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BACKGROUND:For the replacement treatment of long-segment tracheal defects,although tissue engineering research has made some progress in recent years,it is still not perfect,and one of the biggest difficulties is that the hemodynamic reconstruction of the tracheal replacement cannot be achieved rapidly. OBJECTIVE:To preliminarily explore the potential of polycaprolactone scaffolds modified with exosome-loaded hydrogels to construct a rapidly vascularized tracheal substitute. METHODS:Exosomes were extracted from bone marrow mesenchymal stem cells of SD rats.After preparation of hyaluronic acid methacrylate solution,the exosome solution was mixed with hyaluronic acid methacrylate solution at a volume ratio of 1:1.Hyaluronic acid methacrylate hydrogels loaded with exosomes were prepared under ultraviolet irradiation for 5 minutes.The degradation of exosome-unloaded hydrogels and the controlled release of exosome-loaded hydrogels were detected.Polycaprolactone scaffolds were prepared by 3D printing.The pure hyaluronic acid methacrylate solution and the exosome-loaded hyaluronic acid methacrylate solution were respectively added to the surface of the scaffold.Hydrogel-modified scaffolds and exosome-modified scaffolds were obtained after ultraviolet irradiation.Thirty SD rats were randomly divided into three groups with 10 rats in each group and subcutaneously implanted with simple scaffolds,hydrogel-modified scaffolds and exosome-modified scaffolds,respectively.At 30 days after surgery,the scaffolds and surrounding tissues of each group were removed.Neovascularization was observed by hematoxylin-eosin staining and Masson staining and the expression of CD31 was detected by immunofluorescence. RESULTS AND CONCLUSION:(1)As time went by,the hydrogel degraded gradually,and the exosomes enclosed in the hydrogel were gradually released,which could be sustained for more than 30 days.The exosome release rate was faster than the degradation rate of the hydrogel itself,and nearly 20%of the exosomes were still not released after 30 days of soaking.(2)Under a scanning electron microscope,the surface of the simple polycaprolactone scaffold was rough.After hydrogel modification,a layer of gel was covered between the pores of the scaffold,and the scaffold surface became smooth and dense.(3)After 30 days of subcutaneous embedding,hematoxylin-eosin staining and Masson staining showed that more neovascularization was observed inside the scaffolds of the exosome-modified scaffold group compared with the hydrogel-modified scaffold group.The hydrogels on the scaffolds of the two groups were not completely degraded.Immunofluorescence staining showed that CD31 expression in the exosome-modified scaffold group was higher than that in the hydrogel-modified scaffold group(P<0.000 1).(4)These results indicate that hyaluronic acid methacrylate hydrogels can be used as controlled-release carriers for exosomes.The 3D-printed polycaprolactone scaffold modified by hyaluronic acid methacrylate hydrogel loaded with exosomes has good biocompatibility and has the potential to promote the formation of neovascularization.
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BACKGROUND:In recent years,there have been many novel tympanic membrane repair materials,including patches and 3D-printed scaffolds.However,the tympanic membrane repaired by these materials is different from the natural tympanic membrane in terms of thickness and internal structure. OBJECTIVE:To explore the efficacy of bone marrow mesenchymal stem cells-loaded high-porosity polycaprolactone/collagen nanofiber membrane scaffolds in repairing chronic tympanic membrane perforation. METHODS:Polycaprolactone,polycaprolactone-collagen,and high-porosity polycaprolactone-collagen nanofiber membranes were prepared by electrospinning technology,and the surface morphology,porosity and cell compatibility of the scaffolds were characterized.The tympanic membrane perforation model of 50 male SD rats was established by puncturing the posterior lower part of both eardrums with a sterile 23-measure needle combined with mitomycin C and hydrocortisone.After 12 weeks of modeling,the rats were divided into five groups by the random number table method.The blank control group did not receive any treatment.In the other four groups,polycaprolactone nanofiber membrane(polycaprolactone group),polycaprolactone-collagen nanofiber membrane(polycaprolactone-collagen group),high-porosity polycaprolactone-collagen nanofiber membrane(high-porosity polycaprolactone-collagen group)and high-porosity polycaprolactone-collagen nanofiber membrane containing bone marrow mesenchymal stem cells(high-porosity polycaprolactone-collagen group)were implanted at the perforation of the tympanic membrane,respectively.Each group consisted of 10 animals.The healing of the tympanic membrane was examined by otoendoscopy after 1,2,3 and 4 weeks of stent implantation.Hematoxylin-eosin staining,Masson staining,and Ki-67 immunohistochemical staining were performed on the tympanic membrane after 4 weeks of implantation. RESULTS AND CONCLUSION:(1)Scaffold characterization:Scanning electron microscopy showed that compared with other nanofiber membranes,the high-porosity polycaprolactone-collagen nanofiber membranes had more orderly nanofiber structure,larger surface pore size,and higher porosity(P<0.001).Live/dead staining showed that bone marrow mesenchymal stem cells adhered well on the three scaffolds,and the number of living cells on the high-porosity polycaprolactone-collagen nanofiber membrane was more than that on the other two scaffolds.Almarin staining showed that the proliferation rate of bone marrow mesenchymal stem cells on the high-porosity polycaprolactone-collagen nanofiber membrane was higher than that of the other two fiber membranes.(2)Animal experiments:Except for the blank control group,the tympanic membrane of the other four groups healed gradually with the extension of the time of fibrous membrane implantation,among which the healing speed of the cell-loaded high-porosity polycaprolactone-collagen group was the fastest.Hematoxylin-eosin staining,Masson staining,and Ki-67 immunohistochemical staining showed that the tympanic membrane of rats in the cell-carrying high-porosity polycaprolactone-collagen group was moderate in thickness and a three-layer structure with uniform collagen fiber layers,similar to the normal tympanic membrane,and the repair quality of tympanic membrane was better than that of other fiber membrane groups.(3)The results showed that the high-porosity polycaprolactone-collagen nanofiber membrane containing bone marrow mesenchymal stem cells could not only rapidly repair the perforation of the tympanic membrane,but also the newly healed tympanic membrane was similar to normal tympanic membrane in structure and thickness.
