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1.
Pharm Res ; 36(12): 184, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748894

RESUMO

PURPOSE: There is a plethora of studies on recombinant human bone morphogenetic protein-2 (rhBMP-2) application and delivery systems, but surprisingly few reports address the biophysical properties of the protein which are of crucial importance to develop effective delivery systems or to solve general problems related to rhBMP-2 production, purification, analysis and application. METHODS: The solubility, stability and bioactivity of rhBMP-2 obtained by renaturation of E. coli derived inclusion bodies was assessed at different pH and in different buffer systems using (dynamic) light scattering and thermal shift assays as well as intrinsic fluorescence measurements and luciferase based bioassays. RESULTS: rhBMP-2 is poorly soluble at physiological pH and higher. The presence of divalent anions further decreases the solubility even under acidic conditions. Thermal stability analyses revealed that rhBMP-2 precipitates are more stable compared to the soluble protein. Moreover, correctly folded rhBMP-2 is also bioactive as precipitated protein and precipitates readily dissolve under appropriate buffer conditions. Once properly formed rhBMP-2 also retains biological activity after temporary exposure to high concentrations of chaotropic denaturants. However, care should be taken to discriminate bioactive rhBMP-2 precipitates from misfolded rhBMP-2 aggregates, e.g. resolvability in MES buffer (pH 5) and a discrete peak in thermoshift experiments are mandatory for correctly folded rhBMP-2. CONCLUSIONS: Our analysis revealed that E. coli derived rhBMP-2 precipitates are not only bioactive but are also more stable compared to the soluble dimeric molecules. Knowledge about these unusual properties will be helpful to design improved delivery systems requiring lower amounts of rhBMP-2 in clinical applications.


Assuntos
Proteína Morfogenética Óssea 2/química , Escherichia coli/química , Fator de Crescimento Transformador beta/química , Heparina/química , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Oxalatos/química , Tamanho da Partícula , Agregados Proteicos/efeitos dos fármacos , Conformação Proteica , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Cloreto de Sódio/química , Solubilidade/efeitos dos fármacos , Temperatura Ambiente
2.
J Photochem Photobiol B ; 198: 111563, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31369959

RESUMO

Bone Morphogenetic Protein 2 (BMP-2) is an osteoinductive protein which has been overexpressed, refolded (Refolding is often the bottle-neck step in producing recombinant proteins from inclusion bodies of Escherichia coli, especially for dimer proteins) and purified by using Heparin affinity chromatography. Refolding of BMP-2 was based on gradient dialysis in presence of lower urea concentration. The main objective of the present work is to unravel the impact of the extracellular matrix components on BMP-2 conformation and stability. We tried to elucidate the interaction of the bone matrix minerals in the form of nanoparticles with the bone protein. We chose hydroxyapatite nanoparticles (HAp NPs) which is the most abundant bone mineral and the other being a trace mineral in our bones i.e., zinc oxide nanoparticles (ZnO NPs) as the potential nanoparticles for this study. The isolated protein is found to be a ß- sheet type with melting temperature being approximately 70.66 °C. Upon interaction with HAp NPs and ZnO NPs, the absorbance and the fluorescence intensity indicates the interaction with the protein as there was an upsurge in both the cases. Circular Dichroism (CD) spectroscopy revealed that ZnO NPs are having more dominant secondary structure and thermal stabilizing effect as compared to HAp NPs.


Assuntos
Proteína Morfogenética Óssea 2/química , Durapatita/química , Nanopartículas/química , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Dicroísmo Circular , Humanos , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Fluorescência , Temperatura Ambiente , Óxido de Zinco/química
3.
Biomater Sci ; 7(9): 3614-3626, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31210206

