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The integration of precision medicine principles into bone tissue engineering has ignited a wave of research focused on customizing intricate scaffolds through advanced 3D printing techniques. Bioceramics, known for their exceptional biocompatibility and osteoconductivity, have emerged as a promising material in this field. This article aims to evaluate the regenerative capabilities of a composite scaffold composed of 3D-printed gelatin combined with hydroxyapatite/tricalcium phosphate bioceramics (G/HA/TCP), incorporating human dental pulp-derived stem cells (hDPSCs). Using 3D powder printing, we created cross-shaped biphasic calcium phosphate scaffolds with a gelatin layer. The bone-regenerating potential of these scaffolds, along with hDPSCs, was assessed through in vitro analyses and in vivo studies with 60 rats and critical-sized calvarial defects. The assessment included analyzing cellular proliferation, differentiation, and alkaline phosphatase activity (ALP), and concluded with a detailed histological evaluation of bone regeneration. Our study revealed a highly favorable scenario, displaying not only desirable cellular attachment and proliferation on the scaffolds but also a notable enhancement in the ALP activity of hDPSCs, underscoring their pivotal role in bone regeneration. However, the histological examination of calvarial defects at the 12-wk mark yielded a rather modest level of bone regeneration across all experimental groups. The test and cell group exhibited significant bone formation compared to all other groups except the control and cell group. This underscores the complexity of the regenerative process and paves the way for further in-depth investigations aimed at improving the potential of the composite scaffolds.
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Regeneración Ósea , Diferenciación Celular , Proliferación Celular , Cerámica , Osteogénesis , Impresión Tridimensional , Andamios del Tejido , Andamios del Tejido/química , Osteogénesis/efectos de los fármacos , Animales , Humanos , Cerámica/química , Cerámica/farmacología , Regeneración Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ratas , Proliferación Celular/efectos de los fármacos , Ingeniería de Tejidos/métodos , Células Madre/citología , Pulpa Dental/citología , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/química , Cráneo/patología , Masculino , Fosfatasa Alcalina/metabolismoRESUMEN
Background: Repeated Ovum Pick Up (OPU) could have a detrimental effect on ovarian function, reducing In Vitro Embryo Production (IVEP). The present study examined the therapeutic effect of adipose-derived Mesenchymal Stem Cells (MSCs) or its Conditioned Medium (ConM) on ovarian trauma following repeated OPU. Resolvin E1 (RvE1) and Interleukin-12 (IL-12) were investigated as biomarkers. Methods: Jersey heifers (n=8) experienced 11 OPU sessions including 5 pre-treatment and 6 treatment sessions. Heifers received intra-ovarian administration of MSCs or ConM (right ovary) and Dulbecco's Modified Phosphate Buffer Saline (DMPBS; left ovary) after OPU in sessions 5 and 8 and 2 weeks after session 11. The concentrations of RvE1 and IL-12 in follicular fluid was evaluated on sessions 1, 5, 6, 9, and 4 weeks after session 11. Following each OPU session, the IVEP parameters were recorded. Results: Intra-ovarian administration of MSCs, ConM, and DMPBS did not affect IVEP parameters (p>0.05). The concentration of IL-12 in follicular fluid increased at the last session of pre-treatment (Session 5; p<0.05) and remained elevated throughout the treatment period. There was no correlation between IL-12 and IVEP parameters (p>0.05). However, RvE1 remained relatively high during the pre-treatment and decreased toward the end of treatment period (p<0.05). This in turn was associated with decline in some IVEP parameters (p<0.05). Conclusion: Intra-ovarian administration of MSCs or ConM during repeated OPU did not enhance IVEP outcomes in Bos taurus heifers. The positive association between RvE1 and some of IVEP parameters could nominate RvE1 as a promising biomarker to predict IVEP parameters following repeated OPU.
