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Background: Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by heterozygous germline NF1 gene mutations that predispose patients to developing plexiform neurofibromas, which are benign but often disfiguring tumors of the peripheral nerve sheath induced by loss of heterozygosity at the NF1 locus. These can progress to malignant peripheral nerve sheath tumors (MPNSTs). There are no approved drug treatments for adults with NF1-related inoperable plexiform neurofibromas, and only one drug (selumetinib), which is an FDA-approved targeted therapy for the treatment of symptomatic pediatric plexiform neurofibromas, highlighting the need for additional drug screening and development. In high-throughput screening, the effectiveness of drugs against cell lines is often assessed by measuring in vitro potency (AC50) or the area under the curve (AUC). However, the variability of dose-response curves across drugs and cell lines and the frequency of partial effectiveness suggest that these measures alone fail to provide a full picture of overall efficacy. Methods: Using concentration-response data, we combined response effectiveness (EFF) and potency (AC50) into (a) a score characterizing the effect of a compound on a single cell line, S = log[EFF/AC50], and (b) a relative score, ΔS, characterizing the relative difference between a reference (e.g., non-tumor) and test (tumor) cell line. ΔS was applied to data from high-throughput screening (HTS) of a drug panel tested on NF1-/- tumor cells, using immortalized non-tumor NF1+/- cells as a reference. Results: We identified drugs with sensitivity, targeting expected pathways, such as MAPK-ERK and PI3K-AKT, as well as serotonin-related targets, among others. The ΔS technique used here, in tandem with a supplemental ΔS web tool, simplifies HTS analysis and may provide a springboard for further investigations into drug response in NF1-related cancers. The tool may also prove useful for drug development in a variety of other cancers.
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The biocompatibility/inflammation profile of B2A-coated ceramic granules was evaluated using a panel of standard biocompatibility protocols (International Organization for Standardization-10993) including skin irritation and delayed-type hypersensitivity (Kligman maximization test), as well as acute, subacute, and chronic toxicity. Additionally, the potential of B2A-coated granules to elicit inflammatory reactions was also assessed using in vivo air pouch models, and B2A was evaluated using in vitro models of leukocyte recruitment and endothelial cell activation. Overall, the findings demonstrate that B2A-coated ceramic granules exhibit good biocompatibility profiles in the murine air pouch model and in standard subcutaneous implant models, and B2A did not demonstrate evidence of leukocyte recruitment or endothelial cell activation. These findings suggest that B2A and B2A-coated granules have little, if any, propensity to initiate inflammation reactions based on leukocyte recruitment. Thus, traditional biocompatibility and specially designed inflammation models indicate a high degree of biocompatibility and a low possibility of toxicity, inflammation, or edema following the implant of B2A-coated granules.
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Artrodesis , Cerámica , Ensayo de Materiales , Péptidos/síntesis química , Péptidos/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Inflamación/fisiopatología , Mediadores de Inflamación/metabolismo , Leucocitos/citología , Leucocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoprotegerina/farmacología , Prótesis e Implantes , Ligando RANK/metabolismoRESUMEN
B2A is a synthetic peptide that augments osteodifferentiation and improves bony fusion when delivered on ceramic granules during arthrodesis surgery. Nonclinical safety assessments, including genotoxicity, supported the use of B2A-coated granules as a combination medical device in pilot clinical studies. As a prelude to pivotal clinical studies, an assessment of the possibility that B2A-coated granules (and B2A) could enhance tumor growth was undertaken. B2A-coated granules demonstrated no evidence of genotoxicity. Cell culture studies with human tumor cell lines demonstrated that neither exposure of cells to B2A or B2A-coated granules increased cell proliferation or invasive capability relative to controls. In vivo, surgically implanted B2A-coated granules did not increase tumor growth (4 human tumor cell lines) or metastasis (1 cell line) relative to vehicle controls in immune-compromised rodents. Thus, traditional genotoxicity, as well as specially designed tumor growth enhancement studies, indicates that the possibility of tumor enhancement appears unlikely.