RESUMO

Injectable scaffolds have attracted much attention because of their minimum surgical invasiveness. However, limited osteogenic induction property and low mechanical properties hampered their application in bone tissue engineering. CaCO3 microspheres, which possess osteoinductivity, rough surfaces and specific binding sites for BMP-2, were first fabricated; after BMP-2 uploading, microspheres were further entrapped in fibrin-glue hydrogel. CaCO3 microspheres were co-functionalized with casein and heparin. To obtain a high encapsulation of heparin and thus BMP-2 uploading, along with controlled release and simultaneous maintenance of the presence of vaterite which had osteogenic induction property, fabrication parameters were optimized and microspheres were characterized using XRD, FITR and SEM. The formed CaCO3 had a microsphere morphology of ∼1 µm. Both vaterite and calcite phases were present and the relative amount of calcite phase increased with the amount of heparin. Sample 25 mM_4-1Hep with the highest loading amount of heparin was selected as carrier for BMP-2 and BMP-2 loaded CaCO3 microspheres were further entrapped in fibrin-glue hydrogel (FC-B). For the as-prepared composite hydrogel, mechanical properties were characterized and the presence of CaCO3 significantly elevated the tensile strength; controlled release of BMP-2 was sustained until day 21. Based on ALP activity, alizarin red staining and RT-PCR, in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was found to be significantly enhanced under induction of FC-B. Rabbit tibia bone defect model was applied to evaluate its in vivo performance. After implantation for 4 weeks, presence of composite hydrogel was observed in defects. After 8 weeks, bone defects of FC-B group were nearly completely healed. Using the fact that autologous scaffolds can be derived based on fibrin-glue hydrogel, the well-designed BMP-2 loaded fibrin-glue composite hydrogel demonstrated good potential in bone tissue engineering.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Cálcio/farmacologia , Hidrogéis/química , Microesferas , Osteogênese/efeitos dos fármacos , Tíbia/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 2/química , Cálcio/química , Carbonato de Cálcio/síntese química , Carbonato de Cálcio/química , Diferenciação Celular , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Adesivo Tecidual de Fibrina/síntese química , Adesivo Tecidual de Fibrina/química , Hidrogéis/síntese química , Coelhos , Tíbia/patologia , Engenharia Tecidual
4.
PLoS One ; 14(5): e0217766, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150531

RESUMO

Nonautologous bone grafts have limited osteoinductive potential and thus there is substantial interest in reconstituting these graft materials with osteogenic factors such as bone morphogenic protein 2 (BMP2). However, one limitation of this approach is that BMP2 is typically weakly bound to the graft, which can lead to side effects associated with BMP2 dissemination. In the current study we added a hydroxyapatite (HA)-binding domain onto BMP2 to increase coupling to the graft surface. A sequence consisting of eight glutamate residues (E8) was inserted into the C-terminus of BMP2, and the recombinant protein (rBMP2-E8) was expressed in E. coli. Compared with rBMP2, rBMP2-E8 displayed markedly enhanced binding to HA disks and was better retained on the disks following exposure to vigorous wash steps. Furthermore, rBMP2-E8 was purified using a heparin column, and evaluated for its capacity to stimulate osteoblastic cell signaling. Treatment of SAOS2 cells with rBMP2-E8 induced SMAD 1/5 activation, confirming that the protein retains activity. Collectively these results suggest that the E8 domain serves as an effective tool for improving rBMP2 coupling to graft materials. The increased retention of rBMP2-E8 on the graft surface is expected to prolong BMP2's osteoinductive activity within the graft site, while simultaneously reducing off-target effects.


Assuntos
Proteína Morfogenética Óssea 2/química , Durapatita/química , Ácido Glutâmico/genética , Osteogênese/genética , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/farmacologia , Transplante Ósseo/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Ácido Glutâmico/química , Humanos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia
5.
Int J Biol Macromol ; 135: 1123-1133, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31173837

RESUMO

Impact of different monovalent and divalent cationic salts of sulphates and phosphates on conformation and stability of BMP - 2 was unraveled by absorbance, fluorescence and circular dichroism (CD) spectroscopy. Increase in absorbance of protein confirms the ground-state complexation between salt and BMP - 2. Phosphate salts, with the exception of sodium phosphate quenched the fluorescence intensity. The nature of quenching was static, as revealed by temperature-dependent fluorescence studies (Stern-Volmer constant (KSV) decreased with rise in temperature). Moreover, kq (bimolecular quenching constant) was in the range of 1012 M-1 s-1, confirming binding of phosphate salts with the protein. Contrary to this, sulphate salts increased the fluorescence intensity and excited-state lifetime of BMP - 2 (2.668 ns), with the maximum calculated for 300 mM sodium sulphate (3.216 ns). Phosphates reduced the lifetime of protein, with the least observed in presence of 300 mM magnesium phosphate (1.480 ns). Thermal stability of the protein (Tm = 70.66 °C) was altered significantly upon interaction with phosphate salts; however, it did not vary significantly in case of sulphates (exception - magnesium sulphate). Experimental evidences confirm the role played by anionic group on protein conformation and stability and identifies monovalent and divalent cations as insignificant contributor.