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BACKGROUND:Oral and maxillofacial bone tissue defects can seriously affect the physical and mental health of patients.When bone defects occur in diabetic patients,bone metabolism disorders caused by abnormal blood sugar make it more difficult to repair and treat. OBJECTIVE:To attempt to apply AOPDM1,a polypeptide with potential bioactivity to the osteogenic treatment of diabetic patients. METHODS:In normal or high-glucose environment,different concentrations of AOPDM1 were used to interfere with mouse bone marrow mesenchymal stem cells,and cell proliferation,alkaline phosphatase activity,mineralization nodules formation and osteogenic differentiation gene expression were detected.The polycaprolactone scaffold was prepared by electrospinning technology,and the scaffold was modified by polydopamine to prepare the polycaprolactone-polydopamine composite scaffold.Finally,the scaffolds were placed in AOPDM1 solution to prepare polycaprolactone-polydopamine-AOPDM1 scaffolds.The water contact angle and mechanical properties of the scaffolds were tested in the three groups.In normal or high-glucose environment,the three groups of scaffolds were co-cultured with mouse bone marrow mesenchymal stem cells,respectively,and cell adhesion,alkaline phosphatase activity and osteopontin expression were detected. RESULTS AND CONCLUSION:(1)Compared with normal environment,high-glucose environment inhibited the proliferation of bone marrow mesenchymal stem cells.In the same environment,AOPDM1 could promote the proliferation of mouse bone marrow mesenchymal stem cells.When AOPDM1 concentration was the same,alkaline phosphatase activity,mineralization ability and mRNA expression of type Ⅰ collagen,osteopontin,alkaline phosphatase,and Runx2 of bone marrow mesenchymal stem cells were decreased in high-glucose environment compared with normal environment.Under the same environment,AOPDM1 could improve the alkaline phosphatase activity,mineralization ability,and mRNA expression of type Ⅰ collagen,osteopontin,alkaline phosphatase and Runx2 of bone marrow mesenchymal stem cells.(2)The hydrophilicity of polycaprolactone-polydopamine scaffold and polycaprolactone-polydopamine-AOPDM1 scaffold was higher than that of polycaprolactone scaffold(P<0.001),and there was no significant difference in tensile strength and elastic modulus among the three groups(P>0.05).Compared with the other two groups of scaffolds,the cells on the polycaprolactone-polydopamine-AOPDM1 scaffold had better adhesion morphology.When the scaffolds were identical,compared with normal environment,high-glucose environment inhibited alkaline phosphatase activity and osteopontin expression of bone marrow mesenchymal stem cells.When the environment was the same,the alkaline phosphatase activity and osteopontin expression of bone marrow mesenchymal stem cells on the polycaprolactone-polydopamine-AOPDM1 scaffold were higher than those on the other two scaffolds.(3)The above results prove that polycaprolactone-polydopamine-AOPDM composite scaffold can promote the osteogenic properties of bone marrow mesenchymal stem cells in high-glucose environment.
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【Objective】 To solve the problem of insufficient hydrophilicity on the surface of polycaprolactone (PCL)/β-TCP bone tissue engineering scaffolds, NaOH etching method was used to improve the surface microstructure of 3D printed PCL/β-TCP scaffolds, further affecting their hydrophilicity and cell response. 【Methods】 PCL/β-TCP mesh scaffolds were prepared using 3D printing melt deposition molding technology, and the surface roughness of the scaffolds was modified by NaOH etching. The effects of two reaction parameters, NaOH concentration and time, on the microstructure, spectral elements, contact angle, compressive strength, and cell adhesion of the scaffolds before and after modification were observed. 【Results】 After NaOH etching, the surface microporous structure of the mesh scaffold was successfully prepared. With the increase of either NaOH concentration or time, the surface micropores of the scaffold increased while the contact angle of the material surface decreased. However, the compression strength of the etched scaffold treated with NaOH for 1 mol/L (24 h) or 10 mol/L (6 h) was not statistically significant compared to the untreated group (P>0.05). The number of cells on the etched scaffold increased, with a larger spreading area of individual cells, making it more advantageous in the adhesion and proliferation of BMSCs. 【Conclusion】 The use of NaOH etching to improve the hydrophilicity of 3D printed PCL/β-TCP bone tissue engineering scaffolds is a low-cost and effective strategy which can effectively improve the wettability and cell adhesion of the scaffolds.
