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BACKGROUND: Radiotherapy can affect healthy cells, resulting in side effects. This study aimed to assess the impact of radiotherapy on soft tissue in surgical wounds in rats. METHODS: The animals were divided into four groups: control (S) group without irradiation, immediate irradiation (S-IIr) group receiving irradiation right after surgery, late irradiation (S-LIr) group receiving irradiation four weeks after surgery, and early irradiation (Ir-S) group receiving irradiation before surgery. The irradiated groups underwent two fractional stages of 15 Gy. Muscle contractibility (EMG) was evaluated at two different time points, and after 2 and 7 weeks, the animals were euthanized for histological analysis of the muscles and skin. RESULTS: There was no significant difference between the EMG1 and EMG2 values of the S and S-LIr groups, but both S-IIr and Ir-S groups exhibited a statistically significant difference. The S group demonstrated a larger diameter of muscle fiber compared to other groups, showing a significant difference. In terms of skin analysis, the S-IIr group had the least inflammatory infiltrate and the highest amount of red fibers, differing significantly from the other groups. CONCLUSIONS: Regardless of the duration, radiotherapy was found to have effects on the surrounding soft tissues, as concluded by this study.
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Clinically, bone tissue replacements and/or bone repair are challenging. Strategies based on well-defined combinations of osteoconductive materials and osteogenic cells are promising to improve bone regeneration but still require improvement. Herein, we combined polycaprolactone (PCL) fibers, carbon nanotubes (CNT), and hydroxyapatite (nHap) nanoparticles to develop the next generation of bone regeneration material. Fibers formed by rotary jet spinning (RJS) instead of traditional electrospinning (ES) with embedded bone marrow mesenchymal stem cells (BMMSCs) showed the best outcomes to repair rat calvarial defects after 6 weeks. To understand this, it was observed that different morphologies were formed depending on the manufacturing method used. RJS fibers presented a particular topography with rough fibers, which allowed for better cellular growth and cell spreading in vitro around and into a three-dimensional (3D) mesh, while fibers made by ES were more smooth and cellular growth was only measured on the 3D mesh surface. The fibers with incorporated nHap/CNT nanoparticles enhanced in vitro cell performance as indicated by more cellular proliferation, alkaline phosphatase activity, proliferation, and deposition of calcium. Greater bone neoformation occurred by combining three characteristics: the presence of nHap and CNT nanoparticles, the topography of the RJS fibers, and the addition of BMMSCs. RJS fibers with nanoparticles and seeded with BMMSCs showed 10â¯136 mm3 of bone neoformation, meaning a 10-fold increase compared to using RJS only and BMMSCs (0.853 mm3) and a 5-fold increase from using ES only (2054 mm3) after 6 weeks of implantation. Conversely, none of these approaches used individually showed any significant difference for in vivo bone neoformation, suggesting that their combination is essential for optimizing bone formation. In summary, our work generated a potential material composed of well-defined combinations of suitable scaffolds seeded with BMMSCs for enhancing numerous orthopedic tissue engineering applications.
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Células Madre Mesenquimatosas , Nanotubos de Carbono , Animales , Huesos , Durapatita/farmacología , Poliésteres , Ratas , Andamios del TejidoRESUMEN
Endosseous implant surface topography directly affects adherent cell responses following implantation. The aim of this study was to examine the impact of nanoscale topographic modification of titanium implants on Osterix gene expression since this gene has been reported as key factor for bone formation. Titanium implants with smooth and nanoscale topographies were implanted in the femurs of Osterix-Cherry mice for 1-21 days. Implant integration was evaluated using scanning electron microscopy (SEM) to evaluate cell adhesion on implant surfaces, histology, and nanotomography (NanoCT) to observe and quantify the formed bone-to-implant interface, flow cytometry to quantify of Osterix expressing cells in adjacent tissues, and real-time PCR (qPCR) to quantify the osteoinductive and osteogenic gene expression of the implant-adherent cells. SEM revealed topography-dependent adhesion of cells at early timepoints. NanoCT demonstrated greater bone formation at nanoscale implants and interfacial osteogenesis was confirmed histologically at 7 and 14 days for both smooth and nanosurface implants. Flow cytometry revealed greater numbers of Osterix positive cells in femurs implanted with nanoscale versus smooth implants. Compared to smooth surface implants, nanoscale surface adherent cells expressed higher levels of Osterix (Osx), Alkaline phosphatase (Alp), Paired related homeobox (Prx1), Dentin matrix protein 1 (Dmp1), Bone sialoprotein (Bsp), and Osteocalcin (Ocn). In conclusion, nanoscale surface implants demonstrated greater bone formation associated with higher levels of Osterix expression over the 21-day healing period with direct evidence of surface-associated gene regulation involving a nanoscale-mediated osteoinductive pathway that utilizes Osterix to direct adherent cell osteoinduction.