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Artrodesis , Cerámica , Sistemas de Liberación de Medicamentos/métodos , Neoplasias/patología , Péptidos/síntesis química , Péptidos/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular , Daño del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia/tratamiento farmacológico , Prótesis e Implantes , RatasRESUMEN
B2A (B2A2-K-NS) is a synthetic multi-domain peptide that in vitro augments bone morphogenetic protein (BMP)-2-induced cell responsiveness and osteodifferentiation. Augmentation of endogenous BMP-2 is thought to ultimately improve bone repair, and has led to clinical evaluation of B2A in orthopedic applications. In this study, we show that B2A binds to BMP receptor (BMPR)-IB, BMPR-II, and BMPR-IA. B2A reduces the EC50 of rh-BMP-2, thus shifting the response curve to the left. B2A enhances the osteogenic activity of BMP-2, but not growth and differentiation factor-5, BMP-7, or BMP-9, indicating its action is highly BMP-2 selective. Additionally, B2A did not augment Wnt-3a- and retinoic acid-induced differentiation. All three functional domains (receptor-binding domain, hydrophobic-linker domain, heparin-binding domain) of B2A are required for optimal bioactivity. Collectively, the results suggest that B2A, via its unique sequence, acts in a manner consistent with a positive receptor modulator to selectively enhance BMP-2 osteodifferentiation, and yet in the absence of BMP-2, B2A is without cooperative effect.
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Proteína Morfogenética Ósea 2/metabolismo , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Osteoblastos/citología , Péptidos/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Péptidos/genéticaRESUMEN
This study investigated whether the synthetic peptide B2A (B2A2-K-NS) could induce in vitro chondrogenic differentiation and enhance the in vivo repair of damaged cartilage in an osteoarthritis model. In vitro, micromass cultures of murine and human stem cells with and without B2A were used as models of chondrogenic differentiation. Micromasses were evaluated for gene expression using microarray analysis and quantitative PCR; and for extracellular matrix production by Alcian blue staining for sulfated glycosaminoglycan and immunochemical detection of collagen type II. In vivo, osteoarthritis was chemically induced in knees of adult rats by an injection of mono-iodoacetate (MIA) into the synovial space. Treatment was administered at 7- and 14 days after the MIA by injection into the synovial space of B2A or saline and terminated at 21 days, after which knee cartilage damage was determined and scored by histological analysis. In murine C3H10T1/2 micromass culture, B2A induced the expression of more than 11 genes associated with growth factors/receptors, transcription, and the extracellular matrix, including PDGF-AA. B2A also significantly increased the sulfated glycosaminoglycan and collagen of murine- and human micromass cultures. In the knee osteoarthritis model, B2A treatment enhanced cartilage repair compared to untreated knees as determined histologically by a decrease in damage indicators. These findings suggest that B2A induces stem cells chondrogenic differentiation in vitro and enhances cartilage repair in vivo. The results suggest that B2A might be useful to promote cartilage repair.
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Diferenciación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Osteoartritis de la Rodilla/terapia , Péptidos/farmacología , Animales , Cartílago/efectos de los fármacos , Cartílago/fisiología , Línea Celular , Condrogénesis/genética , Colágeno Tipo II/biosíntesis , Matriz Extracelular/metabolismo , Glicosaminoglicanos/biosíntesis , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ácido Yodoacético , Masculino , Ratones , Osteoartritis de la Rodilla/inducido químicamente , Péptidos/uso terapéutico , Proyectos Piloto , RatasRESUMEN
A plasma polymerized tetramethylcyclo-tetrasiloxane (TMCTS) coating was deposited onto a metallic biomaterial, 316 stainless steel, to control the release rate of drugs, including daunomycin, rapamycin and NPC-15199 (N-(9-fluorenylmethoxy-carbonyl)-leucine), from the substrate surface. The plasma-state polymerized TMCTS thin film was deposited in a vacuum plasma reactor operated at a radio-frequency of 13.56 MHz, and was highly adhesive to the stainless steel, providing a smooth and hard coating layer for drugs coated on the substrate. To investigate the influence of plasma coating thickness on the drug diffusion profile, coatings were deposited at various time lengths from 20 s to 6 min, depending on the type of drug. Atomic force spectroscopy (AFM) was utilized to characterize coating thickness. Drug elution was measured using a spectrophotometer or high-performance liquid chromatography (HPLC) system. The experimental results indicate that plasma polymerized TMCTS can be used as an over-coating to control drug elution at the desired release rate. The drug-release rate was also found to be dependent on the molecular weight of the drug with plasma coating barrier on top of it. The in vitro cytotoxicity test result suggested that the TMCTS plasma coatings did not produce a cytotoxic response to mammalian cells. The non-cytotoxicity of TMCTS coating plus its high thrombo-resistance and biocompatibility are very beneficial to drug-eluting devices that contact blood.