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Objective@#To evaluate the bone repair effect of 3D-printed magnesium (Mg)-loaded polycaprolactone (PCL) scaffolds in a rat skull defect model.@*Methods@#PCL scaffolds mixed with Mg microparticles were prepared by using 3D printing technology, as were pure PCL scaffolds. The surface morphologies of the two scaffolds were observed by scanning electron microscopy (SEM), and the surface elemental composition was analyzed via energy dispersive spectroscopy (EDS). The physical properties of the scaffolds were characterized through contact angle measurements and an electronic universal testing machine. This study has been reviewed and approved by the Ethics Committee. A critical size defect model was established in the skull of 15 Sprague-Dawley (SD) rats, which were divided into the PCL group, PCL-Mg group, and untreated group, with 5 rats in each group. Micro-CT scanning was performed to detect and analyze skull defect healing at 4 and 8 weeks after surgery, and samples from the skull defect area and major organs of the rats were obtained for histological staining at 8 weeks after surgery.@*Results@#The scaffolds had a pore size of (480 ± 25) μm, a fiber diameter of (300 ± 25) μm, and a porosity of approximately 66%. The PCL-Mg scaffolds contained 1.0 At% Mg, indicating successful incorporation of Mg microparticles. The contact angle of the PCL-Mg scaffolds was 68.97° ± 1.39°, indicating improved wettability compared to that of pure PCL scaffolds. Additionally, compared with that of pure PCL scaffolds, the compressive modulus of the PCL-Mg scaffolds was (57.37 ± 8.33) MPa, demonstrating enhanced strength. The PCL-Mg group exhibited the best bone formation behavior in the skull defect area compared with the control group and PCL group at 4 and 8 weeks after surgery. Moreover, quantitative parameters, such as bone volume (BV), bone volume/total volume (BV/TV), bone surface (BS), bone surface/total volume (BS/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and bone mineral density (BMD), of skull defects were better than those in the other groups, indicating the best bone regeneration effect. H&E, Goldner, and VG staining revealed more mineralized new bone formation in the PCL-Mg group than in the other groups, and H&E staining of the major organs revealed good biosafety of the material.@*Conclusion@#PCL-Mg scaffolds can promote the repair of bone defects and have clinical potential as a new scaffold material for the repair of maxillofacial bone defects.
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OBJECTIVES@#To explore the physicochemical characteristics and biocompatibility of calcium peroxide (CPO)-loaded polycaprolactone (PCL) microparticle.@*METHODS@#The CPO/PCL particles were prepared. The morphology and elemental distribution of CPO, PCL and CPO/PCL particles were observed with scanning electron microscopy and energy dispersive spectroscopy, respectively. Rat adipose mesenchymal stem cells were isolated and treated with different concentrations (0.10%, 0.25%, 0.50%, 1.00%) of CPO or CPO/PCL particles. The mesenchymal stem cells were cultured in normal media or osteogenic differentiation media under the hypoxia/normoxia conditions, and the amount of released O2 and H2O2 after CPO/PCL treatment were detected. The gene expressions of alkaline phosphatase (ALP), Runt-associated transcription factor 2 (RUNX2), osteopontin (OPN) and osteocalcin (OCN) were detected by realtime RT-PCR. SD rats were subcutaneously injected with 1.00% CPO/PCL particles and the pathological changes and infiltration of immune cells were observed with HE staining and immunohistochemistry at day 7 and day 14 after injection.@*RESULTS@#Scanning electron microscope showed that CPO particles had a polygonal structure, PCL particles were in a small spherical plastic particle state, and CPO/PCL particles had a block-like crystal structure. Energy dispersive spectroscopy revealed that PCL particles showed no calcium mapping, while CPO/PCL particles showed obvious and uniform calcium mapping. The concentrations of O2 and H2O2 released by CPO/PCL particles were lower than those of CPO group, and the oxygen release time was longer. The expressions of Alp, Runx2, Ocn and Opn increased with the higher content of CPO/PCL particles under hypoxia in osteogenic differentiation culture and normal culture, and the induction was more obvious under osteogenic differentiation conditions (all P<0.05). HE staining results showed that the muscle tissue fibers around the injection site were scattered and disorderly distributed, with varying sizes and thicknesses at day 7 after particle injection. Significant vascular congestion, widened gaps, mild interstitial congestion, local edema, inflammatory cell infiltration, and large area vacuolization were observed in some tissues of rats. At day 14 after microparticle injection, the muscle tissue around the injection site and the tissue fibers at the microparticle implantation site were arranged neatly, and the gap size was not thickened, the vascular congestion, local inflammatory cell infiltration, and vacuolization were significantly improved compared with those at day 7. The immunohistochemical staining results showed that the expressions of CD3 and CD68 positive cells significantly increased in the surrounding muscle tissue, and were densely distributed in a large area at day 7 after particle injection. At day 14 of microparticle injection, the numbers of CD3 and CD68 positive cells in peripheral muscle tissue and tissue at the site of particle implantation were lower than those at day 7 (all P<0.01).@*CONCLUSIONS@#CPO/PCL particles have good oxygen release activity, low damage to tissue, and excellent biocompatibility.