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Implantes Dentales , Oseointegración , Animales , Ratones , Osteogénesis , Prótesis e Implantes , Propiedades de Superficie , Titanio/farmacologíaRESUMEN
Implant placement is an important repair method in dentistry and orthopedics. Increasing efforts have focused on optimizing the biocompatibility and osseointegration properties of titanium (Ti) and Ti-based alloys. In this work, Ti-based alloys were modified by the layer-by-layer (LbL) technique, which is a simple and versatile method for surface modification. The morphology and chemical structure of LbL films of poly(sodium 4-styrenesulfonate) (PSS) and Ti dioxide (TiO2) nanoparticles were first characterized employing ultraviolet-visible and Fourier-transform infrared spectroscopies as well as atomic force microscopy for further application in Ti-based alloy implants. The changes provoked by the LbL PSS/TiO2 film on the Ti-based alloy surfaces were then investigated by scanning electron microscopy and micro-Raman techniques. Finally, in vivo tests (immunolabeling and biomechanical analysis) performed with screw implants in rats suggested that PSS/TiO2 multilayers promote changes in both topography and chemical surface properties of the screw, providing beneficial effects for osteoblast activity. This simple and relatively low-cost growth process can open up possibilities to improve dental implants and, probably, bone implants in general.
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Aleaciones/química , Materiales Biocompatibles/química , Implantes Dentales , Poliestirenos/química , Titanio/química , Materiales Biocompatibles/síntesis química , Ensayo de Materiales , Tamaño de la PartículaRESUMEN
OBJECTIVES: This study aimed to evaluate the effects of nanohydroxyapatite (nHAp) particles on the morphological, chemical, physical, and biological properties of chitosan electrospun nanofibers. MATERIALS AND METHODS: nHAp particles with a 1.67 Ca/P ratio were synthesized via the aqueous precipitation method, incorporated into chitosan polymer solution (0.5 wt%), and electrospun into nHAp-loaded fibers (ChHa fibers). Neat chitosan fibers (nHAp-free, Ch fibers) were used as the control. The electrospun fiber mats were characterized using morphological, topographical, chemical, thermal, and a range of biological (antibacterial, antibiofilm, cell viability, and alkaline phosphatase [ALP] activity) analyses. Data were analyzed using ANOVA and Tukey's test (α = 0.05). RESULTS: ChHa fibers demonstrated a bead-like morphology, with thinner (331 ± 110 nm) and smoother (Ra = 2.9 ± 0.3 µm) distribution as compared to the control fibers. Despite showing similar cell viability and ALP activity to Ch fibers, the ChHa fibers demonstrated greater antibacterial potential against most tested bacteria (except for P. intermedia), and higher antibiofilm activity against P. gingivalis biofilm. CONCLUSIONS: The incorporation of nHAp particles did not jeopardize the overall morphology, topography, physical, and biological characteristics of the chitosan nanofibers. CLINICAL RELEVANCE: The combination of nHAp particles with chitosan can be used to engineer bioactive, electrospun composite nanofibers with potential applications in regenerative dentistry.
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Quitosano , Nanofibras , Quitosano/farmacología , Durapatita , PolímerosRESUMEN
Polycaprolactone (PCL) is a biocompatible, biodegradable synthetic polymer which in combination with nanohydroxyapatite (nHAp) can give rise to a low cost, nontoxic bioactive product with excellent mechanical properties and slow degradation. Here we produced, characterized and evaluated in vivo the bone formation of PCL/nHAp scaffolds produced by the rotary jet spinning technique. The scaffolds produced were firstly soaked into simulated body fluid for 21 days to also obtain nHAp onto PCL/nHAp scaffolds. Afterwards, the scaffolds were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy and Raman spectroscopy. For in vivo experiments, 20 male Wistar rats were used and randomly divided in 4 experimental groups (n = 5). A critical defect of 3 mm in diameter was made in the tibia of the animals, which were filled with G1 control (clot); G2-PCL scaffold; G3-PCL/nHAp (5%) scaffold; G4-PCL/nHAp (20%) scaffold. All animals were euthanized 60 days after surgery, and the bone repair in the right tibiae were evaluated by radiographic analysis, histological analysis and histomorphometric analysis. While in the left tibias, the areas of bone repair were submitted to the flexural strength test. Radiographic and histomorphometric analyses no showed statistical difference in new bone formation between the groups, but in the three-point flexural tests, the PCL/nHAp (20%) scaffold positively influenced the flexural mode of the neoformed bone. These findings indicate that PCL/nHAp (20%) scaffold improve biomechanical properties of neoformed bone and could be used for bone medicine regenerative.