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Materiales Biocompatibles Revestidos/química , Portadores de Fármacos/química , Gases em Plasma/química , Polimerizacion , Siloxanos/química , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/toxicidad , Preparaciones de Acción Retardada , Difusión , Ratones , PresiónRESUMEN
STUDY DESIGN: Using a low cell density, hypoxic, alginate-bead culture system, the effects of bone morphogenetic protein-2 (BMP-2) and synthetic peptide B2A on cell proliferation and extracellular matrix (ECM) synthesis were assessed at days 0, 3, 5, and 7, using nucleus pulposus (NP)-like differentiated mesenchymal stem cells (MSCs). OBJECTIVE: This is a preliminary investigation into B2A's potential adjunctive role with MSCs and BMP-2, in NP regeneration. SUMMARY OF BACKGROUND DATA: B2A analogs, alone and in combination with BMP-2, have been shown to promote proliferation and ECM production in chondrocytes and MSCs. Articular chondrocytes and NP cells often respond in a similar manner to growth factor treatments, thus suggesting a potential role for B2A in treating disc degeneration by NP regeneration. METHODS: Using the NP regeneration in vitro model (low cell density, hypoxic, alginate bead culture), B2A and BMP-2 were evaluated alone and in combination, to determine effects on proliferation and ECM synthesis in the presence of transforming growth factor-beta 3 on NP-like differentiated MSCs. RESULTS: B2A administration induced mild proliferation of NP-like differentiated MSCs and diminished an initial wave of low-dose BMP-2-prompted apoptosis. Individually and in combination, B2A and BMP-2 were found to inhibit transforming growth factor-beta 3-permitted collagen accumulation; levels remained similar in their presence. Both collagen I (Col I) and collagen II (Col II) were found in almost all specimens, but increased B2A levels favored Col II unlike BMP-2, which favored Col I. BMP-2 resulted in a minor reduction in aggrecan synthesis, which was unchanged by B2A. CONCLUSION: Using this in vitro model, B2A induced proliferation, continuous aggrecan synthesis, and stabilized collagen accumulation favoring Col II. These characteristics are consistent with cells of the young, healthy NP, indicating potential use of the peptide early in an MSC-based NP-regeneration therapy; whereas, BMP-2 induced apoptosis, Col I accumulation, and aggrecan production hindrance, and was found untherapeutic.