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Ratas , Animales , Osteogénesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Ratas Sprague-Dawley , Peróxido de Hidrógeno/farmacología , Diferenciación Celular , Oxígeno , Hipoxia , Células CultivadasRESUMEN
@#Objective To provide experimental data and theoretical support for further studying the maturity of cardiac patches in other in vitro experiments and the safety in other in vivo animal experiments, through standard chemically defined and small molecule-based induction protocol (CDM3) for promoting the differentiation of human induced pluripotent stem cells (hiPSCs) into myocardium, and preliminarily preparing cardiac patches. Methods After resuscitation, culture and identification of hiPSCs, they were inoculated on the matrigel-coated polycaprolactone (PCL). After 24 hours, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and the stemness of hiPSCs was identified by OCT4 fluorescence. After fixation, electron microscope scanning was performed to observe the cell morphology on the surface of the patch. On the 1st, 3rd, 5th, and 7th days of culture, the cell viability was determined by CCK-8 method, and the growth curve was drawn to observe the cell growth and proliferation. After co-cultured with matrigel-coated PCL for 24 hours, hiPSCs were divided into a control group and a CDM3 group, and continued to culture for 6 days. On the 8th day, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and hiPSCs stemness was identified by OCT4 fluorescence, and cTnT and α-actin for cardiomyocyte marker identification. Results Immunofluorescence of hiPSCs co-cultured with matrigel-coated PCL for 24 hours showed that OCT4 emitted green fluorescence, and hiPSCs remained stemness on matrigel-coated PCL scaffolds. DAPI emitted blue fluorescence: cells grew clonally with uniform cell morphology. Scanning electron microscope showed that hiPSCs adhered and grew on matrigel-coated PCL, the cell outline was clearly visible, and the morphology was normal. The cell viability assay by CCK-8 method showed that hiPSCs proliferated and grew on PCL scaffolds coated with matrigel. After 6 days of culture in the control group and the CDM3 group, immunofluorescence showed that the hiPSCs in the control group highly expressed the stem cell stemness marker OCT4, but did not express the cardiac markers cTnT and α-actin. The CDM3 group obviously expressed the cardiac markers cTnT and α-actin, but did not express the stem cell stemness marker OCT4. Conclusion hiPSCs can proliferate and grow on matrigel-coated PCL. Under the influence of CDM3, hiPSCs can be differentiated into cardiomyocyte-like cells, and the preliminary preparation of cardiac patch can provide a better treatment method for further clinical treatment of cardiac infarction.
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Abstract In an attempt to increase molecular stability and provide controlled release, vascular endothelial growth factor (VEGF) was encapsulated into polycaprolactone (PCL) nanoparticles. Both VEGF-free and VEGF-loaded PCL nanoparticles were formulated by w/o/w double emulsion of the dichloromethane-water system in the presence of polyvinyl alcohol (PVA) and rat serum albumin. To achieve the optimal formulation concerning particle size and monodispersity, studies were carried out with different formulation parameters, including PVA concentration, homogenization time and rate. Scanning electron microscopy and dynamic light scattering analysis showed respectively that particles had a spherical shape with a smooth surface and particle size varying between 58.68-751.9 nm. All of the formulations were negatively charged according to zeta potential analysis. In vitro release study was performed in pH 7.4 phosphate-buffered saline at 37°C and released VEGF amount was measured by enzyme-linked immunosorbent assay (ELISA) method. At the end of the 35th day, 10% of total encapsulated VEGF was released with a sustained-release profile, which fitted the Korsmeyer-Peppas kinetic model. The bioactivation of the nanoparticles was evaluated using XTT and ELISA methods. As a result, the released VEGF was biologically active and also VEGF loaded PCL nanoparticles enhanced proliferation of the human umbilical vein endothelial cells in cell culture.
