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1.
J Mater Sci Mater Med ; 30(6): 68, 2019 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-31165270

RESUMEN

Calcium silicate cements have been considered as alternative bone substitutes owing to its extraordinary bioactivity and osteogenicity. Unfortunately, the major disadvantage of the cements was the slow degradation rate which may limit the efficiency of bone regeneration. In this study, we proposed a facile method to synthesize degradable calcium silicate cements by incorporating strontium into the cements through solid-state sintering. The effects of Sr incorporation on physicochemical and biological properties of the cements were evaluated. Although, our findings revealed that the incorporation of strontium retarded the hardening reaction of the cements, the setting time of different cements (11-19 min) were in the acceptable range for clinical use. The presence of Sr in the CS cements would hampered the precipitation of calcium phosphate products on the surface after immersion in SBF, however, a layer of precipitated calcium phosphate products can be formed on the surface of the Sr-CS cement within 1 day immersion in SBF. More importantly, the degradation rate of the cements increased with increasing content of strontium, consequentially raised the levels of released strontium and silicon ions. The elevated dissolving products may contribute to the enhancement of the cytocompatibility, alkaline phosphatase activity, osteocalcin secretion, and mineralization of human Wharton's jelly mesenchymal stem cells. Together, it is concluded that the strontium-incorporated calcium silicate cement might be a promising bone substitute that could accelerate the regeneration of irregularly shaped bone defects.


Asunto(s)
Cementos para Huesos/química , Regeneración Ósea , Compuestos de Calcio/química , Células Madre Mesenquimatosas/citología , Osteogénesis/efectos de los fármacos , Silicatos/química , Estroncio/química , Fosfatasa Alcalina/metabolismo , Antraquinonas/química , Materiales Biocompatibles/química , Sustitutos de Huesos , Fosfatos de Calcio/química , Adhesión Celular , Proliferación Celular , Humanos , Iones , Osteocalcina/química , Polvos , Regeneración , Células Madre/citología , Resistencia a la Tracción , Gelatina de Wharton/metabolismo
2.
Clin Oral Investig ; 23(3): 1309-1318, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30003342

RESUMEN

OBJECTIVES: The aim of the present study was to evaluate the blood cell content, morphological aspects, gene expression of type I collagen, and release of growth factors on an injectable platelet rich fibrin (i-PRF). MATERIALS AND METHODS: Blood samples were collected from 15 volunteers to prepare i-PRF samples. Peripheral blood was used as a control group. Blood clot and i-PRF samples were cultured for 10 days. The supernatant of the samples was collected for ELISA immunoassay quantification of PDGF and VEGF growth factors over periods of 1, 8, 24, 72, and 240 h. I-PRF and blood clot samples were biologically characterized using histological and immunohistochemistry analysis for IL-10, osteocalcin, and TGF-ß. Scanning electron microscopy (SEM) was used to inspect the fibrin network and distribution of blood platelets and leukocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) method was used to evaluate gene expression for type I collagen. RESULTS: A higher concentration of platelets and lymphocytes was recorded in i-PRF than in peripheral blood (p < 0.05). The release of VEGF was higher in blood clot samples (1933 ± 704) than that for i-PRF (852 ± 376; p < 0.001). Immunohistochemistry showed upregulation of TGF-B, IL-10, and osteocalcin in the i-PRF group. RT-PCR showed increased type I collagen gene expression in i-PRF (p < 0.05). SEM images revealed agglomeration of platelets in some regions, while a fibrin networking was noticeable in the entire i-PRF sample. CONCLUSIONS: Injectable platelet rich fibrin becomes a good approach for soft and mineralized tissue healing considering the formation of a three-dimensional fibrin network embedding platelets, leukocytes, type I collagen, osteocalcin, and growth factors. Indeed, the injectable platelet rich fibrin can be indicated in several medical applications regarding bioactivity, simplied technique, and flowable mixing with other biomaterials. CLINICAL RELEVANCE: Morphological, cell, and protein characterization of platelet rich fibrin provides a better understanding of the clinical effects and improvement of clinical guidelines for several medical applications. Once well physicochemical and biologically characterized, the use of an injectable platelet rich fibrin can be extended to other applications in the field of orthopedics, periodontics, and implant dentistry on the repairing process of both soft and mineralized tissues.