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Líquidos Corporales/química , Durapatita/química , Resistencia Flexional , Osteogénesis , Poliésteres/química , Andamios del Tejido/química , Animales , Fenómenos Biomecánicos/efectos de los fármacos , Líquidos Corporales/fisiología , Regeneración Ósea/efectos de los fármacos , Sustitutos de Huesos/síntesis química , Sustitutos de Huesos/química , Sustitutos de Huesos/farmacología , Materiales Biocompatibles Revestidos/síntesis química , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Durapatita/farmacología , Resistencia Flexional/efectos de los fármacos , Resistencia Flexional/fisiología , Fracturas Óseas/fisiopatología , Fracturas Óseas/terapia , Regeneración Tisular Dirigida/instrumentación , Regeneración Tisular Dirigida/métodos , Masculino , Ensayo de Materiales , Nanoestructuras/química , Osteogénesis/efectos de los fármacos , Poliésteres/farmacología , Polímeros/síntesis química , Polímeros/química , Polímeros/farmacología , Ratas , Ratas Wistar , Estrés Mecánico , Tibia/patología , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodosRESUMEN
Herein, poly(É-caprolactone) (PCL) mats with different amounts of nanohydroxyapatite (nHAp) were produced using rotary-jet spinning (RJS) and evaluated in vitro and in vivo. The mean fiber diameters of the PCL, PCL/nHAp (3%), PCL/nHAp (5%), and PCL/nHAp (20%) scaffolds were 1847 ± 1039, 1817 ± 1044, 1294 ± 4274, and 845 ± 248 nm, respectively. Initially, all the scaffolds showed superhydrophobic behavior (contact angle around of 140oC), but decreased to 80° after 30 min. All the produced scaffolds were bioactive after soaking in simulated body fluid, especially PCL/nHAp (20%). The crystallinity of the PCL scaffolds decreased progressively from 46 to 21% after incorporation of 20% nHAp. In vitro and in vivo cytotoxicity were investigated, as well as the mats' ability to reduce bacteria biofilm formation. In vitro cellular differentiation was evaluated by measuring alkaline phosphatase activity and mineralized nodule formation. Overall, we identified the total ideal amount of nHAp to incorporate in PCL mats, which did not show in vitro or in vivo cytotoxicity and promoted lamellar bone formation independently of the amounts of nHAp. The scaffolds with nHAp showed reduced bacterial proliferation. Alizarin red staining was higher in materials associated with nHAp than in those without nHAp. Overall, this study demonstrates that PCL with nHAp prepared by RJS merits further evaluation for orthopedic applications.
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Materiales Biocompatibles/química , Durapatita/química , Nanoestructuras/química , Poliésteres/química , Animales , Antraquinonas/química , Biopelículas , Médula Ósea/efectos de los fármacos , Regeneración Ósea , Huesos/efectos de los fármacos , Cristalización , Masculino , Nanofibras/química , Osteogénesis , Ratas , Ratas Wistar , Temperatura , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
Poly (lactic acid) (PLA) has been increasingly used in cutaneous tissue engineering due to its low cost, ease of handling, biodegradability, and biocompatibility, as well as its ability to form composites. However, these polymers possess a structure with nanoporous that mimic the cellular environment. In this study, nanocomposites are prepared using PLA and titanium dioxide (TiO2) (10 and 35%-w/w) nanoparticles that also function as an active anti-scarring agent. The nanocomposites were prepared using an electrospinning technique. Three different solutions were prepared as follows: PLA, 10% PLA/TiO2, and 35% PLA/TiO2 (w/w%). Electrospun PLA and PLA/TiO2 nanocomposites were characterized morphologically, structurally, and chemically using electron scanning microscopy, transmission electron microscopy, goniometry, and X-ray diffraction. L929 fibroblast cells were used for in vitro tests. The cytotoxic effect was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Versicam (VCAN), biglicam (BIG), interleukin-6 (IL6), interleukin-10 (IL-10), and type-1 collagen (COL1A1) genes were evaluated by RT-qPCR. In vivo tests using Wistar rats were conducted for up to 15 days. Nanofibrous fibers were obtained for all groups that did not contain residual solvents. No cytotoxic effects were observed for up to 168 h. The genes expressed showed the highest values of versican and collagen-1 (p < 0.05) for PLA/TiO2 nanocomposite scaffolds when compared to the control group (cells). Histological images showed that PLA at 10 and 35% w/w led to a discrete inflammatory infiltration and expression of many newly formed vessels, indicating increased metabolic activity of this tissue. To summarize, this study supported the potential of PLA/TiO2 nanocomposites ability to reduce cutaneous scarring in scaffolds.