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Factor A de Crecimiento Endotelial Vascular , Nanopartículas/clasificación , Técnicas In Vitro/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Microscopía Electrónica de Rastreo/métodos , Técnicas de Cultivo de Célula/métodos , Células Endoteliales de la Vena Umbilical HumanaRESUMEN
Abstract Risperidone is an atypical antipsychotic drug widely prescribed all over the world due to its clinical advantages. The currently available long acting marketed depot formulation of risperidone is a microsphere based preparation using poly-[lactide-co-glycolide] (PLGA) as drug release barrier. It is however, a cold chain product due to thermal instability of PLGA at room temperature. After beginning the depot injection therapy it is administered every two weeks but associated with another drawback of about 3 weeks lag time due to which its tablets are also administered for three weeks so as to attain and maintain therapeutic drug concentration in the body. The present work attempts to develop a long acting depot delivery system of risperidone for once a month administration based on the combination of sucrose acetate isobutyrate and polycaprolactone dissolved in benzyl benzoate to provide an effective drug release barrier for one month without any lag time and which can be stored at room temperature precluding the requirement of cold supply chain. The developed depot formulation showed a sustained in vitro drug release profile with 88.95% cumulative drug release in 30 days with little burst release. The in vivo pharmacokinetic studies of the developed formulation conducted on rats showed attainment of mean peak plasma drug concentration of 459.7 ng/mL in 3 days with a mean residence time of 31.2 days, terminal half-life of 20.6 days, terminal elimination rate constant of 0.0336 per day, and a good in vitro- in vivo correlation.
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Preparaciones Farmacéuticas/administración & dosificación , Risperidona/agonistas , Sacarosa , Técnicas In Vitro/métodos , Liberación de Fármacos/efectos de los fármacosRESUMEN
Objective:For severe skin defects which are deep to dermis, engineered skin with epidermis and dermis (bilayered) is required. Based on the success of engineering epidermis with GT/PCL electrospun membranes, our study was to investigate whether this membrane could be also used for engineering bilayered skin graft.Methods:From 2013 to 2019, we first prepared three GT/PCL electrospun membranes with different proportion (70∶30; 50∶50; 30∶70) in our laboratory; the biocompatibility of the membrane was evaluated in vitro by seeding fibroblasts or keratinocytes on the membranes. Then the outcome of GT/PCL membranes repairing skin defects in the nude mouse was investigated.Results:Cell attachment and proliferation were significantly improved with increase of gelatin. Histological analyses showed that bilayered skin engineered with GT/PCL (70∶30) group could form relatively better structure after 3 weeks of cultivation in vitro. Further in vivo transplantation studies revealed that scaffolds were not degraded in all three groups, indicating that these materials were not suitable for engineering bilayered skin although they had good biocompatibility.Conclusions:The higher gelatin membranes possess better biocompatibility. Further in vivo transplantation studies reveal that bilayered skin engineered with GT/PCL membranes is able to repair skin defects in the nude mouse.
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@#Surgery as an art in rhinoplasty involves grafting techniques wherein materials (usually autologous) are taken from the septum and supplemented by conchal cartilage. However, not all noses have adequate cartilage material. The quest for materials as possible replacement for human tissue have led to invention of synthetic (e.g. silicone, e-PTFE, porous polyethelene) and non-synthetic products (e.g. processed homograft and xenograft). In this era of advanced medical science, tissue engineering has started the use polycaprolactone (PCL) as a template and scaffold for tissue growth. Because of this characteristic feature, PCL as a mesh has a significant role in structural rhinoplasty.
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Rinoplastia , NarizRESUMEN
Objective:To investigate the repair effect of electrospun gelatin polycaprolactone (GT/PCL) nanofiber aerogels (NFA) combined with cartilage extracellular matrix (ECM) for treatment of cartilage injuries in rabbits.Methods:Firstly, the GT/PCL electrospun membrane was prepared by electrospinning and was ground into the short fiber at high speed. ECM was extracted and separated from fresh bovine articular cartilage, which mixed with the short fiber solution (10 ∶1). Subsequently, it was used to prepared GT/PCL/ECM (NFA) three-dimensional scaffold. Finally, the physical characteristics of the three different scaffolds (GT/PCL, ECM and GT/PCL/ECM) were detected by scanning electron microscope and Fourier-transform infrared (FTIR) spectrometer, including the composition, microstructure, swelling rate, porosity, compressive strength and degradation rates. And the biocompatibility research was getting on by co-culturing the scaffold with chondrocytes. Fifteen male New Zealand white rabbits were divided into blank control group (Group A, n=5), ECM group (group B, n=5) and composite scaffold(GT/PCL/ECM)group (Group C, n=5) according to the random number table. An injury model was established and three types of bio-scaffold materials were implanted into different groups. At 3 weeks, the cartilage repair was evaluated among groups by semi quantitative global MRI scoring system (WORMS). After the animals were killed, the knee joints of each group were scored by the international society for cartilage repair histological score (ICRs); the ICRs histological score was performedby HE staining and safranine green staining. Results:Three scaffolds showed a porous three-dimensional structure under the scanning electron microscope. FIRT showed that GT and PCL were introduced into the scaffolds successfully. The GT/PCL NAF was loose and unable to be characterized by materials science. The swelling rate of GT/PCL/ECM scaffold [(1, 092.0±32.2)%] was higher than that of ECM scaffold [(933.6±16.3)%] ( P<0.01). The porosity of GT/PCL/ECM scaffold [(92.3±2.3)%] was higher than that of ECM scaffold [(85.9±2.2)%] ( P<0.05). The compressive strength of ECM scaffold [(2.7±0.1)kPa] and of GT/PCL/ECM scaffold [(2.4±0.1)kPa] showed no statistical difference ( P>0.05). The degradation rate of ECM scaffold was higher than that of GT/PCL/ECM scaffold, but the difference was not statistically significant ( P>0.05). The cytotoxicity rating of GT/PCL/ECM scaffold was grade I, indicating that its biocompatibility was better. At 3 weeks, the MRI WORMS score in Group C [(49.0±11.4)points] was significantly higher than that in Group B [(40.0±6.7)points] and that in Group A [(24.0±6.5) points] ( P<0.05 or 0.01); the general ICRS score of group C was [(7.4±1.1) points], which was significantly higher than that of group B [(4.6±1.1)points] and group A [(3.0±1.2)points] ( P<0.01); The ICRS histological scores of group C and group B were [(6.8±0.8)points] and [(4.2±0.8)points] respectively compared with group A [(2.8±0.8)points] were significantly higher ( P<0.05 or 0.01). Conclusion:GT/PCL/ECM (NFA) scaffold has similar tissue structure to natural cartilage and is superior to traditional ECM scaffold in physical properties and biocompatibility, which provides a stable environment for chondrocyte adhesion and growth, promotes collagen regeneration, and thus accelerates the repair of cartilage injury.