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OBJECTIVE: Cryptorchidism is one of the main causes of infertility and can result in testicular cancer. This study aimed to present quantitative data on the damage caused by cryptorchidism using stereological analysis. METHODS: Thirty newborn rats were randomly divided into control and experimental groups. The experimental group underwent surgery to induce unilateral cryptorchidism in the left testis, whereas the control group underwent a sham surgical procedure 18 days after birth. The testes were removed at designated time points (40, 63, and 90 days after birth) for stereological evaluation and sperm analysis. Total testicular volume, interstitial tissue volume, seminiferous tubule volume and length, and seminiferous epithelium volume and surface area were measured. Other parameters, such as sperm count, sperm morphology, and sperm tail length, were also examined. RESULTS: Statistically significant differences (p<0.05) were observed between the experimental and the control groups at different ages regarding the volumes of various parameters, including the surface area of the germinal layer, the length of the seminiferous tubules, sperm count, and sperm morphology. However, no significant differences were observed in the epithelial volume and the sperm tail length of the groups. CONCLUSION: Given the substantial effect of cryptorchidism on different testicular parameters, as well as the irreversible damage it causes in the testes, it is important to take this abnormality seriously to prevent these consequences.
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Due to the increasing prevalence of bone disorders among people especially in average age, the future of treatments for osseous abnormalities has been illuminated by scaffold-based bone tissue engineering. In this study, in vitro and in vivo properties of 58S bioactive glass-based scaffolds for bone tissue engineering (bare (B.SC), Zein-coated (C.SC), and Zein-coated containing Kaempferol (KC.SC)) were evaluated. This is a follow-up study on our previously published paper, where we synthesized 58S bioactive glass-based scaffolds coated with Kaempferol-loaded Zein biopolymer, and characterized from mostly engineering points of view to find the optimum composition. For this aim, in vitro assessments were done to evaluate the osteogenic capacity and biological features of the scaffolds. In the in vivo section, all types of scaffolds with/without bone marrow-derived stem cells (BMSC) were implanted into rat calvaria bone defects, and potential of bone healing was assessed using imaging, staining, and histomorphometric analyses. It was shown that, Zein-coating covered surface cracks leading to better mechanical properties without negative effect on bioactivity and cell attachment. Also, BMSC differentiation proved that the presence of Kaempferol caused higher calcium deposition, increased alkaline phosphatase activity, bone-specific gene upregulation in vitro. Further, in vivo study confirmed positive effect of BMSC-loaded KC.SC on significant new bone formation resulting in complete bone regeneration. Combining physical properties of coated scaffolds with the osteogenic effect of Kaempferol and BMSCs could represent a new strategy for bone regeneration and provide a more effective approach to repairing critical-sized bone defects.
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Células Madre Mesenquimatosas , Zeína , Ratas , Animales , Ingeniería de Tejidos/métodos , Andamios del Tejido , Estudios de Seguimiento , Quempferoles/farmacología , Zeína/farmacología , Osteogénesis , Regeneración Ósea , Vidrio , Diferenciación Celular , CráneoRESUMEN
The skin undergoes the formation of fine lines and wrinkles through the aging process; also, burns, trauma, and other similar circumstances give rise to various forms of skin ulcers. Induced pluripotent stem cells (iPSCs) have become promising candidates for skin healing and rejuvenation due to not stimulating inflammatory responses, low probability of immune rejection, high metabolic activity, good large-scale production capacity and potentials for personalized medicine. iPSCs can secrete microvesicles (MVs) containing RNA and proteins responsible for the normal repairing process of the skin. This study aimed to evaluate the possibility, safety and effectiveness of applying iPSCs-derived MVs for skin tissue engineering and rejuvenation applications. The possibility was assessed using the evaluation of the mRNA content of iPSC-derived MVs and the behavior of fibroblasts after MV treatment. Investigating the effect of microvesicle on stemness potential of mesenchymal stem cells was performed for safety concerns. In vivo evaluation of MVs was done in order to investigate related immune response, re-epithelialization and blood vessel formation to measure effectiveness. Shedding MVs were round in shape distributed in the range from 100 to 1000 nm in diameter and positive for AQP3, COL2A, FGF2, ITGB, and SEPTIN4 mRNAs. After treating dermal fibroblasts with iPSC-derived MVs, the expressions of collagens Iα1 and III transcripts (as the main fibrous extracellular matrix (ECM) proteins) were upregulated. Meanwhile, the survival and proliferation of MV treated fibroblasts did not change significantly. Evaluation of stemness markers in MV treated MSCs showed negligible alteration. In line with in vitro results, histomorphometry and histopathology findings also confirmed the helpful effect of MVs in skin regeneration in the rat burn wound models. Conducting more investigations on hiPSCs-derived MVs may lead to produce more efficient and safer biopharmaceutics for skin regeneration in the pharmaceutical market.