Assuntos
Proteína Morfogenética Óssea 2/química , Fosfatos/química , Conformação Proteica , Sulfatos/química , Estabilidade Proteica , Espectrometria de Fluorescência , Análise Espectral , Termodinâmica
6.
J Mater Sci Mater Med ; 30(7): 78, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31222566

RESUMO

3D printing is a versatile technique widely applied in tissue engineering due to its ability to manufacture large quantities of scaffolds or constructs with various desired architectures. In this study, we demonstrated that poly (lactic acid) (PLA) scaffolds fabricated via fused deposition not only retained the original interconnected microporous architectures, the scaffolds also exhibited lower lactic acid dissolution as compared to the freeze-PLA scaffold. The 3D-printed scaffolds were then grafted with human bone morphogenetic protein-2 (BMP-2) via the actions of polydopamine (PDA) coatings. The loading and release rate of BMP-2 were monitored for a period of 35 days. Cellular behaviors and osteogenic activities of co-cultured human mesenchymal stem cells (hMSCs) were assessed to determine for efficacies of scaffolds. In addition, we demonstrated that our fabricated scaffolds were homogenously coated with PDA and well grafted with BMP-2 (219.1 ± 20.4 ng) when treated with 250 ng/mL of BMP-2 and 741.4 ± 127.3 ng when treated with 1000 ng/mL of BMP-2. This grafting enables BMP-2 to be released in a sustained profile. From the osteogenic assay, it was shown that the ALP activity and osteocalcin of hMSCs cultured on BMP-2/PDA/PLA were significantly higher when compared with PLA and PDA/PLA scaffolds. The methodology of PDA coating employed in this study can be used as a simple model to immobilize multiple growth factors onto different 3D-printed scaffold substrates. Therefore, there is potential for generation of scaffolds with different unique modifications with different capabilities in regulating physiochemical and biological properties for future applications in bone tissue engineering.


Assuntos
Bivalves , Proteína Morfogenética Óssea 2/química , Osteogênese , Poliésteres/química , Impressão Tridimensional , Tecidos Suporte/química , Animais , Regeneração Óssea , Linhagem Celular , Técnicas de Cocultura , Humanos , Concentração de Íons de Hidrogênio , Indóis/química , Ácido Láctico/química , Células-Tronco Mesenquimais/citologia , Polímeros/química , Porosidade , Engenharia Tecidual/métodos
7.
Mater Sci Eng C Mater Biol Appl ; 101: 148-158, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31029307

RESUMO

Recent studies suggest that nanotopography can trigger colocalization of integrins and bone morphogenetic protein 2 (BMP2) receptors (e.g., BMPR1A), thereby leading to osteogenesis. In this study, the bone marrow homing peptide 1 (BMHP1) motif was bound to a self-assembling peptide core to form a hydrogel-based nanofiber (R-BMHP1). The docking and molecular dynamic study revealed that the R-BMHP1 sequence induced a stronger electrostatic interaction than BMP2 through arginines in the RADA core sequence and through lysine24 in the BMHP1 motif with BMPR1A. Notably, decrease of polar solvation binding energy will enhance the total binding energy and increases bone regeneration even more than BMP2 The enhanced osteogenesis and bone repair potential of R-BMHP1 nanofiber might be related to its chemical interaction with BMPR1A, which triggered downstream signal transduction through osteogenic genes overexpression in osteo-differentiated mesenchymal stem cells (MSCs), as well as implanted critical-sized bone defects in rats. Following that, calcium deposition occurred by osteoblast-like cells, ALP activity increased in osteodifferentiation MSCs and rat serum, and calcium density improved in bone defects (X-ray). The nanofiber was biocompatible and enhanced the cell viability of MSCs, without multinuclear cell infiltration into the defect site. Taking everything into account, not only does nanotopography induce osteogenesis through colocalization of BMPRs and integrins, but also R-BMHP1 nanofibers (considering their chemical structure) induce cell proliferation, osteogenesis, and bone repair through strong electrostatic interaction with BMPR1A and downstream signaling. The entire outcome of this study manifests the plausibility of R-BMHP1 for spine and spinal cord injury repair.


Assuntos
Proteína Morfogenética Óssea 2/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/química , Nanofibras/química , Peptídeos/química , Eletricidade Estática , Fosfatase Alcalina/metabolismo , Motivos de Aminoácidos , Animais , Biomarcadores/metabolismo , Osso e Ossos/patologia , Membrana Celular/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Óxido Nítrico/biossíntese , Osteogênese , Ratos , Tecidos Suporte/química
8.
Biomed Res Int ; 2019: 5697250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31032352