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Objective: To prepare graphene oxide(GO)/polycaprolactone(PCL)composite matrix nanomaterials with different concentrations, and investigate effects of the nanomaterials on the myocardial differentiation of rat brown adipose stem cells (BASC)in vitro. Methods: The GO/PCL composite nanofiber materials were prepared by electrospinning technology. The biocompatibility of the nanomaterials was tested by the CCK-8 assay after cultivation of the BASC on the pure PCL and GO/PCL composite nanofibers, and the characterization of the nanofiber materials was performed by the scanning electron microscopy(SEM)and electrical conductivity measurement. The expression of cardiomyocyte-related proteins was examined by the cytological method and immunofluorescence staining. The GO/PCL composite nanofiber materials were systematically evaluated for their effect on the viability, proliferation and myocardial differentiation of BASC. Results: The GO/PCL composite nanofiber materials showed no obvious cytotoxicity at the 0.1 mg/ml GO concentration. The statistical results for the protein fluorescence intensities showed that the expression of the cardiomyocyte specific protein, α-actinin, and the intercalated disc-related protein, connexin-43(CX-43), was significantly increased in the GO/PCL group than in the PCL group(P<0.05). The cytoskeletal staining results showed that, compared with the PCL group, the cells in the GO/PCL group showed a long spindle-like stretch similar to the natural myocardial cell bundle, and the growth direction had a certain polarity. Conclusion: This study successfully prepared GO/PCL composite nanomaterials, which could promote the differentiation of BASCs into cardiomyocytes and the expression of intercalated disc-related proteins.
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BACKGROUND: Functional tracheal reconstruction remains a surgical challenge due to the lack of satisfactory tracheal substitutes. OBJECTIVE: To review the research hotspot, clinical application, and main obstacles of tissue-engineered trachea METHODS: A computer-based search of PubMed, Medline, and WanFang databases was performed to retrieve relevant articles published from 2004 to 2019 with the search terms “3D printing, tissue-engineered trachea, trachea reconstruction, tracheal replacement” in English and Chinese. A total of 47 literatures were included in the final analysis. RESULTS AND CONCLUSION: At present, the methods of tracheal reconstruction mainly include artificial tracheal transplantation, allotransplantation, autologous tissue transplantation and tissue-engineered tracheal transplantation. Artificial trachea transplants often fail due to rupture, infection and narrowing of the trachea. Allotransplantation requires long-term immunosuppressive therapy, and death is often caused by necrosis and infection because of insufficient angiogenesis after transplantation. Autogenous tissue has limited ability to replicate the structure and function of the trachea and also has surgical trauma. Tissue-engineered trachea can simulate the biological structure and function similar to natural trachea by selecting suitable scaffold materials and implanting seed cells evenly in the scaffold. It seems to be an ideal tracheal substitute. An intact tracheal scaffold was prepared with biodegradable material using 3D printing technology combined with tissue engineering technology and then implanted into the tissue-engineered trachea cultured with mesenchymal stem cells. This provides a new approach to long-segment tracheal defect reconstruction.