Asunto(s)
Fibrina Rica en Plaquetas/química , Fibrina Rica en Plaquetas/citología , Adulto , Plaquetas/citología , Colágeno Tipo I/química , Fibrina/química , Humanos , Interleucina-10/química , Leucocitos/citología , Masculino , Osteocalcina/química , Factor de Crecimiento Transformador beta1/química , Factor A de Crecimiento Endotelial Vascular/química
3.
J Mater Sci Mater Med ; 28(1): 3, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27878735

RESUMEN

Prevention of infection and enhanced osseointegration are closely related, and required for a successful orthopaedic implant, which necessitate implant designs to consider both criteria in tandem. A multi-material coating containing 1:1 ratio of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite as the top functional layer, and hydroxyapatite as the base layer, was produced via the drop-on-demand micro-dispensing technique, as a strategic approach in the fight against infection along with the promotion of bone tissue regeneration. The homogeneous distribution of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite micro-droplets at alternate position in silicon-substituted hydroxyapatite-silver-substituted hydroxyapatite/hydroxyapatite coating delayed the exponential growth of Staphylococcus aureus for up to 24 h, and gave rise to up-regulated expression of alkaline phosphatase activity, type I collagen and osteocalcin as compared to hydroxyapatite and silver-substituted hydroxyapatite coatings. Despite containing reduced amounts of silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite micro-droplets over the coated area than silicon-substituted hydroxyapatite and silver-substituted hydroxyapatite coatings, silicon-substituted hydroxyapatite-silver-substituted hydroxyapatite/hydroxyapatite coating exhibited effective antibacterial property with enhanced bioactivity. By exhibiting good controllability of distributing silicon-substituted hydroxyapatite, silver-substituted hydroxyapatite and hydroxyapatite micro-droplets, it was demonstrated that drop-on-demand micro-dispensing technique was capable in harnessing the advantages of silver-substituted hydroxyapatite, silicon-substituted hydroxyapatite and hydroxyapatite to produce a multi-material coating along with enhanced bioactivity and reduced infection.


Asunto(s)
Apatitas/química , Materiales Biocompatibles Revestidos/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Adipocitos/citología , Fosfatasa Alcalina/metabolismo , Antibacterianos/farmacología , Regeneración Ósea , Proliferación Celular , Colágeno/química , Humanos , Hidroxiapatitas/química , Nanopartículas del Metal/química , Pruebas de Sensibilidad Microbiana , Microscopía Confocal , Oseointegración/efectos de los fármacos , Osteocalcina/química , Polvos , Silicio/química , Plata/química , Propiedades de Superficie
4.
J Mater Sci Mater Med ; 28(2): 25, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-28070691

RESUMEN

Craniofacial bone defects such as alveolar cleft affect the esthetics and functions that need bone reconstruction. The advanced techniques of biomaterials combined with stem cells have been a challenging role for maxillofacial surgeons and scientists. PCL-coated biphasic calcium phosphate (PCL-BCP) scaffolds were created with the modified melt stretching and multilayer deposition (mMSMD) technique and merged with human dental pulp stem cells (hDPSCs) to fulfill the component of tissue engineering for bone substitution. In the present study, the objective was to test the biocompatibility and biofunctionalities that included cell proliferation, cell viability, alkaline phosphatase activity, osteocalcin, alizarin red staining for mineralization, and histological analysis. The results showed that mMSMD PCL-BCP scaffolds were suitable for hDPSCs viability since the cells attached and spread onto the scaffold. Furthermore, the constructs of induced hDPSCs and scaffolds performed ALP activity and produced osteocalcin and mineralized nodules. The results indicated that mMSMD PCL-BCP scaffolds with hDPSCs showed promise in bone regeneration for treatment of osseous defects.


Asunto(s)
Materiales Biocompatibles/química , Regeneración Ósea/efectos de los fármacos , Pulpa Dental/citología , Células Madre/efectos de los fármacos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Adolescente , Adulto , Fosfatasa Alcalina/química , Fosfatos de Calcio/química , Línea Celular , Proliferación Celular , Supervivencia Celular , Pulpa Dental/efectos de los fármacos , Humanos , Inmunofenotipificación , Microscopía Confocal , Microscopía Electrónica de Rastreo , Osteocalcina/química , Poliésteres/química , Células Madre/citología , Adulto Joven
5.
J Biol Chem ; 288(11): 7885-7893, 2013 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23362258

RESUMEN

Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration.