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Proteína Morfogenética Ósea 2/farmacología , Disco Intervertebral/citología , Células Madre Mesenquimatosas/citología , Péptidos/farmacología , Agrecanos/metabolismo , Alginatos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Hipoxia de la Célula , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/metabolismo , Disco Intervertebral/fisiología , Masculino , Células Madre Mesenquimatosas/metabolismo , Microesferas , Modelos Biológicos , Péptidos/síntesis química , Ratas , Ratas Wistar , Regeneración/efectos de los fármacos , Ingeniería de Tejidos/métodos , Factor de Crecimiento Transformador beta3/farmacologíaRESUMEN
OBJECT: New generations of devices for spinal interbody fusion are expected to arise from the combined use of bioactive peptides and porous implants. The purpose of this dose-ranging study was to evaluate the fusion characteristics of porous ceramic granules (CGs) coated with the bioactive peptide B2A2-K-NS (B2A) by using a model of instrumented lumbar interbody spinal fusion in sheep. METHODS: Instrumented spinal arthrodesis was performed in 40 operative sites in 20 adult sheep. In each animal, posterior instrumentation (pedicle screw and rod) and a polyetheretherketone cage were placed in 2 single-level procedures (L2-3 and L4-5). All cages were packed with graft material prior to implantation. The graft materials were prepared by mixing (1:1 vol/vol) CGs with or without a B2A coating and morselized autograft. Ceramic granules were coated with B2A at 50, 100, 300, and 600 microg/ml granules (50-B2A/CG, 100-B2A/CG, 300-B2A/CG, and 600-B2A/CG, respectively), resulting in 4 B2A-coated groups plus a control group (uncoated CGs). Graft material from each of these groups was implanted in 8 operative sites. Four months after arthrodesis, interbody fusion status was assessed with CT, and the interbody site was further evaluated with quantitative histomorphometry. RESULTS: All B2A/CG groups had higher CT-confirmed interbody fusion rates compared with those in controls (CGs only). Seven of 8 sites were fused in the 50-B2A/CG, 100-B2A/CG, and 300-B2A/CG groups, whereas 5 of 8 sites were fused in the group that had received uncoated CGs. New woven and lamellar bone spanned the fusion sites with excellent osseointegration. There was no heterotopic ossification or other untoward events attributed to the use of B2A/CG in any group. Each B2A/CG treatment produced more new bone than that in the CG group. CONCLUSIONS: Bioactive treatment with B2A effectively enhanced the fusion capacity of porous CGs. These findings suggest that B2A/CG may well represent a new generation of biomaterials for lumbar interbody fusion and indicate that additional studies are warranted.
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Trasplante Óseo/métodos , Cerámica , Vértebras Lumbares/cirugía , Proteínas/farmacología , Fusión Vertebral/métodos , Animales , Benzofenonas , Fenómenos Biomecánicos , Clavos Ortopédicos , Tornillos Óseos , Trasplante Óseo/instrumentación , Materiales Biocompatibles Revestidos , Cámaras de Difusión de Cultivos , Estudios de Factibilidad , Disco Intervertebral/diagnóstico por imagen , Disco Intervertebral/cirugía , Cetonas , Vértebras Lumbares/diagnóstico por imagen , Ensayo de Materiales , Modelos Animales , Polietilenglicoles , Polímeros , Prótesis e Implantes , Ovinos , Fusión Vertebral/instrumentación , Tomografía Computarizada por Rayos X , Trasplante AutólogoRESUMEN
A plasma cross-linking process was employed to improve the surface lubricity of different types of biomaterials, including stainless steel (SS), nitinol, polyethylene and nylon. To investigate the influence of monomers containing double bonds on top-layer cross-linking of poly(ethylene oxide) compound (PEOC), five different monomers, N-trimethylsilyl-allylamine (TMSAA), ethylene, propylene, allyl alcohol and ethane, were used in the study to produce a cross-linked coating layer on sample surfaces. Before the plasma cross-linking, samples underwent plasma treatment followed by wet chemical coating. The plasma treatment consists of plasma etching in NH(3)/O(2), Tetramethylcyclo-tetrasiloxane (TMCTS) coating and TMSAA grafting. The wet coating process includes dip-coating in a solution of poly(oxyethylene)-compound bis(1-hydroxy-benzotriazolyl carbonate) (HPEOC), then dip-coating in a solution of PEOC. By application of plasma processing, HPEOC and PEOC wet coating to sample surfaces, the lubricity was increased by 83% compared to clean samples. The plasmas of TMSAA, ethylene, propylene and allyl alcohol, all containing a C=C double bond, produced a cross-linking layer on the PEOC surface. Consequently the surface lubricity was improved by 20% to 37% in comparison to no cross-linking. The favorable condition for plasma cross-linking was found to be high power and long time. Ethane plasma also reduced the pulling force although it has no double bond in the molecular structure, which indicated a thin plasma coating from saturated hydrocarbons deposited on HPEOC or PEOC surfaces could also cause cross-linking and improve lubricity. It was found that the TMSAA cross-linking also worked on HPEOC and HEPOC/PEOC, even though the prior plasma coating process was skipped.
