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BACKGROUND: Montmorillonite (MMT) is a biocompatible nanoclay and its incorporation into polymeric matrix not only improves the polymer's wettability/biodegradability, but also enhances cellular proliferation, and differentiation. On the other hand, the positive effect of boron (B) on the healing cascade and its antibacterial properties have drawn the attention of researchers. MATERIALS & METHODS: In this regard, B compounds in different chemical structures, boron nitride (BN), zinc borate (ZB), and phenylboronic acid (PBA), were adsorbed onto MMT and then, poly (lactic acid) (PLA) based MMT/B including micron/submicron fibers were fabricated by electrospinning. RESULTS: The incorporation of MMT nanoparticles into the PLA demonstrated a porous fiber topography with enhanced thermal properties, water uptake capacity, and antibacterial effect. Furthermore, the composites including BN, ZB, and PBA showed bacteriostatic effects against Gram-negative and Gram-positive pathogenic bacteria (Escherichia coli and Staphylococcus aureus). In-vitro cell culture studies performed with human dermal fibroblasts (HDF) indicated the non-toxic effect of B compounds. The results showed that incorporation of MMT supported cell adhesion and proliferation, and further addition of B compounds especially PBA increased cell viability for 14 days. CONCLUSION: The results illustrated the acceptable characteristics of the B-containing composites and their favorable effect on the cells, demonstrating their potential as a skin tissue engineering product.
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Nanofibras , Polímeros , Humanos , Polímeros/farmacología , Polímeros/química , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/química , Nanofibras/química , Arcilla , Antibacterianos/farmacología , Antibacterianos/química , Poliésteres/farmacología , Poliésteres/química , Compuestos de Boro/farmacología , VendajesRESUMEN
Corneal opacities are a major cause of vision loss worldwide. However, the current therapies are suboptimal to manage the corneal wound healing process. Therefore, there is an obvious need to develop new treatment strategies that are efficient in promoting wound healing in patients with severe corneal disorders. In this study, we investigated and compared the efficacy of adipose-derived mesenchymal stem cells (ADMSCs) and photobiomodulation (PBM) with polychromatic light in the NIR (600-1200 nm) alone and in combination, on corneal opacity, inflammatory response, and tissue architecture in a rat corneal opacity model created by mechanical injury. All animals were divided into four groups randomly following the injury: injury only (no treatment), ADMSCs treatment, PBM treatment and combined (ADMSCs+PBM) treatment (n = 12 eyes per group). At the 10th and 30th day following injury, corneal opacity formation, neovascularization, and corneal thickness were assessed. On the 30th day the harvested corneas were analyzed by transmission electron microscopy (TEM), histological evaluation, immunohistochemical (IHC) staining and real-time polymerase chain reaction (RT-PCR). On day 30, the corneal opacity score, neovascularization grade, and corneal thickness in all treatment groups were significantly lower in comparison with the untreated injured corneas. The TEM imaging and H&E staining together clearly revealed a significant enhancement in corneal regeneration with improved corneal microenvironment and reduced vascularization in the combined administration of PBM and ADMSCs compared to treatment of PBM and ADMSCs alone. In addition, the IHC staining, and RT-PCR analysis supported our hypothesis that combining ADMSCs therapy with PBM alleviated the inflammatory response, and significantly decreased scar formation compared to either ADMSCs or PBM alone during the corneal wound healing.