Asunto(s)
Durapatita/química , Osteocalcina/química , Secuencia de Aminoácidos , Animales , Desarrollo Óseo , Calcio/química , Química Física/métodos , Dicroismo Circular , Cristalización , Humanos , Microscopía Electrónica/métodos , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión/métodos , Datos de Secuencia Molecular , Oseointegración , Péptidos/química , Prótesis e Implantes , Estructura Terciaria de Proteína , Regeneración
6.
Artículo en Inglés | MEDLINE | ID: mdl-22075542

RESUMEN

Nutritional status including vitamin A could explain some of the developmental deformities observed in cultivated teleosts, including Atlantic cod (Gadus morhua). In the present study we aimed to investigate the transcriptional effect of retinoic acid (RA) on bone related genes using Atlantic cod craniofacial explants tissue cultures. Two different osteoblast specific osteocalcin/bone gla protein isoforms were discovered in cod. Transcription of both isoforms was up-regulated following RA treatment of 65 dph cod lower jaw explants. In contrast, transcripts coding for genes related to bone resorption and osteoclast activity, matrix metalloproteinase 9 and cathepsin K were down-regulated following RA treatment. This could be linked to the decreased transcriptional ratio between receptor activator of nuclear factor kappa-B ligand rankl and osteoprotegerin observed in the same tissue samples. RA treatment of juvenile explants had no effect on runt-related transcription factor 2 and osterix mRNA levels. However, osterix was significantly down-regulated in 25 dph cod head explants following RA treatment. In situ hybridizations revealed differential spatial distribution of the two isoforms and the predominant expression of cathepsin K in bone surrounding tissues. The present study indicates that RA causes a shift in the balance between osteoclast activity and osteoblast activity in favor of the latter.


Asunto(s)
Gadus morhua/metabolismo , Osteocalcina/metabolismo , Osteoclastos/metabolismo , Cráneo/metabolismo , Tretinoina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biomarcadores/metabolismo , Resorción Ósea/genética , Resorción Ósea/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Análisis por Conglomerados , Cara , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Gadus morhua/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hibridación in Situ , Datos de Secuencia Molecular , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteocalcina/química , Osteocalcina/genética , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Cráneo/efectos de los fármacos , Técnicas de Cultivo de Tejidos , Transcripción Genética/efectos de los fármacos
7.
J Biomed Mater Res B Appl Biomater ; 108(3): 1129-1140, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31397056

RESUMEN

In the present study, scaffolds based on alginate-pullulan-bioactive glass-ceramic with 0.5 and 1.5 mol % copper oxide were orthotopically implanted in experimental rat models to assess their ability to heal an induced bone defect. By implying magnetic resonance and imaging scans together with histological evaluation of the processed samples, a progressive healing of bone was observed within 5 weeks. Furthermore, as the regenerative process continued, new bone tissue was formed, enhancing the growth of irregular bone spicules around the scaffolds. A significantly higher amount of new bone was formed (37%) in the defect that received the composite with 1.5 mol % CuO (in glass-ceramic matrix) content implant. Nevertheless, the bone regeneration obtained by scaffold with 0.5 mol % CuO implanted is comparable with the alginate-pullulan-ß-tricalcium phosphate/hydroxiapatite composite implant. The assessed amount of new bone formed was found to be between 29.75 and 37.15% for all the composition involved in the present study. During this process a regeneration process was shown when the alginate-pullulan composite materials were involved, fact that indicate the great potential of these materials to be used in tissue engineering.


Asunto(s)
Alginatos/química , Regeneración Ósea , Cerámica/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Materiales Biocompatibles/química , Sustitutos de Huesos , Huesos , Durapatita , Electroquímica , Técnicas In Vitro , Luminiscencia , Imagen por Resonancia Magnética , Masculino , Microscopía Electrónica de Rastreo , Osteocalcina/química , Manejo del Dolor , Polímeros/química , Ratas , Ratas Wistar
8.
J Mater Chem B ; 8(30): 6378-6389, 2020 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-32633309

RESUMEN

A demineralized bone matrix (DBM) scaffold has good biocompatibility, low antigenicity, a natural porous structure and no cytotoxicity, and so it is an appropriate material for bone regeneration. However, osteoinductive growth factors are often removed during preparation, which destroys the osteoinductive capacity of the DBM scaffold. Biomaterials combined with gene therapy is a promising approach to effectively avoid this adverse side effect. This study develops a human bone morphogenetic protein 2 (hBMP2) gene-activated DBM scaffold to enhance the osteoinductive capacity of DBM and improve bone repair. Bone marrow mesenchymal stem cell (MSC)-derived microvesicles (MVs) were obtained, and polyethyleneimine (PEI) and human bone morphogenetic protein 2 (hBMP2) plasmids (phBMP2) were sequentially coated on the MVs by layer-by-layer (LBL) self-assembly to form an MVs-PEI/phBMP2 non-viral gene vector. Finally, the gene-activated scaffold (DBM/MVs-PEI/phBMP2) was prepared by loading MVs-PEI/phBMP2 onto a DBM scaffold. The experimental results show that the MVs-PEI/phBMP2 exhibits higher transfection efficiency and lower cytotoxicity to MSCs when the MVs/PEI weight ratio = 5, and could enhance the osteogenic differentiation of MSCs in vitro. Subcutaneous implantation into rats showed that the DBM/MVs-PEI/phBMP2 scaffold could efficiently enhance the deposition of: collagen fibers, osteocalcin, osteopontin and CD34 endogenous proteins. Rabbit femoral condyle defect experiments proved that the DBM/MVs-PEI/phBMP2 scaffold could significantly promote bone repair. This study presents a novel, highly efficient and low cytotoxicity gene delivery vector based on MVs. The gene-activated DBM scaffold based on MVs not only could promote bone formation but also angiogenesis, implying that this kind of gene-activated scaffold is a promising bone substitute material.