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Micropartículas Derivadas de Células , Células Madre Pluripotentes Inducidas , Humanos , Ratas , Animales , Células Madre Pluripotentes Inducidas/metabolismo , Transcriptoma , Rejuvenecimiento , Piel/patología , Micropartículas Derivadas de Células/metabolismoRESUMEN
Objectives: Recent investigations indicate that canine periodontal ligament-derived stem cells (cPDLSCs) may reveal a reliable strategy for repair of periodontal tissues via cell-based tissue engineering approaches. Due to limited research, this study aimed to demonstrate the phenotypic characterization of cPDLSc in comparison with canine bone marrow-derived mesenchymal stem cells (cBMSCs) in vitro. Methods: Mesenchymal stem cells (MSCs) were obtained from PDL and BM of five male adult Mongrel dogs. In vitro isolation and expansion as well as biologic characterization including colony unit formation (CFU), osteogenic and adipogenic differentiation, flow cytometric analysis of CD34 and CD44, and RT-PCR of alkaline phosphatase (ALP), osteocalcin (OCN), periostin (POSTN) and S100A4 were performed. Furthermore, electron microscopy analysis was done to complement the comparative research. Results: CFU assay revealed that colonies of cPDLSCs presented 70% confluency with a more finite lifespan than BM-MSCs, showing a significant increase in cPDLSCs. Both types of MSCs showed osteogenic and adipogenic phenotypic characterized with clusters of mineralized depositions and lipid vacuoles, respectively. Both types of MSCs expressed CD44 with limited expression of CD34. RT-PCR of cPDLSCs revealed that expression of ALP, POSTN, OCN and S100A4 genes were significantly higher than those of BMSCs. In addition, comparison of SEM and revealed that cPDLSCs expressed more extracellular collagen fibers. Conclusions: The current study indicated that cPDLSCs show potency as a novel cellular therapy for periodontal regeneration a large animal model.
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ATP is a crucial molecule for every energy-dependent process in cells. In ischemic tissues, ATP production declines, and it finally results in cell death. One of the most common strategies in burn wound management is saving the zone of ischemia. In the current study, Mg-ATP-containing nanoliposomes were formulated and studied in vitro and in vivo. The particle size of the vesicles was between 50 and 100 nm and the mean zeta potential was -4.05 ± 0.52 mV as evaluated by dynamic light scattering and Zeta sizer instrument, respectively. The encapsulation efficiency of ATP in the nanoliposomes was found to be 9.3%. The morphology and size of nanoliposomes were further studied by transmission electron microscopy. The standard MTT assay revealed no cytotoxicity of the nanoliposomes when tested on the rat fibroblast cells. Forty rats were randomly divided into four groups (N = 10 each). Burn wounds were created by burn comb model on the back of the rats and the zone of stasis in each group was treated every 12 h for 3 days by injecting them with the Mg-ATP-nanoliposomes. Control samples included empty nanoliposomes, unencapsulated Mg-ATP and the Krebs-Henseleit buffer. Laser Doppler flowmetry results revealed that blood perfusion in the zone of ischemia in rats treated with Mg-ATP-nanoliposomes was more than in the other groups (p < 0.05). Histopathology revealed saving zone of stasis by Mg-ATP-nanoliposomes. Findings obtained in this study demonstrated that the formulated Mg-ATP-nanoliposome has the potential to save the stasis zone in burn wounds.