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Materiales Biocompatibles/química , Reactivos de Enlaces Cruzados/química , Metales/química , Polietilenglicoles/química , Polímeros/química , Aleaciones/química , Materiales Biocompatibles/síntesis química , Reactivos de Enlaces Cruzados/síntesis química , Ensayo de Materiales , Nylons/síntesis química , Nylons/química , Polietileno/síntesis química , Polietileno/química , Polietilenglicoles/síntesis química , Polímeros/síntesis química , Acero Inoxidable/química , Propiedades de Superficie , Factores de TiempoRESUMEN
A multi-domain peptide, PAB2-1c, was designed and synthesized as a bioactive mimic of PDGF. PBA2-1c bound to both alpha- and beta-PDGF receptors as determined by surface plasmon resonance (SPR). The equilibrium dissociation constant (Kd) of binding to alpha-PDGF receptors by PAB2-1c (1.7 x 10(-8) M) compared favorably rhPDGF-AA (1.34 x 10(-8) M). Binding to -PDGF receptor by PAB2-1c (2.2 x 10(-8) M) was less favorable than, that of recombinant human PDGFBB (1.59 x 10(-9) M). Interestingly, PBA2-1c bound to these two receptors with similar affinity suggesting that, PBA2-1c was not PDGF receptor selective. In a murine myoblast cell line C2C12, PBA2-1c increased the tyrosine phosphorylation on PDGF receptors and the phosphorylation of AKT and ERK1/2 in a concentration-related manner. PBA2-1c also stimulated an increase in cell proliferation, cell migration, and collagen gel contraction. In these cell-based assays, PAB2-1c was effective at 1 microg/ml or lesser. The results support the hypothesis that PBA2-1c is a mimetic of PDGF, although it has a more promiscuous receptor interaction.
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Factor de Crecimiento Derivado de Plaquetas/química , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/química , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/química , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cinética , Sistema de Señalización de MAP Quinasas , Ratones , Datos de Secuencia Molecular , Péptidos/química , Fosforilación , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
Recombinant fibroblast growth factors (FGFs) maintain the integrity of the gut epithelium and reduce mucosal injury in experimental inflammatory bowel disease (IBD). Chemically synthesized FGF mimetics could potentially extend the utility of FGFs by tailoring them for optimal bioactivity and oral administration, for example. Here, F2A4-K-NS (Fibratide), a synthetic FGF mimetic peptide, alleviated dextran sulfate sodium (DSS)-induced ulcerative colitis in mice when delivered systemically and, to a lesser extent, orally. Intraperitoneal injection of Fibratide (1 or 5 mg/kg/day) ameliorated DSS-induced ulcerative colitis, resulting in reduced weight loss, decreased colon wall thickening, and increased colon length. Fibratide also improved epithelial integrity by reducing histological-detectable crypt damage and inflammation. Orally administered Fibratide (1 mg/kg/day) was also effective in ameliorating symptoms with effects generally similar to those of intraperitoneal injection. In vitro studies were conducted to help clarify how Fibratide might act in vivo. Fibratide exhibited a modest enhancement of epithelial cell proliferation. On the other hand, Fibratide doubled the rate of epithelial cells migration and restitution in a cell culture model of wound repair. Collectively, the results indicate that Fibratide reduced the severity of experimental ulcerative colitis and may be potentially useful in the treatment of IBD.