Asunto(s)
Materiales Biocompatibles/química , Proteína Morfogenética Ósea 2/genética , Regeneración Ósea/genética , Células Madre Mesenquimatosas/metabolismo , Plásmidos/genética , Andamios del Tejido/química , Animales , Antígenos CD34/química , Sustitutos de Huesos/metabolismo , Diferenciación Celular , Células Cultivadas , Colágeno/química , Fémur/trasplante , Técnicas de Transferencia de Gen , Humanos , Células Madre Mesenquimatosas/citología , Osteocalcina/química , Osteogénesis , Osteopontina/química , Polietileneimina/química , Prótesis e Implantes , Conejos , Ratas , Transfección
9.
Mater Sci Eng C Mater Biol Appl ; 109: 110491, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32228965

RESUMEN

Multifunctional scaffolds have recently attained superior significance in tissue regeneration due to combinational activity profile that is usually accomplished by separate sequential therapy. Here, we present a dual system comprised of surface-phosphorylated PET fibrous matrix coated with ciprofloxacin-impregnated biodegradable polymer poly (hydroxyethyl methacrylate) aiming at regeneration of bone tissue deprived of bacterial infections, particularly osteomyelitis. The ATR, XPS and FESEM/EDX results provided confirmative evidences for surface phosphorylation of PET and in situ coating of the polymer. Swelling and contact angle measurements demonstrated improved hydrophilicity which is further corroborated by in vitro degradation profile in PBS. Preliminary evaluation by MTT and actin staining proved its biocompatibility while enhanced in vitro mineralization in 1.5X SBF by FESEM/EDX clearly indicate the primary nucleation and secondary growth of beautiful apatite crystals with Ca/P ratio similar to human bone. Alizarin red S and von Kossa staining validated the biomineralization in MG-63 cells. The sequential expression of early and late biomarkers -alkaline phosphatase (ALP) and osteocalcin (OSN)- of osteoblast differentiation in rat bone marrow mesenchymal cells (BMC) has demonstrated osteoinductive nature of the system. The second functionality of the scaffold has been proven by step-wise ciprofloxacin-release profile (in vitro) with ~60% release within 120 h. In addition, antibacterial studies of ciprofloxacin- eluted from the scaffold have shown apparent zones of inhibition against Staphylococcus aureus (3.6 ± 0.3 cm) and Escherichia coli (3.0 ± 0.8 cm). Hence, the surface-transformed PET scaffold function as a dual system as localized antibiotic delivery vehicle against bone infections and undergo self-biomineralization leading to osteoinduction.


Asunto(s)
Tereftalatos Polietilenos/química , Fosfatasa Alcalina/metabolismo , Animales , Antibacterianos/química , Antibacterianos/farmacología , Regeneración Ósea/efectos de los fármacos , Huesos/citología , Línea Celular Tumoral , Ciprofloxacina/química , Ciprofloxacina/farmacología , Escherichia coli/efectos de los fármacos , Humanos , Osteocalcina/química , Fosforilación/efectos de los fármacos , Tomografía de Emisión de Positrones , Ratas , Staphylococcus aureus/efectos de los fármacos
10.
Int J Oral Maxillofac Implants ; 22(4): 542-50, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17929514

RESUMEN

PURPOSE: To investigate the effects of different chemically modified titanium surfaces on protein adsorption and the osteoblastic differentiation of human embryonic palatal mesenchymal (HEPM) cells. MATERIALS AND METHODS: Three different surfaces were evaluated. The first, a machined surface (Ti-M), was considered a control. The second surface was acid etched (Ti-AE). The third surface was prepared by exposing the Ti-AE samples to sodium hydroxide (NaOH) solution (Ti-AAE). The surface characteristics of chemically modified titanium were investigated by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and profilometry. To evaluate the production of biomarkers, commercial kits were utilized. RESULTS: Surface composition and morphology affected the kinetics of protein adsorption. Ti-AE surfaces manifested a greater affinity for fibronectin adsorption compared to Ti-M or Ti-AAE surfaces. It was observed that Ti-AE and Ti-AAE surfaces promoted significantly greater cell attachment compared to Ti-M surfaces. Statistically significant differences were also observed in the expression of alkaline phosphatase (ALP) activity, osteocalcin, and osteopontin on all 3 titanium surfaces. ALP activity and osteocalcin production up to day 12 suggested that differentiation of the cells into osteoblasts had occurred and that cells were expressing a bone-forming phenotype. CONCLUSIONS: It was thus concluded from this study that surface morphology and composition play a critical role in enhancing HEPM cell proliferation and differentiation into osteoblast cells.