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Quemaduras , Cicatrización de Heridas , Ratas , Animales , Quemaduras/patología , Isquemia , Modelos Animales de Enfermedad , Adenosina Trifosfato/farmacologíaRESUMEN
Magnesium (Mg) plays an important role in controlling bone apatite structure and density and is a potential bioactive material in repairing critical-sized bone defects. In this study, we aimed to evaluate the effect of adding NanoMgO to polycaprolactone/beta-tricalcium phosphate (PCL/ß-TCP) scaffolds on bone regeneration. Novel 3D-printed porous PCL/ß-TCP composite scaffolds containing 10% nanoMgO were fabricated by fused deposition modeling (FDM) and compared with PCL/ß-TCP (1:1) scaffolds (control). The morphology and physicochemical properties of the scaffolds were characterized by ATR-FTIR, XRD, scanning electron microscope-energy dispersive X-ray analysis (SEM-EDX), transmission-electron-microscopy (TEM), water contact angle, and compressive strength tests and correlated to its cytocompatibility and osteogenic capacity in-vitro. To evaluate in-vivo osteogenic capacity, bone-marrow-derived stem cell (BMSC)-loaded scaffolds were implanted into 8 mm rat critical-sized calvarial defects for 12 weeks. The hydrophilic scaffolds showed 50% porosity (pore size = 504 µm). MgO nanoparticles (91.5 ± 27.6 nm) were homogenously dispersed and did not adversely affect BMSCs' viability and differentiation. Magnesium significantly increased elastic modulus, pH, and degradation. New bone formation (NBF) in Micro-CT was 30.16 ± 0.31% and 23.56 ± 1.76% in PCL/ß-TCP/nanoMgO scaffolds with and without BMSCs respectively, and 19.38 ± 2.15% and 15.75 ± 2.24% in PCL/ß-TCP scaffolds with and without BMSCs respectively. Angiogenesis was least remarkable in PCL/ß-TCP compared with other groups (p < .05). Our results suggest that the PCL/ß-TCP/nanoMgO scaffold is a more suitable bone substitute compared to PCL/ß-TCP in critical-sized calvarial defects.
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Nanopartículas , Ingeniería de Tejidos , Ratas , Animales , Andamios del Tejido/química , Óxido de Magnesio/farmacología , Magnesio , Fosfatos de Calcio/farmacología , Fosfatos de Calcio/química , Poliésteres/farmacología , Poliésteres/química , Impresión TridimensionalRESUMEN
Bone-marrow derived stem cells (BMSCs) can differentiate into several mesenchymal cell lines that are suitable for bone and dental tissue engineering. This study was aimed to assess the efficacy of cell therapy in direct pulp capping (DPC) of canine teeth using autologous BMSCs along with collagen/hydroxyapatite hybrid scaffold in terms of the quantity and quality of calcified bridge formation. The teeth were randomly divided into three groups of DPC with mineral trioxide aggregate (MTA), hydroxyapatite/collagen hybrid scaffold alone and BMSCs with hydroxyapatite/collagen hybrid scaffold. DPC was performed under general anesthesia in cavities prepared on the buccal surfaces of mandibular and maxillary premolars of the same dogs from which, stem cells had been isolated. All cavities were then restored with light-cure resin modified glass ionomer cement. Histomorphometric assessments after 12 weeks showed formation of dentinal bridge following DPC with BMSCs and MTA. The efficacy of MTA for calcified bridge formation following DPC was significantly higher than that of BMSCs plus hybrid scaffold. According to the present study, we concluded DPC using BMSCs and hybrid scaffold did not provide clinically noticeable results in canine patients.
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The aim of this study was to introduce a new animal model of fecal incontinence (FI) by injecting abobotulinumtoxinA in the external anal sphincter (EAS) muscle of dogs which replaces models based on anal sphincter destructions that are invasive, mostly require surgical procedures, expensive, permanent, and painful to the animals. 4 healthy mongrel dogs were used in this study. First, they were received NaCl 0.09% (as control) injections in EAS muscle and effects were assessed by means of Electromyography (EMG) and clinically evaluated by sphincter pinch test and presence of leakage of feces for 2 weeks. Then, they received abobotulinumtoxinA in EAS muscle and reevaluated for 6 weeks to see short-term and medium-term effects of abobotulinumtoxinA injection. Saline had no significant changes in results obtained from EMG, however, there were significant decreases in amplitudes of action potentials after receiving abobotulinumtoxinA in comparison with no injection or saline injection in EAS muscle. Pinch tests were normal after saline injection assessment period, however, then started to be negative, ranging from two days after abobotulinumtoxinA injection to seven days after receiving abobotulinumtoxinA. Animals also had significant presentations of fecal incontinence (leakage of feces and cage contamination with feces) from the 1st week after receiving abobotulinumtoxinA until the 6th week after receiving abobotulinumtoxinA. AbobotulinumtoxinA caused paralysis in the EAS and producd FI conditions in dogs. This animal model was an appropriate substitute to the various invasive, expensive and also complicated procedures with an easy, feasible, noninvasive and non-painful single-stage abobotulinumtoxinA injection.