RESUMO

Although titanium (Ti) alloys have been widely used as implant materials, the bioinertness of pristine Ti impairs their bioactivity and early osseointegration. In the present work, we prepared TiO2 nanotubes (TNT) layer on the titanium (Ti) surface by anodic oxidation. The anodized surface was functionalized with human bone morphogenetic protein-2 coating to form the hBMP-2/TNT surface. The release behavior of hBMP-2 on the hBMP-2/TNT surface displayed a controlled and sustained pattern, compared to that on the hBMP-2/Ti surface, which showed a rapid release. In vitro cellular activity tests demonstrated that both TNT and hBMP-2/Ti surfaces, particularly the hBMP-2/TNT surface, enhanced adhesion, proliferation, and differentiation of osteoblast cells. Increased cell adhesion, improved cytoskeleton organization, and immunofluorescence staining of vinculin were observed on the modified surfaces. The TNT, hBMP-2/Ti, and hBMP-2/TNT surfaces, especially the hBMP-2/TNT surface, further displayed an upregulated gene expression of adhesion and osteogenic markers vinculin, collagen type 1, osteopontin, and osteocalcin, compared to the pristine Ti surface. In vivo experiments using a rat model demonstrated that the TNT and hBMP-2/Ti surfaces, in particular the hBMP-2/TNT surface, improved osseointegration and showed a superior bone bonding ability compared to Ti. Our study revealed a synergistic role played by TiO2 nanotubes nanotopography and hBMP-2 in promoting initial osteoblast adhesion, proliferation, differentiation, and osseointegration, thus suggesting a promising method for better modifying the implant surface.


Assuntos
Proteína Morfogenética Óssea 2/administração & dosagem , Diferenciação Celular/efeitos dos fármacos , Osseointegração/efeitos dos fármacos , Titânio/administração & dosagem , Animais , Proteína Morfogenética Óssea 2/química , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Nanotubos/química , Osteoblastos/efeitos dos fármacos , Próteses e Implantes , Ratos , Propriedades de Superfície , Titânio/química
9.
Artif Cells Nanomed Biotechnol ; 47(1): 1662-1673, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31032645

RESUMO

The purpose of this research is to assess the feasibility of poly(lactic-co-glycolic) acid (PLGA) incorporating gelatin microspheres (PLGA/GMs scaffold) for enhancing osteogenesis in vitro and at a radius defect of rabbits after X-ray radiation in vivo. After incorporating gelatin microspheres, PLGA scaffold demonstrated improved mechanical properties. Moreover, a sustained release property of recombinant human bone morphogenetic protein-2 (BMP-2) was achieved in BMP-2-releasing PLGA/GMs scaffold. BMP-2-releasing PLGA/GMs scaffold also enhanced proliferation and osteogenesis of rabbit bone mesenchymal stem cells (BMSCs) in vitro, indicating the bioactivity of BMP-2. After finishing X-ray radiation of the radius bone, 20-mm radius bone defects were generated, followed by being implanted with BMP-2-releasing PLGA/GMs scaffolds with or without bone marrow. Both PLGA/GMs scaffolds containing bone marrow or BMP-2 showed more obvious enhancement for bone regeneration than the empty scaffolds (control) at the radius defect. In the X-ray radiated groups, however, the bone regeneration was inhibited either with bone marrow or BMP-2. When combined with bone marrow, the BMP-2 showed significantly high osteogenic effect, regardless of X-ray radiation. It is considered that it is a promising way to repair bone defects even after X-ray radiation by a combination of bone marrow with the BMP-2-releasing PLGA/GMs scaffold.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Portadores de Fármacos/química , Gelatina/química , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Rádio (Anatomia)/efeitos dos fármacos , Tecidos Suporte/química , Animais , Proteína Morfogenética Óssea 2/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cinética , Masculino , Coelhos , Rádio (Anatomia)/patologia , Rádio (Anatomia)/efeitos da radiação , Raios X/efeitos adversos
10.
Carbohydr Polym ; 213: 27-38, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30879669

RESUMO

Physical properties of scaffolds such as nanofibers and aligned structures have been reported to exert profound effects on the growth and differentiation of stem cells due to their homing-effect features and contact guidance. However, the biological function of aligned nanofibers utilized as bone-scaffold has not been rigorously characterized. In the present study, aligned electrospun cellulose/CNCs nanocomposite nanofibers (ECCNNs) loaded with bone morphogenic protein-2 (BMP-2) were used for the first time to investigate (1) in vitro osteogenic differentiation of human mesenchymal stem cells (BMSCs) and (2) in vivo collagen assembly direction and cortical bone regeneration. Aligned ECCNNs scaffolds loaded with BMP-2 possess good biological compatibility. The growth orientation of BMSCs followed the underlying aligned nanofiber morphology, accompanied with increased alizarin red stain, alkaline phosphatase (ALP) activity and calcium content in vitro while, a rabbit calvaria bone defect model was used in an in vivo study with micro CT and histology analyses.