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Colitis Ulcerosa/tratamiento farmacológico , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Administración Oral , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/patología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Factor 2 de Crecimiento de Fibroblastos/síntesis química , Inyecciones Intraperitoneales , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Sustitutos del Plasma/toxicidad , Proteínas Recombinantes/síntesis química , Resultado del TratamientoRESUMEN
These studies evaluated whether F2A4-K-NS, a peptide mimetic of FGF-2, could augment ectopic bone production following the subcutaneous implant of human demineralized bone matrix (DBM). DBM was formulated into a gel with and without F2A4-K-NS, and injected subcutaneously into athymic rats. After 28 days the resultant tissue was excised and fixed. The tissue was examined with soft X-rays and microcomputerized tomography (micro-CT), and by histological methods. Inclusion of F2A4-K-NS with DBM resulted in an increased mineral deposition as determined by soft X-ray and micro-CT analysis and von Kossa staining. DBM-containing tissues showed extensive mineralization compared to the carrier alone, which was poorly mineralized. The mineralization was qualitatively and quantitatively the most extensive in the samples containing F2A4-K-NS plus DBM. Additionally, the highest amount of von Kossa staining for calcium was observed in tissues from animals that had received DBM plus F2A4-K-NS. In these studies, 100 ng of peptide per 0.2 mL of injectable DBM gel generated the most optimal results. The synthetic peptide F2A4-K-NS augmented DBM-induced ectopic mineralization in athymic animals.
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Matriz Ósea/efectos de los fármacos , Sustitutos de Huesos , Calcificación Fisiológica/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Oligopéptidos/farmacología , Animales , Técnica de Desmineralización de Huesos , Matriz Ósea/diagnóstico por imagen , Matriz Ósea/trasplante , Trasplante Óseo , Calcificación Fisiológica/fisiología , Calcio/análisis , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Radiografía , Ratas , Ratas Desnudas , Coloración y EtiquetadoRESUMEN
UNLABELLED: A multidomain, synthetic peptide designated B2A2 synergizes the activity of BMP-2. B2A2 interacts with BMP receptor isoforms, potentiating the action of BMP-2 in activating alkaline phosphatase and triggering Smad and MAPK signaling. B2A2's design permits its delivery as a local surface coating as well as a soluble co-factor, thus broadening potential bioengineering applications. INTRODUCTION: BMP-2 induces osteogenic differentiation and accelerates bone repair. Although BMP-2 inhibitors have been discovered, no BMP-2 mimetics or enhancers that function in the physiological range have yet been found. Here we report that a synthetic peptide designated B2A2, consisting of (1) a BMP receptor-targeting sequence, (2) a hydrophobic spacer, and (3) a heparin-binding sequence, is a positive modulator of recombinant BMP-2. MATERIALS AND METHODS: Cultures of mesenchymal cell lines C2C12 and C3H10T1/2 were given B2A2, recombinant BMP-2, or both. Alkaline phosphatase (ALP) activity was assayed by conversion of paranitrophenol phosphate (PNPP). Signaling through Smad and MAP kinase pathways was monitored by Western blot. Receptor binding was assessed by incubating immobilized B2A2 with soluble recombinant receptor-Fc chimeras and detecting bound receptor by anti-Fc antibody ELISA. Surface coating of medical device materials was done by first dip-coating with silyl-heparin, followed by B2A2. RESULTS AND CONCLUSIONS: Treatment of cells with B2A2 alone marginally increased ALP activity. However, B2A2 plus BMP-2 resulted in 5- to 40-fold augmentation of ALP compared with BMP-2 alone in C3H10T1/2 or C2C12 cells, respectively. This synergistic enhancement was observed over a broad concentration range (4-1000 ng/ml BMP-2). B2A2 interacted directly with BMP receptor isoforms (preferentially to BMPR-Ib and ActivinR-II). In cells, B2A2 + BMP-2 led to a repression of MAP kinase and an increase of Smad activation, consistent with known activation pathways of BMP-2. B2A2 was ineffective when paired with other cytokine/growth factors (basic fibroblast growth factor [FGF-2], TGF-beta1, vascular endothelial growth factor [VEGF]). Simultaneous co-administration was not strictly required. Pulse-chase experiments revealed that temporal separations up to 1 h were still effective. B2A2 was also effective when delivered in a polystyrene- or stainless steel-coated surface through a heparin platform (silyl-heparin) while BMP-2 was added exogenously in solution. These results suggest that B2A2 might promote aggregation of receptor subunits, enabling BMP-2 to activate signaling pathways at effectively lower concentrations. Synthetic multidomain constructs like B2A2 may be useful to accelerate bone repair/deposition through augmentation of endogenous levels of BMP-2 or through local BMP-2 contained in artificial or engineered matrices.