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In this study, polyvinyl alcohol hydrogel chains were crosslinked by polyurethane in order to synthesize a suitable substrate for cartilage lesions. The substrate was fully characterized, and in vitro and in vivo investigations were conducted based on a sheep model. In vitro tests were performed based on the chondrocyte cells with the Alcian Blue and safranin O staining in order to prove the presence of proteoglycan on the surface of the synthesized substrate, which has been secreted by cultures of chondrocytes. Furthermore, the expression of collagen type I, collagen type II, aggrecan, and Sox9 was presented in the chondrocyte cultures on the synthesized substrate through RT-PCR. In addition, the H&E analysis and other related tests demonstrated the formation of neocartilage tissue in a sheep model. The results were found to be promising for cartilage tissue engineering and verified that the isolated chondrocyte cultures on the synthesized substrate retain their original composition.
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Condrocitos , Poliuretanos , Agrecanos/metabolismo , Azul Alcián/metabolismo , Animales , Cartílago , Células Cultivadas , Condrocitos/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo II , Poliuretanos/metabolismo , Proteoglicanos/metabolismo , Ovinos , Ingeniería de Tejidos/métodos , Andamios del TejidoRESUMEN
In vivo bioreactors serve as regenerative niches that improve vascularization and regeneration of bone grafts. This study has evaluated the masseter muscle as a natural bioreactor for ßTCP or PCL/ßTCP scaffolds, in terms of bone regeneration. The effect of pedicle preservation, along with sole, or MSC- or rhBMP2-combined application of scaffolds, has also been studied. Twenty-four mongrel dogs were randomly placed in six groups, including ßTCP, ßTCP/rhBMP2, ßTCP/MSCs, PCL/ßTCP, PCL/ßTCP/rhBMP2, and PCL/ßTCP/MSCs. During the first surgery, the scaffolds were implanted into the masseter muscle for being prefabricated. After 2 months, each group was divided into two subgroups prior to mandibular bone defect reconstruction; one with a preserved vascularized pedicle and one without. After 12 weeks, animals were euthanized, and new bone formation was evaluated using histological analysis. Histological analysis showed that all ß-TCP scaffold groups had resulted in significantly greater rates of new bone formation, either with a pedicle surgical approach or non-pedicle surgical approach, comparing to their parallel groups of ßTCP/PCL scaffolds (p ≤ .05). Pedicled ß-TCP scaffold groups that were treated with either rhBMP2 (48.443% ± 0.250%) or MSCs (46.577% ± 0.601%) demonstrated the highest rates of new bone formation (p ≤ .05). Therefore, masseter muscle can be used as a local in vivo bioreactor with potential clinical advantages in reconstruction of human mandibular defects. In addition, scaffold composition, pedicle preservation, and treatment with MSCs or rhBMP2, influence new bone formation and scaffold degradation rates in the prefabrication technique.