Assuntos
Proteína Morfogenética Óssea 2/química , Celulose/química , Engenharia Tecidual , Fator de Crescimento Transformador beta/química , Animais , Regeneração Óssea , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Coelhos , Proteínas Recombinantes/química
11.
Int J Pharm ; 562: 151-161, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30853482

RESUMO

Bone tissue engineering is an emerging medical field that has been developed in recent years to address pathologies with limited ability of bones to regenerate. Here we report the fabrication and characterization of microbial transglutaminase crosslinked gelatin-based scaffolds designed for serving as both cell substrate and growth factor release system. In particular, morphological, biomechanical and biological features have been analyzed. The enzyme ratio applied during the fabrication of the scaffolds affects the swelling capacity and the mechanical properties of the final structure. The developed systems are not cytotoxic according to the biocompatibility tests. The biological performance of selected formulations was studied using L-929 fibroblasts, D1 MSC and MG63 osteoblasts. Moreover, scaffolds allowed efficient osteogenic differentiation and signaling of MSCs. MSC cultured on the scaffolds not only presented lower proliferative and stemness profile, but also increased expression of osteoblast-related genes (Col1a1, Runx2, Osx). Furthermore, the in vitro release kinetics of vascular endothelial growth factor (VEGF) and bone morphogenetic protein -2 (BMP-2) from the scaffolds were also investigated. The release of the growth factors produced from the scaffolds followed a first order kinetics. These results highlight that the scaffolds designed and developed in this work may be suitable candidates for bone tissue regeneration purposes.


Assuntos
Gelatina/química , Tecidos Suporte , Transglutaminases/química , Animais , Proteína Morfogenética Óssea 2/química , Osso e Ossos , Linhagem Celular , Liberação Controlada de Fármacos , Humanos , Camundongos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Fator de Transcrição Sp7/genética , Engenharia Tecidual , Fator A de Crescimento do Endotélio Vascular/química
12.
Adv Mater ; 31(17): e1900291, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30844123

RESUMO

The controlled fabrication of gradient materials is becoming increasingly important as the next generation of tissue engineering seeks to produce inhomogeneous constructs with physiological complexity. Current strategies for fabricating gradient materials can require highly specialized materials or equipment and cannot be generally applied to the wide range of systems used for tissue engineering. Here, the fundamental physical principle of buoyancy is exploited as a generalized approach for generating materials bearing well-defined compositional, mechanical, or biochemical gradients. Gradient formation is demonstrated across a range of different materials (e.g., polymers and hydrogels) and cargos (e.g., liposomes, nanoparticles, extracellular vesicles, macromolecules, and small molecules). As well as providing versatility, this buoyancy-driven gradient approach also offers speed (<1 min) and simplicity (a single injection) using standard laboratory apparatus. Moreover, this technique is readily applied to a major target in complex tissue engineering: the osteochondral interface. A bone morphogenetic protein 2 gradient, presented across a gelatin methacryloyl hydrogel laden with human mesenchymal stem cells, is used to locally stimulate osteogenesis and mineralization in order to produce integrated osteochondral tissue constructs. The versatility and accessibility of this fabrication platform should ensure widespread applicability and provide opportunities to generate other gradient materials or interfacial tissues.


Assuntos
Materiais Biocompatíveis/química , Proteína Morfogenética Óssea 2/química , Nanocompostos/química , Fenômenos Físicos , Tecidos Suporte/química , Células Cultivadas/química , Reagentes para Ligações Cruzadas/química , Gelatina/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células-Tronco Mesenquimais , Metacrilatos/química , Osteogênese , Propriedades de Superfície , Engenharia Tecidual/métodos
13.
Int J Nanomedicine ; 14: 733-751, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30705589

RESUMO

Introduction: Bone tissue engineering has become one of the most effective methods to treat bone defects. Silk fibroin (SF) is a natural protein with no physiological activities, which has features such as good biocompatibility and easy processing and causes minimal inflammatory reactions in the body. Scaffolds prepared by electrospinning SF can be used in bone tissue regeneration and repair. Graphene oxide (GO) is rich in functional groups, has good biocompatibility, and promotes osteogenic differentiation of stem cells, while bone morphogenetic protein-2 (BMP-2) polypeptide has an advantage in promoting osteogenesis induction. In this study, we attempted to graft BMP-2 polypeptide onto GO and then bonded the functionalized GO onto SF electrospun scaffolds through electrostatic interactions. The main purpose of this study was to further improve the biocompatibility of SF electrospun scaffolds, which could promote the osteogenic differentiation of bone marrow mesenchymal stem cells and the repair of bone tissue defects. Materials and methods: The successful synthesis of GO and functionalized GO was confirmed by transmission electron microscope, X-ray photoelectron spectroscopy, and thermogravimetric analysis. Scanning electron microscopy, atomic force microscopy, mechanical test, and degradation experiment confirmed the preparation of SF electrospun scaffolds and the immobilization of GO on the fibers. In vitro experiment was used to verify the biocompatibility of the composite scaffolds, and in vivo experiment was used to prove the repairing ability of the composite scaffolds for bone defects. Results: We successfully fabricated the composite scaffolds, which enhanced biocompatibility, not only promoting cell adhesion and proliferation but also greatly enhancing in vitro osteogenic differentiation of bone marrow stromal cells using either an osteogenic or non-osteogenic medium. Furthermore, transplantation of the composite scaffolds significantly promoted in vivo bone formation in critical-sized calvarial bone defects. Conclusion: These findings suggested that the incorporation of BMP-2 polypeptide-functionalized GO into chitosan-coated SF electrospun scaffolds was a viable strategy for fabricating excellent scaffolds that enhance the regeneration of bone defects.