Asunto(s)
Materiales Dentales/química , Células Madre Mesenquimatosas/fisiología , Osteoblastos/fisiología , Proteínas/química , Titanio/química , Grabado Ácido Dental , Adsorción , Fosfatasa Alcalina/química , Óxido de Aluminio/química , Biomarcadores/análisis , Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Grabado Dental/métodos , Células Madre Embrionarias/fisiología , Fibronectinas/química , Humanos , Ácido Clorhídrico/química , Ensayo de Materiales , Osteocalcina/química , Osteopontina/química , Hueso Paladar/embriología , Fenotipo , Hidróxido de Sodio/química , Propiedades de Superficie
11.
Biomaterials ; 26(30): 5991-8, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15878198

RESUMEN

Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg-Gly-Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 mm) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 mm RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 mm being optimal concentration.


Asunto(s)
Materiales Biocompatibles/química , Células de la Médula Ósea/citología , Hidrogeles/química , Oligopéptidos/farmacología , Polietilenglicoles/química , Células del Estroma/citología , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Animales , Médula Ósea/metabolismo , Adhesión Celular , ADN/química , Cabras , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Modelos Estadísticos , Oligopéptidos/química , Osteocalcina/química , Osteocalcina/metabolismo , Osteogénesis , Péptidos/química , Fósforo/química , ARN/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Ingeniería de Tejidos/métodos
12.
J Biomed Mater Res A ; 73(4): 377-87, 2005 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-15957205

RESUMEN

A novel hybrid coating of biomimetic apatite(BAp) and osteocalcin (OC) was prepared by incubating BAp-coated Ti6A14V coupons in an osteocalcin-containing medium. A significant amount (up to 1.0 wt %) of OC was adsorbed by the BAp coating within 3 h of incubation as demonstrated by high-performance liquid chromatography. Characterizations of the hybrid coating with environmental scanning microscopy and X-ray diffraction indicated that protein adsorption does not alter the microstructure of the coating. The presence of OC in the hybrid coating was visualized with fluorescence microscopy using an immuno-labeling procedure. The affinity of OC to the BAp coating was examined using a 20-h elution test in phosphate-buffered saline and only a minimal amount (<10%) of the loaded OC was eluted out. When the coating was fully dissolved in hydrochloric acid solution after elution, about 78% of the loaded OC could be recovered. Enzyme-linked immunosorbent assay and peptide sodium dodecylsulfate-polyacrylamide gel electrophoresis confirmed the integrity and activity of OC molecules throughout the tests. The preliminary cell culture tests showed a significant effect of OC on the attachment and proliferation of osteoblasts. The quick loading profile and high affinity of OC to the BAp coating make it an ideal candidate for the hybrid coating preparation in clinical environment.


Asunto(s)
Apatitas/química , Materiales Biomiméticos/química , Materiales Biocompatibles Revestidos/farmacología , Osteocalcina/química , Animales , Materiales Biocompatibles , Cromatografía Líquida de Alta Presión , Materiales Biocompatibles Revestidos/química , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Microscopía Electrónica de Rastreo , Osteoblastos/metabolismo , Osteoblastos/ultraestructura , Osteoclastos/metabolismo , Péptidos/química , Ratas , Ratas Wistar , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Factores de Tiempo , Difracción de Rayos X
13.
J Biomed Mater Res A ; 73(4): 445-55, 2005 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-15900611