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Músculo Masetero , Andamios del Tejido , Animales , Reactores Biológicos , Regeneración Ósea , Perros , Mandíbula/cirugíaRESUMEN
This study was based on in vivo assessment of bone regeneration capacity of synthesized porous ß-tricalcium phosphate (ß-TCP) nanocomposite granules and aimed to explore the effects of fabricated ß-TCP granules reinforced with layered double hydroxides (LDH) nanoclay compared to ß-TCP granules, in terms of osteoconductivity and biodegradability. Granules with diameters of 2-3 mm were implanted into cavities drilled in rabbit distal femur and were left in situ for up to 3 months. The mechanical study demonstrated that the presence of LDH nanoparticles in ß-TCP granules resulted in a significant increase in compressive modulus from 174.4 to 231.4 MPa, while the porosity was constant at 76%-80%. The results revealed that the obtained granules showed no cytotoxicity. In this study, x-ray radiographic, micro-computed tomography, and histological staining analysis were taken to evaluate the percentage of bone ingrowth and biodegradability of the porous granules. The results exhibited that both granules support bone regeneration and also the amount of new bone formation in the bone defect filled with both granules was almost six times higher than the empty defects. Although no significant difference in bone formation for two different granules was observed, a higher biodegradability was detected in ß-TCP granules in comparison to ß-TCP/LDH granules. Overall, the addition of LDH nanoclay (10%) enhanced the physicochemical and mechanical properties of ß-TCP granules while it is biological and osteoconductity properties have been maintained and its biodegradation rate has been decreased.
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Sustitutos de Huesos , Nanocompuestos , Animales , Regeneración Ósea , Sustitutos de Huesos/farmacología , Fosfatos de Calcio/química , Fosfatos de Calcio/farmacología , Hidróxidos/farmacología , Conejos , Microtomografía por Rayos XRESUMEN
Burns is a critical fatal event due to the risk of infection and complex inflammatory cascades. This study aimed to fabricate and characterize a new antibacterial and anti-inflammatory dressing for second-degree burns by the immobilization of bromelain and zinc oxide nanoparticles on silk fibroin nanofibers. Thus, electrospun silk nanofibers with an average fiber diameter of 345 nm were prepared and then grafted with acrylic acid after exposure to O2 plasma. Next, bromelain was immobilized on the modified SF nanofibers (SF-Br). Subsequently, different amounts of ZnO NPs coated with polydopamine were immobilized on the SF-Br nanofibers. The successful immobilization of bromelain and ZnO NPs on the SF nanofibers was proved by SEM, EDS, and FTIR analysis. The loading efficiency of bromelain was 85.63%, and activity ranged between 88% and 92%. The crystallinity of SF nanofibers decreased after the addition of bromelain and ZnO NPs, which increased the bromelain and zinc ions released from the dressing. Antibacterial activity has improved with the addition of ZnO NPs. The amounts of bromelain released from the dressings are not toxic to fibroblasts. Moreover, fibroblast attachment and proliferation enhanced at lower ZnO amounts, while there was an inverse trend at high doses of ZnO NPs. In vivo studies showed that treating the burn with silk fibroin-bromelain-ZnO NPs enhanced the healing process and considerably lowered the inflammatory response at the wound. Overall, the dressing presented here offers excellent potential for burn management.
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Quemaduras , Fibroínas , Nanofibras , Nanopartículas , Óxido de Zinc , Antibacterianos/uso terapéutico , Antiinflamatorios/uso terapéutico , Vendajes , Bromelaínas , Quemaduras/tratamiento farmacológico , Humanos , SedaRESUMEN
Wound healing is a complex pathophysiological process, highlighting the importance of effective and thorough wound care along with the prevention of wound infection, a major barrier that can slow down or even disrupt the healing process. To date, there are plenty of herbal plants well known and historically supernatural, showing profound wound healing effects. Application of such herbal extracts/ingredients in electrospun nanofiber platforms has shown promising outcomes in improving wound healing process. Based on these facts, we loaded Calendula officinalis extract (CO) in chitosan/polyethylene oxide scaffolds (CS/PEO) by electrospinning. Using SEM, morphology of electrospun scaffolds showed a narrow range of fiber diameter, around 143--252 nm, with uniform and bead-free appearance. FT-IR spectroscopy confirmed the presence of CO extract in nanofibrous scaffolds. Of importance, incorporation of CO extract improved mechanical properties of CS/PEO nanofibers. A 1602 cP reduction in viscosity and a 0.892 ms/cm increase in the conductivity of the solution was observed after addition of the CO extract. CO extract showed strong antibacterial properties with 96% and 94% reduction in Gram positive and Gram negative bacteria, respectively. In vitro studies with fibroblast cells confirmed enhanced proliferation, growth and attachment of the cells. The in vivo and histological analysis of rat wounds, revealed excellent wound healing ability of CS/PEO/CO dressings (87.5 % wound closure after 14 days) via improving collagen synthesis, re-epithelization and remodeling of the tissue. In sum, our findings show that CS/PEO/CO scaffolds can be used as a promising dressing for the treatment of skin wounds.
