypoxic PASMCs.

RESULTS

The level of BMP4 mature protein was increased for ∼183% in distal pulmonary arterial smooth muscle (PA) from CH (10% O2 for 21 days; CH) exposed rats, and 143% in PASMCs cultured under prolonged hypoxia (4% O2 for 60 h). In rat PASMCs, HIF-1α overexpression up-regulated, whereas HIF-1α knockdown under hypoxia decreased BMP4 expression; site-mutation identified two functional HIF-1-binding sites in Bmp4 gene promoter; noggin or BMP4 siRNA treatment blocked hypoxia-induced increases of TRPC1 and TRPC6 expression and basal [Ca(2+)]i. Likewise, in mice, exposure to CH increased BMP4 expression in distal PA for ∼80%, which was absent in HIF-1α heterozygous mutant mice. Comparing with wild-type littermates, BMP4 heterozygous mutant mice exposed to CH displayed lower BMP4 and TRPC levels in PA, decreased basal [Ca(2+)]i in PASMCs, and attenuated CHPH. In human PASMCs, HIF-1α knockdown attenuated hypoxia-induced BMP4 expression and knockdown of either HIF-1α or BMP4 abolished hypoxia-induced TRPC expression and basal [Ca(2+)]i.

CONCLUSIONS

BMP4 acts downstream of HIF-1 and mediates hypoxia-induced up-regulation of TRPC, leading to increased basal [Ca(2+)]i in PASMCs, promoting CHPH pathogenesis.

OBJECTIVE

Bone morphogenetic proteins (BMPs) have a diverse role and they act in a time, concentration and cell type specific manner. They regulate cellular motility and the cells ability to invade. Recently, BMP molecules have further been shown to have an impact on the biological behaviour of breast cancer cells. In this study, we looked, for the first time, at the expression of a panel of BMPs in breast carcinomas.

METHODS

Fresh frozen primary human breast cancer tissues (n = 120) and non-neoplastic mammary tissues (n = 32) were used. The distribution and location of BMPs was assessed using immunohistochemical methods and the transcript levels of BMPs (BMP-1, -2, -3, -4, -5, -6, and -7) were determined using quantitative RT-PCR. The results were analysed against the clinical, pathological and follow-up (10 years) data.

RESULTS

BMP-2 and BMP-7 exhibited contrasting patterns of expression in normal and tumour tissues, wherein BMP-2 transcript level was lower and BMP-7 was higher in breast tumours than normal tissues. BMP-2 transcript was also significantly lower in tumours from patients with a moderate and poor prognosis than from those with a good prognosis (p = 0.04). BMP-2 and BMP-7 also showed a significant difference between node positive and node negative tumours (p = 0.033 and p = 0.031 respectively). BMPs 1, 3, 4, 5 and 6 showed an inconsistent variation in transcript levels within the cancer subgroups with no statistically significant results.

CONCLUSIONS

This study has demonstrated a differential pattern of expression of BMP molecules in breast cancer and reveals a potential prognostic value of BMP-2 and BMP-7 for patients. The findings also suggest that these BMPs may be potential therapeutic targets.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2) is a human recombinant <em>bone</em>-inducing factor that stimulates <em>bone</em> formation within 14 days. Twenty-six dogs underwent reconstruction of 3-cm full-thickness mandibular defects. After stabilizing the defects with stainless steel reconstruction plates, test implants composed of inactive dog <em>bone</em> matrix carrier and human recombinant BMP-2 were placed in defects of 12 animals (group 1). Control implants (carrier without BMP-2) were used in <em>10</em> animals (group 2), and no implants were placed in mandibular defects of four animals (group 3). Animals were killed at 3 and 6 months. The reconstructed segments were evaluated by roentgenography, analysis of functional stability, histology, histomorphometry, and analysis of biomechanical strength using three-point bend testing. In group 1, reconstruction plates were removed at <em>10</em> weeks because stiff, noncompressible mineralized <em>bone</em> formed across the defects, allowing the animals to chew a solid diet. The defects from groups 2 and 3 showed minimal, if any, <em>bone</em> formation and remained grossly unstable, prohibiting plate removal or advancement to a solid diet. Histomorphometric analysis at 6 months revealed that 68% of the group 1 implants were replaced by mineralized <em>bone</em>, whereas mineralized <em>bone</em> occupied less than 4% of the implants in groups 2 and 3. Biomechanical testing at 6 months revealed that the average bending strength of the reconstructed hemimandibles (expressed as a percentage of the contralateral hemimandible) was 27% for group 1 and 0% for group 2. The biomechanical strength of the defects reconstructed with BMP-2 increased significantly from 3 to 6 months and was related to degree of mineralization and thickness of <em>bone</em> bridging the defect.

The purpose of this study was to examine the regeneration of periodontal tissue after the application of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) to horizontal circumferential defects created by experimental periodontitis. Twelve mandibular second premolars in 6 adult beagle dogs were subjected to experimental periodontal breakdown by placing silk ligatures around the teeth until the <em>bone</em> loss exceeded half of the root length. Flap surgery was then performed and the exposed cementum removed. The distance between the <em>bone</em> crest and cemento-enamel junction (CEJ) was about 5 mm. RhBMP-2 (40 micrograms/<em>10</em>0 microliters) with a sponge-type carrier material made of gelatin and polylactic acid polyglycolic acid copolymer was placed in the furcation area (5 mm x 5 mm x 5 mm) and around the roots (<em>10</em> mm x 5 mm x 2.5 mm x 2 pieces). In the control group, the same carrier material without rhBMP-2 was placed in the same manner. The flaps were replaced and sutured to cover these materials completely. Twelve weeks after surgery, the animals were sacrificed and serial sections were prepared in a bucco-lingual plane. Considerable new <em>bone</em> formation was observed in the rhBMP-2-treated sites. New cementum with Sharpey's fibers was observed on the instrumented root surface. On histometric analysis, the amount of new <em>bone</em>, new cementum, and connective tissue attachment was significantly greater in the rhBMP-2-treated group (paired t test; P < 0.01). These results indicate that suitable application of rhBMP-2 can produce considerable periodontal tissue regeneration, even in cases of horizontal circumferential defects.