Assuntos
Proteína Morfogenética Óssea 2/química , Regeneração Óssea/efeitos dos fármacos , Fibroínas/farmacologia , Grafite/química , Óxidos/química , Peptídeos/química , Tecidos Suporte/química , Animais , Osso e Ossos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Eletricidade , Fibroínas/química , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ratos , Engenharia Tecidual
14.
Colloids Surf B Biointerfaces ; 177: 242-252, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30763789

RESUMO

Bacterial infection and surface biointertness are two main causes for titanium (Ti)-based orthopedic implants failure. To improve the antibacterial ability and enhance poor osteogenic property of Ti substrates, in this work, we employed titania nanotubes (TNTs) as Bone Morphogenetic Protein 2 (BMP2) carrier, and a pH-responsive multilayer film composed of alginate dialdehyde-gentamicin (ADA-Gen) and chitosan (Chi) was constructed onto BMP2-loaded TNTs substrates through layer-by-layer (LBL) assembly technique, resulting in TNT-BMP2-LBLg samples. The release experiments revealed that acidic environment could trigger the release of Gen from the multilayer films and in turn accelerate the release of BMP2 from TNTs. Moreover, antibacterial assay against E. coli and S. aureus confirmed that the TNT-BMP2-LBLg had excellent antibacterial capacity both in early (6 h) and in long-term (72 h). Meanwhile, in vitro cellular tests demonstrated that TNT-BMP2-LBLg had good cytocompatibility toward osteoblasts even co-cultured with S. aureus. Importantly, the obtained TNT-BMP2-LBLg promoted differentiation of osteoblasts, including enhanced alkaline phosphatase activity, improved mineralization capability and stimulated osteogenic-relative gene expression. This study thus provides a promising strategy to develop pH-responsive antibacterial and enhance bone integrative Ti-based implants for potential orthopedic application.


Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Proteína Morfogenética Óssea 2/metabolismo , Nanotubos/química , Osteogênese/efeitos dos fármacos , Titânio/farmacologia , Antibacterianos/química , Proteína Morfogenética Óssea 2/química , Configuração de Carboidratos , Diferenciação Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Tamanho da Partícula , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Titânio/química
15.
Mater Sci Eng C Mater Biol Appl ; 97: 602-612, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30678947

RESUMO

Biocompatible scaffolds have been used to promote cellular growth and proliferation in order to develop grafts, prostheses, artificial skins and cartilage. Electrospinning is widely studied as a method capable of producing nanofibers which enables cell attachment and proliferation, generating a functional scaffold that is suitable for many types of organs or tissues. In this study, electrospinning was used to obtain core-shell and monolithic fibers from the biocompatible poly (lactic acid) and poly (vinyl alcohol) polymers. The main purpose of this work is to produce core-shell nanofiber based scaffolds that works as a sustained delivery vehicle for BMP-2 protein, allowing those fibers to be used in the recovery of alveolar bone tissue without further bone surgery. Then, polymer nanofibers were manufactured by optimizing process parameters of coaxial electrospinning with emphasis on the most relevant ones: voltage, internal and external flows in an attempt to correlate fibers properties with protein releasing abilities. All nanofibers were characterized according to its morphology, thermal behaviour, crystallinity and release profile. For the release tests, bovine albumin was added into internal fiber for future periodontal restorage application. Obtained results demonstrate that fibers were formed with diameters up to 250 nm. According to electronic microscopy images, one could observe surface of nanofibers, thickness and core-shell morphology confirmed. X-ray diffraction analysis and contact angle tests showed fibers with low crystal degree and low hydrophobicity. Nanofibers structure affected in vitro release model tests and consequently the cellular assays.


Assuntos
Materiais Biocompatíveis/química , Proteína Morfogenética Óssea 2/química , Nanofibras/química , Poliésteres/química , Álcool de Polivinil/química , Regeneração , Fator de Crescimento Transformador beta/química , Animais , Materiais Biocompatíveis/farmacologia , Proteína Morfogenética Óssea 2/farmacologia , Osso e Ossos/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Humanos , Camundongos , Nanofibras/toxicidade , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Regeneração/efeitos dos fármacos , Engenharia Tecidual , Fator de Crescimento Transformador beta/farmacologia
16.
ACS Appl Mater Interfaces ; 11(7): 6741-6750, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30676016

RESUMO

We present a nanoengineered system for sustained and prolonged delivery of protein therapeutics, which has the potential to impact current orthopedic regeneration strategies. Specifically, we introduce two-dimensional nanosilicates with a high surface area and charged characteristics for delivery of active proteins for more than 30 days. The nanosilicates show high binding efficacy without altering the protein conformation and bioactivity. The released proteins are able to maintain high activity as demonstrated by enhanced differentiation of human mesenchymal stem cells at 10-fold lower concentration compared to the exogenous control. Utilizing the nanosilicates as a delivery vehicle could minimize the negative side effects observed because of the use of supraphysiological dosages of protein therapeutics for orthopedic regeneration strategies.