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Calendula , Quitosano , Nanofibras , Animales , Antibacterianos/uso terapéutico , Bacterias Gramnegativas , Bacterias Grampositivas , Extractos Vegetales , Ratas , Espectroscopía Infrarroja por Transformada de Fourier , Cicatrización de HeridasRESUMEN
Synergistic promotion of angiogenesis and osteogenesis in bone tissue-engineered constructs remains a crucial clinical challenge, which might be overcome by simultaneous employment of superior techniques including coculture systems, differentiation-stimulated factors, combinatorial scaffolds and bioreactors.Current study investigated the effect of flow perfusion along with coculture of human adipose stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs) on osteogenic and angiogenic differentiation.Pre-treated hASCs with 1,25-dihydroxyvitamin D3 were seeded onto poly(lactic-co-glycolic acid)/ß-tricalcium phosphate/polycaprolactone (PLGA/ß-TCP/PCL) scaffold with/without HUVECs, and cultured for 14 days within a flask or modified perfusion bioreactor. Analysis of osteogenic and angiogenic gene expression, alkaline phosphatase (ALP) activity and ALP staining indicates a synergistic effect of perfusion flow and coculture system on osteogenic and angiogenic differentiation. The advantage of modified perfusion bioreactor is its five-branch flow distributor which directly connect to the porous PCL hollow fibers embedded in the 3D scaffold to improve flow and flow-induced shear stress uniformity.Dynamic coculture increased VEGF165 by 6-fold, VEGF189 by 2-fold, and Endothelin-1 by 4-fold, relative to dynamic monoculture. Static coculture enhanced osteogenic and angiogenic differentiation, compared with static monoculture. Although dynamic coculture is in preference to static coculture due to significant increase in ALP activity and promoted angiogenic marker expression. Our finding is the first to indicate that the modified perfusion bioreactor combined with the beneficial cell-cell crosstalk in pre-treated hASC/HUVEC cocultures provides a synergy between osteogenic and angiogenic differentiation of the accumulation of cells, suggesting that it represents a promising approach for regeneration of critical-sized bone defects.
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Osteogénesis , Células Madre , Reactores Biológicos , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Perfusión , Andamios del TejidoRESUMEN
ABSTRACT: Alternative treatment approaches to improve the regeneration ability of damaged peripheral nerves are currently under investigation. The aim of the current study was to evaluate the effects of leucocyte/platelet-rich fibrin (L-PRF) with or without a collagen membrane as a supporter on crushed sciatic nerve healing in a rat model. Recovery of motor function and electrophysiologic measurements were evaluated at 4 weeks postoperatively. The whole number of myelinated axons, peripheral nerve axon density, average nerve fiber diameter (µm), and G-ratio were analyzed and compered among the groups. Functional, electrophysiological, and histological evaluations showed no significant difference among the groups with the exception of the L-PRF with collagen membrane groups that showed relatively positive effects on the functional and histological nerve recovery. In addition, the collagen membrane with L-PRF can be effect in nerve regeneration.
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Fibrina Rica en Plaquetas , Animales , Axones , Colágeno , Regeneración Nerviosa , Ratas , Nervio CiáticoRESUMEN
Separating cells from the body and cultivating them in vitro will alter the function of cells. Therefore, for optimal cell culture in the laboratory, conditions similar to those of their natural growth should be provided. In previous studies, it has been shown that the use of cellular shape at the culture surface can regulate cellular function. In this work, the efficiency of the imprinting method increased by using microfluidic chip design and fabrication. In this method, first, a cell-imprinted substrate of chondrocytes was made using a microfluidic chip. Afterwards, stem cells were cultured on a cell-imprinted substrate using a second microfluidic chip aligned with the substrate. Therefore, stem cells were precisely placed on the chondrocyte patterns on the substrate and their fibroblast-like morphology was changed to chondrocyte's spherical morphology after 14-days culture in the chip without using any chemical growth factor. After chondrogenic differentiation and in vitro assessments (real-time PCR and immunocytotoxicity), differentiated stem cells were transferred on a collagen-hyaluronic acid scaffold and transplanted in articular cartilage defect of the rabbit. After 6 months, the post-transplantation analysis showed that the articular cartilage defect had been successfully regenerated in differentiated stem cell groups in comparison with the controls. In conclusion, this study showed the potency of the imprinting method for inducing chondrogenicity in stem cells, which can be used in clinical trials due to the safety of the procedure.