RESUMEN

The ability to generate new bone for reconstructive surgery use is a major clinical need. Tissue engineering with osteoprogenitor cells isolated from the patient's periosteum and seeded into bioresorbable scaffolds offers a promising approach to the generation of skeletal tissue. To our knowledge, there is no description about the expression of Ets2 in tissue engineered "bone neotissue." The aim of our study was to manufacture cell-seeded three-dimensional bone constructs with human periosteal cells on poly (lactic-co-glycolic acid) polymer fleeces to describe the expression pattern of Ets2 and its target genes osteocalcin and osteopontin; expression analysis of type I collagen, core-binding factor-1, alkaline phosphatase, and osteonectin; the ability of matrix mineralization and ALP enzymatic activity showed the osteogenic character of the constructs. A significant correlation between the expression of Ets2 and osteopontin mRNA (r = -0.70; p < 0.05) could be shown. A 1.35-fold increase of Ets2 expression from days 1 to 9 was detected, followed by a slight decrease from days 11 to 15. Until the end of the culture period, the expression of Ets2 reached a comparable high level as detected on day 9. In contrast, the expression level of osteopontin mRNA reached a maximum at day 7, followed by a progressive 3.04-fold decrease until day 21. This study shows for the first time that Ets2 gene and its transcriptional target genes are expressed in tissue-engineered bone constructs. These findings have the potential to provide much-needed information about the role and function of Ets2 in human osteogenesis processes and creation of "bone neotissue."


Asunto(s)
Materiales Biocompatibles/química , Sustitutos de Huesos/química , Huesos/metabolismo , Cromosomas Humanos Par 21 , Proteínas Proto-Oncogénicas/biosíntesis , Transactivadores/biosíntesis , Fosfatasa Alcalina/metabolismo , Huesos/patología , Proliferación Celular , Células Cultivadas , Factores de Unión al Sitio Principal , Cartilla de ADN/química , Regulación de la Expresión Génica , Glicolatos/química , Humanos , Ácido Láctico/química , Mesodermo/citología , Mesodermo/patología , Microscopía Electrónica de Rastreo , Proteínas de Neoplasias/metabolismo , Osteoblastos/metabolismo , Osteocalcina/química , Osteocalcina/metabolismo , Osteogénesis , Osteonectina/química , Osteopontina , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/química , Proteína Proto-Oncogénica c-ets-2 , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sialoglicoproteínas/química , Factores de Tiempo , Ingeniería de Tejidos , Factores de Transcripción/metabolismo
14.
Comp Biochem Physiol B Biochem Mol Biol ; 140(4): 665-72, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15763522

RESUMEN

Northern blotting, RT-PCR, and Western blotting techniques were used to characterize the matrix constituents of avian cortical and medullary bone. Extracts of bone tissue were found to contain multiple isoforms of bone sialoprotein (BSP), osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and dentin matrix protein-1 (DMP-1). Only single transcripts were observed with Northern blotting; therefore it was concluded that the isoforms were due to differences in post-translational modifications. Since medullary bone is rich in keratan sulfate (KS), RT-PCR was used to investigate the expression of known keratan sulfate-containing proteoglycans (KSPGs). Although this tissue was found to express lumican and osteoglycin/mimecan, there was little evidence to suggest that these proteoglycans were a major source of the keratan sulfate glycosaminoglycans. Treatment of medullary bone extracts with keratanase resulted in the appearance of a BSP immunoactive band of approximately 59 kDa. However, it was not possible to isolate and identify the intact keratan sulfate proteoglycan.


Asunto(s)
Aves/anatomía & histología , Huesos/química , Colágeno/química , Proteínas/química , Animales , Northern Blotting , Western Blotting , Sialoproteína de Unión a Integrina , Sulfato de Queratano/química , Osteocalcina/química , Osteonectina/química , Osteopontina , Fosfoproteínas/química , Proteoglicanos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sialoglicoproteínas/química
15.
Biomed Mater Eng ; 15(3): 127-36, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15911994

RESUMEN

In an attempt to reduce complications in cases of severe open fracture, we developed a bio-artificial periosteum composed of osteogenic cells and collagen sponge. In the present study, we evaluated the osteogenic potential of the bio-artificial periosteum in vivo and in vitro. After 4-week incubation in vitro, the bio-artificial periosteum had high alkaline phosphatase activity and osteocalcin content. Moreover, energy dispersive X-ray analysis revealed numerous crystal structures consisting of P and Ca on the surface of the bio-artificial periosteum. Using a rat model for severe bone injury, we examined the bone formation process in defect sites covered with the bio-artificial periosteum. New bone formation occurred in the central part of the bone defect as well as at the bone edge. We conclude that by using the bio-artificial periosteum, the fracture site benefited from an improved osteogenic environment. These results indicate that a clinical trial to further evaluate this technique should be conducted.