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BACKGROUND: At present, there are many types of bone defect repair scaffolds, but a single type of material is difficult to meet the requirements of bone tissue engineering scaffold materials. Several suitable materials can be combined into a composite material by appropriate methods, taking into account the advantages and disadvantages of various materials. It is the focus of scholars in recent years. OBJECTIVE: To construct nano-hydroxyapatite/chitosan/polycaprolactone composite scaffolds and analyze characterization of composite scaffolds. METHODS: Nano-hydroxyapatite/chitosan/polycaprolactone porous ternary composite scaffold material was prepared by 3D printing and molding technology. The characterization of scaffold material was studied from X-ray diffraction analysis, stent water absorption rate, stent compressive strength, stent degradation performance in vitro, stent aperture analysis, scanning electron microscope analysis and other dimensions. RESULTS AND CONCLUSION: (1) X-ray diffraction analysis showed that the crystal-shaped peak map of nano-hydroxyapatite/chitosan/ polycaprolactone scaffold materials was similar to the hydroxyapatite powder diffraction standard card, suggesting that the scaffold materials were integrated with each other through physical interaction, and did not affect the biological function of hydroxyapatite. (2) The average water absorption rate of the scaffold was 18.28%, and the hydrophilicity was good. The maximum pressure that the scaffold could withstand was 1 415 N, and the degradation rate was similar to the osteogenic rate. (3) Under a microscope, a ternary scaffold material with an aperture of 250 µm was successfully produced. The pore size was uniform and distributed regularly. (4) Scanning electron microscope demonstrated that the fibers composed of chitosan and polycaprolactone were arranged orderly and grid like, hydroxyapatite was distributed uniformly on the fiber surface in granular form, and the ternary composite material presented uniform and loose microporous structure. (5) Nano-hydroxyapatite/chitosan/polycaprolactone ternary composite scaffold material can be successfully prepared through 3D printing and molding technology, which has moderate compressive strength, certain porosity, appropriate degradation rate and water absorption rate, and can lay a foundation for repairing bone defects.
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BACKGROUND: It is still difficult to construct tissue-engineered anulus fibrosus scaffolds which have bionic structure, suitable biodegradability and good biocompatibility. OBJECTIVE: To fabricate bionic biodegradable scaffolds with polycaprolactone (PCL) and polydioxanone (PDS) and evaluate the feasibility as a tissue-engineered annulus fibrosus scaffold. METHODS: Five groups of scaffolds at different PCL/PDS proportions were prepared by melt spinning technique: PCL, PCL/PDS70/30, PCL/PDS50/50, PCL/PDS30/70, and PDS groups. Scanning electron microscopy was used to observe the structure and measure the fiber diameter and pore size of these prepared scaffolds. The mechanical properties and contact angle of the scaffolds were measured. The in vitro and in vivo biodegradation of the scaffolds were observation by in vitro simulation and subcutaneous implantation. The expression of inflammatory factors interleukin-1β and tumor necrosis factor-α in the biodegraded tissues was detected. Human Wharton’s jelly mesenchymal stem cells were cultured for 7 days. Cell viability and proliferation was determined by live/dead cell staining. This study was approved by the Medical Ethics Committee of Tianjin Hospital, China on March 2, 2016. RESULTS AND CONCLUSION: Scanning electron microscopy results showed that the thickness of the scaffold fibers was uniform and the angle between fibers was 60°. The mechanical properties analysis showed that the tensile and compressive modulus of the PDS group was the lowest, which did not meet the mechanical requirements of the anulus fibrosus; the tensile and compressive modulus in the PCL group was the highest, and those in the PCL/PDS70/30 and PCL/PDS50/50 group were moderate. Hydrophilicity test showed that higher PDS proportion led to better hydrophilicity. Biodegradation test showed that the biodegradation of pure PDS and PCL/PDS30/70 was too fast, that of PCL was too slow, and that of PCL/PDS70/30 and PCL/PDS50/50 was appropriate. Analysis of inflammatory response around the biodegraded tissue showed that higher proportion of PCL in the scaffold resulted in more severe inflammatory response. CCK-8 and live/dead cell staining showed that human Wharton’s jelly mesenchymal stem cells had good proliferative activity and high survival rate in the PCL/PDS70/30, PCL/PDS50/50, and PCL/PDS30/70 groups. These results suggest that scaffolds in the PCL/PDS70/30 and PCL/PDS50/50 groups can simulate the structure of natural annulus fibrosus, have appropriate biodegradability, excellent mechanical properties and good biocompatibility, which make it a suitable candidate for tissue-engineered annulus fibrosus scaffold.