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Cartílago Articular , Células Madre Mesenquimatosas , Animales , Diferenciación Celular , Células Cultivadas , Condrocitos , Condrogénesis , Dispositivos Laboratorio en un Chip , Conejos , Regeneración , Ingeniería de TejidosRESUMEN
Production of a 3D bone construct with high-yield differentiated cells using an appropriate cell source provides a reliable strategy for different purposes such as therapeutic screening of the drugs. Although adult stem cells can be a good source, their application is limited due to invasive procedure of their isolation and low yield of differentiation. Patient-specific human-induced pluripotent stem cells (hiPSCs) can be an alternative due to their long-term self-renewal capacity and pluripotency after several passages, resolving the requirement of a large number of progenitor cells. In this study, a new biphasic 3D-printed collagen-coated HA/ß-TCP scaffold was fabricated to provide a 3D environment for the cells. The fabricated scaffolds were characterized by the 3D laser scanning digital microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and mechanical test. Then, the osteogenesis potential of the hiPSC-seeded scaffolds was investigated compared to the buccal fat pad stem cell (BFPSC)-seeded scaffolds through in vitro and in vivo studies. In vitro results demonstrated up-regulated expressions of osteogenesis-related genes of RUNX2, ALP, BMP2, and COL1 compared to the BFPSC-seeded scaffolds. In vivo results on calvarial defects in the rats confirmed a higher bone formation in the hiPSC-seeded scaffolds compared to the BFPSC-seeded groups. The immunofluorescence assay also showed higher expression levels of collagen I and osteocalcin proteins in the hiPSC-seeded scaffolds. It can be concluded that using the hiPSC-seeded scaffolds can lead to a high yield of osteogenesis, and the hiPSCs can be used as a superior stem cell source compared to BFPSCs for bone-like construct bioengineering.
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Tejido Adiposo/diagnóstico por imagen , Células Madre Pluripotentes Inducidas/metabolismo , Osteogénesis/fisiología , Impresión Tridimensional/normas , Andamios del Tejido/normas , Tejido Adiposo/fisiopatología , Animales , Diferenciación Celular , Proliferación Celular , Humanos , Masculino , Ratas , Ratas WistarRESUMEN
Core-sheath nanofibrous scaffolds from polyvinyl alcohol (PVA)-strontium ranelate (SrR)-Polycaprolactone (PCL) were prepared by water in oil electrospinning method. Thus, PCL (the oil phase) was used as the shell part and a mixture of PVA and SrR (the water phase) was inserted in the core. The amounts of SrR was varied from 0 to 15 wt.% Mussel-inspired dopamine-gelatin coating was done on the nanofibrous to improve their hydrophilicity and cellular attachment. The effect of the SrR content on morphology, mechanical, physicochemical, in vitro release behaviors, and biological properties as well as in vivo bone regeneration was investigated. Morphological observations revealed that continuous nanofibers with a core/shell structure were successfully obtained and the fibers diameter increased as the SrR content rose. X-ray diffraction (XRD) analysis revealed that SrR was molecularly distributed in the nanofibers and increasing the amount of the SrR decreased the crystallinity of the nanofibers. Moreover, the SrR release was regulated through the mechanism of Fickian diffusion and it was assumed as fast as possible in the samples with higher SrR content. The mesenchymal stem cell culturing showed improved cell proliferation by adding SrR and accelerating the expression of ALP, Runx2, Col I, and OCN genes. Besides, the SrR-loaded nanofibers improved bone formation of calvarial defects in a rat model as revealed by in vivo investigations.