Asunto(s)
Materiales Biocompatibles/química , Ingeniería Biomédica/métodos , Sustitutos de Huesos/química , Colágeno/química , Curación de Fractura , Fracturas Abiertas/terapia , Periostio/química , Fosfatasa Alcalina/metabolismo , Animales , Órganos Bioartificiales , Células de la Médula Ósea/citología , Huesos/diagnóstico por imagen , Huesos/ultraestructura , Técnicas In Vitro , Masculino , Microscopía Electrónica de Rastreo , Osteocalcina/química , Osteocalcina/metabolismo , Osteogénesis , Periostio/diagnóstico por imagen , Radiografía , Ratas , Ratas Endogámicas F344 , Factores de Tiempo , Cicatrización de Heridas
16.
Biomed Res ; 26(2): 51-9, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15889618

RESUMEN

The purpose of the current study was to evaluate the bone formation when beta-tricalcium phosphate (TCP) was implanted in bone defects of rat femurs. beta-TCP granules were applied to defects created in the femurs of 65 male rats who were sacrificed 3, 7, 10, 14 or 30 days later. Bone tissues were embedded in paraffin, serial sections were cut and then stained with hematoxylin-eosin. Histomorphometric analyses were also conducted. Furthermore, total mRNAs were extracted, homogenized, and reverse transcribed, after which quantitative PCR assays were conducted with a LightCycler using the double-stranded DNA dye Syber Green I with primers for either rat osteopontin or osteocalcin. Tissues in defects without beta-TCP were used as controls. The amount of newly formed bone tissue in the beta-TCP implanted group was significantly greater in both the side areas and the central area of defects than in the control group. Expressions of osteopontin and osteocalcin mRNAs of cells in the defects of the experimental group were up-regulated compared with the control group at all time periods. Taken together, these results prove that beta-TCP is an appropriate material for osteoconduction and promotes bone formation in bone defects.


Asunto(s)
Materiales Biocompatibles , Sustitutos de Huesos/química , Fosfatos de Calcio/química , Fémur/patología , Animales , Benzotiazoles , Regeneración Ósea , Huesos/metabolismo , Cartilla de ADN/química , Diaminas , Colorantes Fluorescentes/farmacología , Masculino , Compuestos Orgánicos/farmacología , Osteocalcina/química , Osteopontina , Quinolinas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sialoglicoproteínas/química , Factores de Tiempo , Regulación hacia Arriba , Cicatrización de Heridas
17.
Endocrine ; 48(2): 394-404, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25158976

RESUMEN

Osteocalcin (OC) is the main non-collagenous hydroxyapatite-binding protein synthesized by osteoblasts, odontoblasts, and hypertrophic chondrocytes. It has a regulatory role in mineralization and it is considered a marker of bone cell metabolism. Recent findings evidenced new extra-skeletal roles for OC, depicting it as a real hormone. OC shares many functional features with the common hormones, such as tissue-specific expression, circadian rhythm, and synthesis as a pre-pro-molecule. However, it has some peculiar features making it a unique molecule: OC exists in different forms based on the degree of carboxylation. Indeed, OC has three glutamic acid residues, in position 17, 21, and 24, which are subject to γ-carboxylation, through the action of a vitamin K-dependent γ-glutamyl carboxytransferase. The degree of carboxylation, and thus the negative charge density, determines the affinity for the calcium ions deposited in the extracellular matrix of the bone. The modulation of the carboxylation could, thus, represent the mechanism by which the body controls the circulating levels, and hence the hormonal function, of OC. There are evidences linking OC, and the bone metabolism, with a series of endocrine (glucose metabolism, energy metabolism, fertility) physiological (muscle activity) and pathological functions (ectopic calcification). Aim of this review is to give a full overview of the physiological roles of OC by collecting the newest experimental findings on this intriguing molecule.


Asunto(s)
Osteocalcina/fisiología , Humanos , Osteocalcina/química , Osteocalcina/metabolismo
18.
Biomed Mater ; 10(1): 015019, 2015 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-25668049

RESUMEN

To investigate the potential application of graphene oxide (GO) in bone repair, this study is focused on the preparation, characterization and cell behavior of graphene oxide coatings on quartz substrata. GO coatings were prepared on the substrata using a modified dip-coating procedure. Atomic force microscopy (AFM), scanning electron microscopy (SEM) and Raman spectroscopy results demonstrated that the as-prepared coatings in this study were homogeneous and had an average thickness of ~67 nm. The rapid formation of a hydroxyapatite (HA) layer in the simulated body fluid (SBF) on GO coated substrata at day 14, as proved by SEM and x-ray diffraction (XRD), strongly indicated the bioactivity of coated substrata. In addition, MC3T3-E1 cells were cultured on the coated substrata to evaluate cellular activities. Compared with the non-coated substrata and tissue culture plates, no significant difference was observed on the coated substrata in terms of cytotoxicity, viability, proliferation and apoptosis. However, interestingly, higher levels of alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion were observed on the coated substrata, indicating that GO coatings enhanced cell differentiation compared with non-coated substrata and tissue culture plates. This study suggests that GO coatings had excellent biocompatibility and more importantly promoted MC3T3-E1 cell differentiation and might be a good candidate as a coating material for orthopedic implants.