Assuntos
Proteína Morfogenética Óssea 2 , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/farmacocinética , Proteína Morfogenética Óssea 2/farmacologia , Linhagem Celular , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia
17.
Biomed Mater ; 14(3): 035001, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30699400

RESUMO

INTRODUCTION: Bone tissue regeneration requires a three-dimensional biological setting. An ideal scaffold should enable cell proliferation and differentiation by mimicking structure and mechanical properties of the compromised defect as well as carrying growth factors. Two-photon polymerization (2PP) allows the preparation of 3D structures with a micrometric resolution. METHODS: In this study, 2PP was applied to design scaffolds made from biocompatible methacrylated D,L-lactide-co-ε-caprolactone copolymers (LC) with a controlled porous architecture. Proliferation and differentiation of bone marrow mesenchymal stromal cells on LC was analyzed and compared to a standard inorganic urethane-dimethacrylate (UDMA) matrix. To functionalize LC and UDMA surfaces we analyzed a biomimetic, layer-by-layer coating, which could be modified in stiffness and integration of bone morphogenetic protein 2 (BMP2) and evaluated its effect on osteogenic differentiation. RESULTS: On LC surfaces, BMSC demonstrated an optimal proliferation within pore sizes of 60-100 µm and showed a continuous expression of Vimentin. On the polyelectrolyte multilayer coating a significant increase in BMSC proliferation and differentiation as marked by Osteonectin expression was achieved using stiffness modification and BMP2 functionalization. CONCLUSION: Combining 3D-Design with biofunctionalization, LC offers a promising approach for future regenerative applications in osteogenic differentiation of BMSCs.


Assuntos
Proteína Morfogenética Óssea 2/química , Regeneração Óssea , Células-Tronco Mesenquimais/citologia , Poliésteres/química , Engenharia Tecidual/métodos , Osso e Ossos , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Eletrólitos , Escherichia coli , Humanos , Osteogênese , Fótons , Polimerização , Porosidade , Medicina Regenerativa , Estresse Mecânico , Tecidos Suporte , Uretana/química
18.
Protein J ; 38(1): 12-22, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30604108

RESUMO

Transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) signaling plays a fundamental role in embryonic skeletal development and postnatal bone homeostasis. The signaling pivot protein BMP-2 belongs to the TGF-ß superfamily and has been implicated in the pathogenesis of osteonecrosis of femoral head (ONFH). The biologically functional BMP-2 is a homodimer that has two tightly packed cores at its dimerization interface; each core is defined by the intermolecular interaction between a helical arm from one monomer and a hydrophobic pocket from another monomer. Inhibition and disruption of BMP-2 dimerization have been recognized as an attractive therapeutic strategy against ONFH. Here, we investigate the self-binding behavior of helical arm-derived peptides to the BMP-2 dimerization interface. The native BMP-2 helical arm and its several grafted versions from BMP-4, BMP-6 and BMP-7 are stripped from the intact dimerization interface to generate a number of isolated helical peptides. Computational simulations demonstrate that the stripping does not substantially influence the direct intermolecular interaction between BMP-2 monomer and these helical peptides or desolvation effect upon the interaction. However, the C-terminus of stripped peptides is found to have an intrinsic disorder and large flexibility in the isolated state, which would impair the rebinding of stripped peptides to BMP-2. Next, we rationally design a hydrocarbon bridge across the C-terminal residues 65 and 69 of helical peptides, which can effectively constrain peptide conformational flexibility in the isolated state, thus considerably promoting the binding potency of stripped helical peptides. Circular dichroism (CD) spectroscopy reveals that the peptide helicity increases from 51.8 to 67.9% upon hydrocarbon stapling. Fluorescence polarization assays substantiate that, as designed, the stapling can convert these helical peptides from weak binders to moderate or good binders of BMP-2 protein; their Kd values are improved by up to ~ fourfold.