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Proteínas Morfogenéticas Óseas/farmacología , Péptidos/farmacología , Factor de Crecimiento Transformador beta/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 2 , Células Cultivadas , Cricetinae , Regulación hacia Abajo , Sinergismo Farmacológico , Humanos , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Péptidos/química , Isoformas de Proteínas/farmacologíaRESUMEN
OBJECTIVES: Our purpose was to improve the performance of carbon-coated expanded polytetrafluoroethylene vascular grafts by bonding the grafts with silyl-heparin, a biologically active heparin analog, using polyethylene glycol as a cross-linking agent. Material and method Silyl-heparin-bonded carbon-coated expanded polytetrafluoroethylene vascular grafts (Bard Peripheral Vascular, Tempe, Ariz), were evaluated for patency and platelet deposition 2 hours, 7 days, and 30 days after graft implantation in a canine bilateral aortoiliac artery model. Platelet deposition was determined by injection of autologous, (111)Indium-radiolabeled platelets, followed by a 2-hour circulation period prior to graft explantation. Histologic studies were performed on a 2-mm longitudinal strip of each graft (7-day and 30-day groups). Heparin activity of the explanted silyl-heparin grafts was determined by using an antithrombin-III based thrombin binding assay. RESULTS: Overall chronic graft patency (7-day and 30-day groups) was 100% for the silyl-heparin bonded (16/16) grafts versus 68.75% for control (11/16) grafts (P =.043). Acute 2-hour graft patency was 100% for the silyl-heparin bonded (6/6) grafts versus 83.3% for control (5/6) grafts. Radiolabeled platelet deposition studies revealed a significantly lower amount of platelets deposited on the silyl-heparin grafts as compared with control grafts in the 30-day group (13.8 +/- 7.18 vs 28.4 +/- 9.73, CPM per cm2 per million platelets, mean +/- SD, P =.0451, Wilcoxon rank sum test). In the 2-hour group of dogs, a trend towards a lower deposition of platelets on the silyl-heparin grafts was observed. There was no significant difference in platelet deposition between the two grafts in the 7-day group. Histologic studies revealed a significant reduction in intraluminal graft thrombus present on the silyl-heparin grafts as compared with control grafts in the 30-day group of animals. In contrast, there was no difference in amount of graft thrombus present on both graft types in the 7-day group of dogs. Pre-implant heparin activity on the silyl-heparin bonded grafts was 2.0 IU/cm(2) (international units[IU]/cm(2)). Heparin activity remained present on the silyl-heparin grafts after explantation at all 3 time points (2 hours: above upper limit of assay, upper limit = 0.57, n = 6; 7 days: 0.106 +/- 0.015, n = 5; 30 days: 0.007 +/- 0.001, n = 5; mean +/- SD, IU/cm(2)). CONCLUSION: Silyl-heparin bonding onto carbon-coated expanded polytetrafluoroethylene vascular grafts resulted in (1) improved graft patency, (2) increased in vivo graft thromboresistance, and (3) a significant reduction in intraluminal graft thrombus. This graft may prove to be useful in the clinical setting.