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BACKGROUND: Polycaprolactone/nano-hydroxyapatite composite is a new composite scaffold material prepared based on common bone tissue engineering materials using 3D printing technology. At present, little is reported on the in vitro biocompatibility of the composite material. OBJECTIVE: To investigate the cytocompatibility of 3D printed polycaprolactone/nano-hydroxyapatite composite scaffolds. METHODS: Polycaprolactone and polycaprolactone/nano-hydroxyapatite composite scaffolds were prepared by 3D printing technology to characterize the microstructure, porosity and mechanical properties of the two materials. Rat bone marrow mesenchymal stem cells were inoculated on the surface of the 3D-printed polycaprolactone and polycaprolactone/nano-hydroxyapatite composite scaffolds. Cell proliferation rate was detected by CCK-8 method. Cell growth on the scaffolds was observed by scanning electron microscopy and Live/Dead cell staining. RESULTS AND CONCLUSION: Two kinds of scaffolds had a three-dimensional network and interconnected structure. The fibers were arranged in a regular order and interlaced. There was no gap on the fiber surface, and the fiber spacing and diameter were relatively uniform. There was no significant difference in the porosity between two kinds of scaffolds (P > 0. 05). The elastic modulus of the composite scaffold was higher than that of the simple polycaprolactone scaffold (P < 0. 05). There was no significant difference in cell proliferation between two kinds of scaffolds after 1 day of culture. After 4 and 7 days of culture, cell proliferation on the composite scaffold was significantly faster than that on the simple polycaprolactone scaffold (P < 0. 05). Live/Dead cell staining showed that both polycaprolactone and polycaprolactone/nano-hydroxyapatite composite scaffolds had good cytocompatibility and high cell viability. A larger number of cells adhered to the polycaprolactone/nano-hydroxyapatite composite scaffolds. Scanning electron microscopy showed that cells grew well on two kinds of scaffolds and distributed on the surface and micropores of the scaffold. The secreted extracellular matrix appeared in filaments and surrounded the cells. These findings suggest that the polycaprolactone/nano-hydroxyapatite composite material prepared by 3D printing technology has abundant pores, exhibit good mechanical properties, and have good cytocompatibility and can be used as a scaffold material for tissue engineering.
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Abstract Mesenchymal stem cells and osteoblasts play important roles in bone formation. Achatina fulica mucus presented the property of osteoinduction. This study aimed to examine the effects of A. fulica mucus on human mesenchymal stem cell (hMSC) and human fetal osteoblastic cell line (HFOB) differentiation. The integrated effects of A. fulica mucus and polycaprolactone (PCL) on the differentiation of hMSCs were tested. The cell viability of hMSCs treated with A. fulica mucus was investigated by the MTT assay. The cell mineralization was observed by Alizarin Red S staining, the gene expression was investigated using RT-PCR, and the PI3K activation was studied using flow cytometry. The results indicated that A. fulica mucus induced osteogenic differentiation in hMSCs and HFOBs by upregulation of the osteogenic markers; osteopontin (OPN) and osteocalcin (OCN). The results of the Alizarin Red S staining indicated that A. fulica mucus supported mineralization in both hMSCs and HFOBs. The hMSCs cultured on PCL supplemented with A. fulica mucus showed significantly increased RUNX2 and OPN expressions. A. fulica mucus was observed to increase PI3K activation in hMSCs. The findings of this study suggested that A. fulica mucus and biomaterials could be applied together for use in bone regeneration in the future.
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Humanos , Animales , Osteogénesis/fisiología , Regeneración Ósea , Células Madre Mesenquimatosas/citología , Moluscos/química , Moco/química , Pruebas de Toxicidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Citometría de FlujoRESUMEN
BACKGROUND: Statins plays a significant role in regulating blood lipids, treating and preventing cardiovascular and cerebrovascular diseases. Studies have shown that statins has certain potential in promoting bone formation and treating osteoporosis. OBJECTIVE: To prepare the drug release scaffolds for the sustained release of atorvastatin calcium, which consist of bovine serum albumin microspheres and polycaprolactone electrostatic spinning fibers, and to investigate the effects of the drug sustained release scaffolds on osteoblast adhesion and proliferation. METHODS: Bovine serum albumin microspheres containing atorvastatin calcium were prepared by desolvation. A layer of chitosan was coated on the surface of the bovine serum albumin microspheres by electrostatic adsorption, which can increase the stability of the microspheres. Bovine serum albumin microspheres were purified and lyophilized for later use. The lyophilized powder of microspheres was dissolved in organic solvent. An appropriate amount of hydroxyapatite was added in the solvent. The nanofiber scaffolds for sustained release of atorvastatin calcium were prepared via electrospinning. The micromorphology, degradation performance, and sustained-release performance of the nanofiber scaffolds were characterized. The prepared nanofiber scaffolds for sustained-release of atorvastatin calcium were co-cultured with MC3T3-E1 cells to observe cell adhesion and proliferation. RESULTS AND CONCLUSION: (1) Transmission electron microscopy revealed that the shape of the bovine serum albumin nanospheres was regular and circular. Bovine serum albumin nanospheres were discarded in the electrostatic spinning fibers. The basic morphology of the microspheres was retained. (2) Scanning electron microscopy revealed that the nanofibers used for preparation of nanofiber scaffolds for sustained-release of atorvastatin calcium were composed of filaments with uniform diameters and continuous smooth surface. Filaments were intertwined to form a network structure. (3) The nanofiber scaffolds exhibited the fastest degradation in the first month. The material was incomplete when degraded for 3 months. (4) The nanofiber scaffolds had the ability to slow down the release of drugs. The effect could last for more than 1 month. The overall process of drug release was similar to the zero-order kinetic process. (5) The nanofiber scaffolds for sustained-release of atorvastatin calcium can promote MC3T3-E1 cell adhesion and proliferation. (6) These results suggest that the nanofiber scaffolds for sustained-release of atorvastatin calcium have good biocompatibility and can promote the adhesion and proliferation of osteoblasts.
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Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.
Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.