Assuntos
Proteína Morfogenética Óssea 2/química , Necrose da Cabeça do Fêmur , Peptídeos/química , Multimerização Proteica , Proteína Morfogenética Óssea 2/metabolismo , Humanos , Peptídeos/metabolismo , Estrutura Secundária de Proteína
19.
Biomed Mater ; 14(2): 025008, 2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30609417

RESUMO

The tissue engineering applications of coaxial electrospinning are growing due to the potential increased functionality of the fibres compared to basic electrospinning. Previous studies of core and shell scaffolds have placed the active elements in the core, however, the surface response to a biomaterial affects the subsequent behaviour, thus here hydroxyapatite (HA) was added to the shell. Coaxial electrospun polycaprolactone (PCL)-polylactic acid (PLA)/HA (core-shell) scaffolds were produced in 2D sheets using a plate collector, or 3D tubes for bone tissue engineering using a rotating needle collector. The scaffolds include high hydroxyapatite content while retaining their structural and mechanical integrity. The effect of the collector type on fibre diameter, fibre alignment and mechanical properties have been evaluated, and the impact of HA incorporation on bioactivity, BMP-2 release, cell behaviour and mechanical properties for up to 12 weeks degradation were assessed. Fibre uniformity in coaxial electrospinning depends on the relative flow rate of the core and shell solutions. Using a rotating needle collector increased fibre alignment compared to a stationary collector, without affecting fibre diameter significantly, while HA content increased fibre non-uniformity. Coaxial PCL-PLA/HA fibres exhibited significantly higher bioactivity compared to PCL-PLA scaffolds due to the surface exposure of the HA particles. Apatite formation increased with increasing SBF immersion time. Coaxial tubular scaffolds with and without HA incorporation showed gradual reductions in their mechanical properties over 12 weeks in PBS or SBF but still retained their structural integrity. Coaxial scaffolds with and without HA exhibited gradual and sustained BMP-2 release and supported MSCs proliferation and differentiation with no significant difference between the two scaffolds types. These materials therefore show potential applications as bone tissue engineering scaffolds.


Assuntos
Proteína Morfogenética Óssea 2/química , Osso e Ossos/metabolismo , Poliésteres/química , Engenharia Tecidual/métodos , Tecidos Suporte/química , Fator de Crescimento Transformador beta/química , Materiais Biocompatíveis , Proteína Morfogenética Óssea 2/metabolismo , Adesão Celular , Diferenciação Celular , Proliferação de Células , Durapatita/química , Eletroquímica , Humanos , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Proteínas Recombinantes/química , Estresse Mecânico , Resistência à Tração
20.
Mater Sci Eng C Mater Biol Appl ; 96: 329-336, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30606540

RESUMO

OBJECTIVES: (1) To determine whether the biocompatibility of coralline hydroxyapatite (CHA) granules could be improved by using an octacalcium phosphate (OCP) coating layer, and/or functionalized with bone morphogenetic protein 2 (BMP-2), and (2) to investigate if BMP-2 incorporated into this coating is able to enhance its osteoinductive efficiency, in comparison to its surface-adsorbed delivery mode. METHODS: CHA granules (0.25 g per sample) bearing a coating-incorporated depot of BMP-2 (20 µg/sample) together with the controls (CHA bearing an adsorbed depot of BMP-2; CHA granules with an OCP coating without BMP-2; pure CHA granules) were implanted subcutaneously in rats (n = 6 animals per group). Five weeks later, the implants were retrieved for histomorphometric analysis to quantify the volume of newly generated bone, bone marrow, fibrous tissue and foreign body giant cells (FBGCs). The osteoinductive efficiency of BMP-2 and the rates of CHA degradation were also determined. RESULTS: The group with an OCP coating-incorporated depot of BMP-2 showed the highest volume and quality or bone, and the highest osteoinductive efficacy. OCP coating was able to reduce inflammatory responses (improve biocompatibility), and also simple adsorption of BMP-2 to CHA achieved this. CONCLUSIONS: The biocompatibility of CHA granules (reduction of inflammation) was significantly improved by coating with a layer of OCP. Pure surface adsorption of BMP-2 to CHA also reduced inflammation. Incorporation of BMP-2 into the OCP coatings was associated with the highest volume and quality of bone, and the highest biocompatibility degree of the CHA granules. CLINICAL SIGNIFICANCE: Higher osteoinductivity and improved biocompatibility of CHA can be obtained when a layer of BMP-2 functionalized OCP is deposited on the surfaces of CHA granules.


Assuntos
Materiais Biomiméticos , Proteína Morfogenética Óssea 2 , Cerâmica , Materiais Revestidos Biocompatíveis , Hidroxiapatitas , Teste de Materiais , Osteogênese/efeitos dos fármacos , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/farmacologia , Cerâmica/química , Cerâmica/farmacologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Hidroxiapatitas/química , Hidroxiapatitas/farmacologia , Masculino , Ratos , Ratos Wistar
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