Asunto(s)
Prótesis Vascular , Carbono , Materiales Biocompatibles Revestidos , Oclusión de Injerto Vascular/prevención & control , Heparina/análogos & derivados , Politetrafluoroetileno , Trombosis/prevención & control , Animales , Anticoagulantes/farmacología , Implantación de Prótesis Vascular , Perros , Oclusión de Injerto Vascular/etiología , Heparina/farmacología , Adhesividad Plaquetaria , Trombosis/etiología , Grado de Desobstrucción VascularRESUMEN
The plasma generated from a gas mixture of NH3 plus O2 (NH3 + O2) has been used to impart unique chemical and biological characteristics to polytetrafluoroethylene (PTFE). PTFE treated with NH3 + O2 plasma was physiochemically distinct from surfaces treated with plasma of either NH3 or O2 alone, as determined by electron spectroscopy for chemical analysis (ESCA). The contact angle analysis revealed that the PTFE surfaces became less hydrophobic after plasma treatments. ESCA results indicate the presence of oxygen-containing groups and nitrogen-containing groups at the plasma-treated surfaces. PTFE treated with NH3 + O2 plasma resisted the attachment of platelets and leukocytes in a manner similar to untreated PTFE; however, the attachment of bovine aorta endothelial cells was substantially increased. Once attached, these cells grew to confluency. The increased endothelial cell attachment was higher than that observed following plasma treatment with each gas used separately, which could be attributed to the considerable amount of CF(OR)2-CF2 formed on the NH3 + O2 plasma-treated PTFE surface. At 14 days after subcutaneous implantation in rats, the PTFE wafers treated with NH3 + O2 plasma demonstrated less encapsulation and lower levels of inflammatory cells compared to controls. Collectively, the results suggest that NH3 + O2 plasma treatment imparts a unique character to PTFE and could be useful in certain in vivo applications.
Asunto(s)
Amoníaco/química , Materiales Biocompatibles/química , Plaquetas/citología , Células Endoteliales/citología , Leucocitos/citología , Oxígeno/química , Politetrafluoroetileno/química , Implantes Absorbibles , Adsorción , Animales , Proteínas Sanguíneas/química , Bovinos , Adhesión Celular , División Celular , Línea Celular , Humanos , Ratas , Análisis Espectral , Propiedades de SuperficieRESUMEN
Stainless steel treated with a mixed gas plasma of NH(3) plus O(2) had chemical and biologic characteristics distinct from untreated stainless steel or stainless steel treated with NH(3) or O(2) plasmas used separately. NH(3)/O(2) plasmas deposited nitrogen as both -CN (organic) and -NO (nitrate, nitrite)--materials not found on untreated stainless steel--and the contact angle changed from 44 degrees to 23 degrees. Treatment of stainless steel (and titanium) resulted in surfaces with enhanced resistance to platelet and leukocyte attachment. A gas plasma of N(2)O/O(2) also was found to reduce platelet and leukocyte attachment, suggesting that these properties may be common to surfaces coated with oxynitrites (nitrides). Upon subcutaneous implantation, no inflammation, hemolysis, or untoward thrombosis was noted in the tissue surrounding the wafers treated with the NH(3)/O(2) plasmas, although the cellular density was considerably reduced by 2 weeks after implant. Collectively, the results suggest that NH(3)/O(2) plasmas impart a unique character to stainless steel that may be useful in the construction of medical devices.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Implantes Experimentales , Acero Inoxidable/química , Amoníaco/química , Animales , Células Sanguíneas/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Materiales Biocompatibles Revestidos/química , Gases , Humanos , Oxígeno/química , Ratas , Ratas Endogámicas F344RESUMEN
A growth factor delivery system was developed that is based on the use of silyl-heparin, a chemically modified analogue of heparin. The silyl-heparin was adsorbed onto surfaces by hydrophobic interaction via the prosthetic unit and can then be used as a solid-phase adsorbent for bFGF. All the coating steps were performed by adsorption, a process that allowed preparation of surfaces by immersion or "dip-coating". In this study a series of silyl-heparins were synthesized and each of the analogues found to function similar to unmodified heparin relative to their binding of antithrombin III and also the binding of bFGF. The silyl-heparins were found to be adsorbed onto a wide variety of substrates including polystyrene and lactide:glycolide copolymer. Enzyme-linked immumosorbant assay (ELISA) was used to establish that bFGF was readily bound to surface adsorbed silyl-heparin, and that the amount bound was directly related to amount offered for binding. Once adsorbed the silyl-heparin/FGF was able to induce capillary tube formation of endothelial cells and to increase the growth of endothelial cells. When coated onto suture material and implanted in muscle, the FGF/silyl-heparin coating caused an increased density of mesenchymal cells in the area of the implant. This coating method could prove to be useful in a number of tissue engineering applications for the local delivery of FGF and other growth factors.