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The modification of biomaterials approved by the Food and Drug Administration could be an alternative to reduce the period of use in humans. Porous bioceramics are widely used as support structures for bone formation and repair. This composite has essential characteristics for an implant, including good mechanical properties, high chemical stability, biocompatibility and adequate aesthetic appearance. Here, three-dimensional porous scaffolds of Al2 O3 containing 5% by volume of ZrO2 were produced by the replica method. These scaffolds had their surfaces chemically treated with phosphoric acid and were coated with calcium phosphate using the biomimetic method simulated body fluid (SBF, 5×) for 14 days. The scaffolds, before and after biomimetic coating, were characterized mechanically, morphologically and structurally by axial compression tests, scanning electron microscopy, microtomography, apparent porosity, X-ray diffractometry, near-infrared spectroscopy, inductively coupled plasma optical emission spectroscopy, energy dispersive X-ray spectroscopy and reactivity. The in vitro cell viability and formation of mineralization nodules were used to identify the potential for bone regeneration. The produced scaffols after immersion in SBF were able to induce the nodules formation. These characteristics are advantaged by the formation of different phases of calcium phosphates on the material surface in a reduced incubation period. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2615-2624, 2018.
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Óxido de Aluminio , Materiales Biomiméticos , Cerámica , Materiales Biocompatibles Revestidos , Ensayo de Materiales , Circonio , Óxido de Aluminio/química , Óxido de Aluminio/farmacología , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Línea Celular Tumoral , Cerámica/química , Cerámica/farmacología , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Humanos , Porosidad , Circonio/química , Circonio/farmacologíaRESUMEN
Nanomaterials based on graphene oxide nanoribbons (GNR) and nanohydroxyapatite (nHAp) serve as attractive materials for bone tissue engineering. Herein, we evaluated the potential of nHAp/GNR toward in vitro analysis of specific genes related to osteogenesis and in vivo bone regeneration using animal model. Three different concentrations of nHAp/GNR composites were analyzed in vitro using a cytotoxicity assay, and osteogenic potential was determined by ALP, OPN, OCN, COL1, and RUNX2 genes and alkaline phosphatase assays. In vivo bone neoformation using a well-established in vivo rat tibia defect model was used to confirm the efficiency of the optimized composite. The scaffolds were nontoxic, and the osteogenesis process was dose-dependent (at 200 µg mL-1 of nHAp/GNR) compared to controls. The in vivo results showed higher bone neoformation after 15 days of nHAp/GNR implantation compared to all groups. After 21 days, both nHAp/GNR composites showed better lamellar bone formation compared to control. We attributed this enhanced bone neoformation to the high bioactivity and surface area presented by nHAp/GNR composites, which was systematically evaluated in previous studies. These new in vivo results suggest that nHAp/GNR composites can be exploited for a range of strategies for the improved development of novel dental and orthopedic bone grafts to accelerate bone regeneration.