Asunto(s)
Materiales Biocompatibles/química , Durapatita/química , Grafito/química , Ortopedia , Óxidos/química , Células 3T3 , Fosfatasa Alcalina/química , Animales , Apoptosis , Líquidos Corporales/química , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Medios de Cultivo/química , Ratones , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Osteocalcina/química , Cuarzo , Sincalida/química , Espectrometría Raman , Propiedades de Superficie , Difracción de Rayos X
19.
Biomaterials ; 56: 46-57, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25934278

RESUMEN

Biomaterial surface design with biomimetic proteins holds great promise for successful regeneration of tissues including bone. Here we report a novel proteinaceous hybrid matrix mimicking bone extracellular matrix that has multifunctional capacity to promote stem cell adhesion and osteogenesis with excellent stability. Osteocalcin-fibronectin fusion protein holding collagen binding domain was networked with fibrillar collagen, featuring bone extracellular matrix mimic, to provide multifunctional and structurally-stable biomatrices. The hybrid protein, integrated homogeneously with collagen fibrillar networks, preserved structural stability over a month. Biological efficacy of the hybrid matrix was proven onto tethered surface of biopolymer porous scaffolds. Mesenchymal stem cells quickly anchored to the hybrid matrix, forming focal adhesions, and substantially conformed to cytoskeletal extensions, benefited from the fibronectin adhesive domains. Cells achieved high proliferative capacity to reach confluence rapidly and switched to a mature and osteogenic phenotype more effectively, resulting in greater osteogenic matrix syntheses and mineralization, driven by the engineered osteocalcin. The hybrid biomimetic matrix significantly improved in vivo bone formation in calvarial defects over 6 weeks. Based on the series of stimulated biological responses in vitro and in vivo the novel hybrid proteinaceous composition will be potentially useful as stem cell interfacing matrices for osteogenesis and bone regeneration.


Asunto(s)
Materiales Biocompatibles/química , Huesos/patología , Ingeniería de Proteínas/métodos , Ingeniería de Tejidos/métodos , Animales , Biopolímeros/química , Regeneración Ósea , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Colágeno/química , Fibronectinas/química , Masculino , Células Madre Mesenquimatosas/citología , Osteocalcina/química , Osteogénesis , Fenotipo , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/química , Propiedades de Superficie , Andamios del Tejido/química
20.
Biomaterials ; 25(20): 4911-9, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15109851

RESUMEN

Calcium phosphate coated titanium and titanium alloy are widely used as dental and orthopaedic implants. This study examines the effect of novel calcium titanium and calcium titanium zirconium phosphates suitable for plasma-spraying onto titanium substrata on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that on native titanium and hydroxyapatite-coated titanium. Test materials were an acid etched and sand-blasted titanium surface (Ti-DPS), a plasma-sprayed hydroxyapatite coating (HA), and five materials which were created from CaTi(4)(PO(4))(6) (CTP) and CaZr(4)(PO(4))(6) (CZP): sintered CaTi(4)(PO(4))(6) (CTP-S1), sintered 46CaO.23TiO(2).31P(2)O(5) (CTP-S2), sintered CaTiZr(3)(PO(4))(6), (CTZP-S1), sintered 46CaO.23ZrO(2).31P(2)O(5) (CTZP-S2) and sintered 55CaO.20TiO(2).31P(2)O(5) (CTP-S3). HBDC were grown on the substrata for 3, 7, 14 and 21 d, counted and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrates significantly affected cellular growth and the temporal expression of an array of bone-related genes and proteins. At 14 and 21 d, cells on CTP-S3 displayed significantly enhanced expression of all osteogenic mRNAs. Surfaces of CTP-S1 and CTP-S3 had the most effect on osteoblastic differentiation inducing a greater expression of an array of osteogenic markers than recorded for cells grown on Ti-DPS and HA, suggesting that these novel materials may possess a higher potency to enhance osteogenesis.


Asunto(s)
Sustitutos de Huesos/química , Calcio/química , Osteogénesis , Titanio/química , Fosfatasa Alcalina/química , Anticuerpos/química , Fosfatos de Calcio/química , Proliferación Celular , Células Cultivadas , Colágeno/química , Durapatita/química , Humanos , Inmunohistoquímica , Hibridación in Situ , Sialoproteína de Unión a Integrina , Osteoblastos/química , Osteocalcina/química , Osteonectina/química , Osteopontina , Prótesis e Implantes , ARN Mensajero/metabolismo , Sialoglicoproteínas/química , Factores de Tiempo
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