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Combining polyester scaffolds with synthetic nanohydroxyapatite (nHAp), which is bioactive and osteoconductive, is a plausible strategy to improve bone regeneration. Here, we propose the combination of PBAT [poly(butylene-adipate-co-terephthalate)] and synthetic nHAp (at 3 and 5wt%). PBAT is a relatively a new polymer with low crystallinity and attractive biodegradability and mechanical properties for orthopedic applications, however, with a still underexplored potential for in vivo applications. Then, we performed a careful biological in vitro and in vivo set of experiments to evaluate the influence of PBAT containing two different nHAp loads. For in vitro assays, osteoblast-like MG63 cells were used and the bioactivity and gene expression related to osteogenesis were evaluated by qRT-PCR. For in vivo experiments, twenty-four male rats were used and a tibial defect model was applied to insert the scaffolds. Micro-computed tomography (Micro-CT) and histological analysis were used to assess e bone neoformation after 6 weeks of implantation. Three point flexural tests measured the mechanical properties of the neoformed bone. All scaffolds showed promising in vitro properties, since they were not cytotoxic against MG-63 cells and promoted high cell proliferation and formation of mineralized nodules. From a mechanistic point-of-view, nHAp loading increased hydrophilicity, which in turn allowed for a better adsorption of proteins and consequent changes in the phenotypic expression of osteoblasts. nHAp induced better cellular responses on/in the scaffolds, which was mainly attributed to its osteoconductive and osteoinductive properties. Micro-CT images showed that nHAp at 3% and 5wt% led to more effective bone formation, presenting the highest bone volume after 6 weeks of implantation. Considering the three point flexural tests, 5wt% of nHAp positively influenced the flexural mode of the neoformed bone, but the stiffiness was similar between the 3% and 5wt% groups. In summary, this investigation demonstrated great potential for the application of these novel scaffolds towards bone regeneration and, thus, should be further studied.
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Durapatita/química , Nanofibras/química , Poliésteres/química , Andamios del Tejido , Animales , Regeneración Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/fisiopatología , Línea Celular Tumoral , Durapatita/farmacología , Conductividad Eléctrica , Galvanoplastia/métodos , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Poliésteres/farmacología , Ratas , Tibia/efectos de los fármacos , Tibia/fisiopatologíaRESUMEN
This study analysed the initial effects of the combination of oestrogen deficiency with a calcium-deficient diet on alveolar bone repair. Sixty-three 3-month-old female rats were either ovariectomized (OVX, n = 42) or sham-operated (SHAM, n = 21). Among the 42 ovariectomized rats, 21 received standard commercial food (OVX) and 21 received food with low calcium content (ESP). The mandibular first molars were extracted bilaterally 15 days after ovariectomy or sham surgery. The rats were weighed and killed at 7, 21 and 45 days after tooth extraction. The results were evaluated by descriptive microscopic analysis, histomorphometry of the trabecular and osteoid volume and mast cell counts. Analysis of the results indicated that trabecular volume and mast cell counts increased significantly over time, while osteoid volume decreased over time. Comparisons between the SHAM and OVX groups demonstrated no statistical differences, while comparison between the OVX and ESP groups indicated differences in trabecular volume and the number of mast cells. The data suggest that hormonal deficiency does not delay alveolar bone repair in OVX rats; however, oestrogen deficiency associated with calcium deficiency can lead to bone resorption through the activation of mast cells.
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Regeneración Ósea , Calcio de la Dieta/administración & dosificación , Hipocalcemia/patología , Mandíbula/patología , Osteoporosis/patología , Alveolo Dental/patología , Animales , Resorción Ósea/patología , Resorción Ósea/fisiopatología , Modelos Animales de Enfermedad , Estrógenos/deficiencia , Femenino , Hipocalcemia/complicaciones , Hipocalcemia/fisiopatología , Mandíbula/fisiopatología , Diente Molar , Osteoporosis/complicaciones , Osteoporosis/fisiopatología , Ovariectomía , Ratas , Ratas Wistar , Extracción DentalRESUMEN
The fixation and the bone ingrowth at the interface of porous cylindrical implants (total porosity of 37% and average pores diameter of 480 microm) were compared in vivo to rough cylindrical implants (R (a) = 5.3 microm), both of commercially pure titanium, made by powder metallurgy. The implants were inserted into the tibias of 20 rabbits and the animals were sacrificed 4 and 8 weeks after surgery. The percentage of bone-implant contact observed in porous implant was significantly larger than in the rough ones for all of sacrifice periods, respectively, 57% vs. 46% after 4 weeks, and 59% vs. 50% after 8 weeks. The mechanical tests showed a significant increase in the shear strength of the porous implants for the two analyzed periods, 4 and 8 weeks (14 and 20 MPa), when compared with rough ones (4 and 13 MPa). These results suggest that porous implants improve the contact at the implant-bone interface and increase the fixation to the bone, improving the osseointegration. Thus, the porous implant might be an alternative to dental implant in less favorable conditions, and appear to be better fixed to bone, offering promising alternatives.