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Opacidad de la Córnea , Células Madre Mesenquimatosas , Ratas , Humanos , Animales , Cicatrización de Heridas , Células Madre , Opacidad de la Córnea/terapia , CórneaRESUMEN
Premature ovarian insufficiency (POI) is defined as the development of hypergonadotropic hypogonadism before the age of 40 with definitive treatment being absent. In the current study, we aim to compare the efficacy of the cell sheet method with an intravenous (IV) application of adipose-derived mesenchymal stem cells (AdMSCs) to the POI with an animal model. In the current prospective study, 6-to-8-week-old Sprague Dawley rats were generated four groups: (i) a control group in which only PBS was administered; (ii) an only-POI group generated by cyclophosphamide; (iii) a POI group treated by way of IV AdMSCs; and (iv) a POI group treated by way of the cell sheet method. Twenty-eight days after an oophorectomy was performed, intracardiac blood was taken. Follicle count, immunohistochemical examination for GDF9, BMP15, and TUNEL were conducted, gene expressions of GDF9 and BMP15 were examined, and E2 was measured in the serum samples. With hematoxylin-eosin, in the third group, multi oocytes follicles were the most remarkable finding. In the fourth group, most of the follicles presented normal morphology. GDF9 involvement was similar between the first and fourth groups. BMP-15 immunoreactivity, in contrast to fourth group, was weak in all stages in the second and third groups. The current attempt represents a pioneer study in the literature in which a cell sheet method is used for the first time in a POI model. These results suggest that the cell sheet method may be a feasible and efficient method for the stem cell treatment of models with POI and could be a new treatment approach in POI.
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Insuficiencia Ovárica Primaria , Ratas , Humanos , Femenino , Animales , Estudios Prospectivos , Ratas Sprague-Dawley , Insuficiencia Ovárica Primaria/terapia , Insuficiencia Ovárica Primaria/metabolismo , Folículo Ovárico/metabolismo , TecnologíaRESUMEN
The CRISPR/Cas9 mechanism offers promising therapeutic approaches for bone regeneration by stimulating or suppressing critical signaling pathways. In this study, we aimed to increase the activity of BMP-2 signaling through knockout of Noggin, thereby establishing a synergistic effect on the osteogenic activity of cells in the presence of BMP-2. Since Noggin is an antagonist expressed in skeletal tissues and binds to subunits of bone morphogenetic proteins (BMPs) to inhibit osteogenic differentiation, here Noggin expression was knocked out using the CRISPR/Cas9 system. In accordance with this purpose, C2C12 (mouse myoblast) cells were transfected with CRISPR/Cas9 plasmids. Transfection was achieved with Lipofectamine and confirmed with intense fluorescent signals in microscopic images and deletion in target sequence in Sanger sequencing analysis. Thus, Noggin knockout cells were identified as a new cell source for tissue engineering studies. Then, the transfected cells were seeded on highly porous silk scaffolds bearing BMP-2-loaded silk nanoparticles (30 ng BMP-2/mg silk nanoparticle) in the size of 288 ± 62 nm. BMP-2 is released from the scaffolds in a controlled manner for up to 60 days. The knockout of Noggin by CRISPR/Cas9 was found to synergistically promote osteogenic differentiation in the presence of BMP-2 through increased Coll1A1 and Ocn expression and mineralization. Gene editing of Noggin and BMP-2 increased almost 2-fold Col1A1 expression and almost 3-fold Ocn expression compared to the control group. Moreover, transfected cells produced extracellular matrix (ECM) containing collagen fibers on the scaffolds and mineral-like structures were formed on the fibers. In addition, mineralization characterized by intense Alizarin red staining was detected in transfected cells cultured in the presence of BMP-2, while the other groups did not exhibit any mineralized areas. As has been demonstrated in this study, the CRISPR/Cas9 mechanism has great potential for obtaining new cell sources to be used in tissue engineering studies.
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Osteogénesis , Seda , Animales , Ratones , Osteogénesis/genética , Ratones Noqueados , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/genéticaRESUMEN
In this study, nanofibrous polymeric matrices were successfully developed with nanoclay, montmorillonite (MMT) and various boron (B) compounds, which were known to have positive effects on the wound healing with elevated antibacterial properties. For this purpose, MMT was modified with quaternary ammonium salt, trimethyl octadecyl ammonium bromide (TMOD), and boron compounds, boron nitride (BN), zinc borate (ZB), or phenylboronic acid (PBA) were adsorbed on organomodified MMT (OMMT). Then, poly (lactic acid) (PLA) based nanofibrous PLA-OMMT/B composites were fabricated via electrospinning. Modification of MMT nanoparticles with TMOD occurred through ion-exchange reaction and led to better homogenous fibrous structures which exhibited dramatic inhibition for gram-positive bacteria. Moreover, composites with ZB and PBA demonstrated both bacteriostatic and bactericidal effects for gram-positive and gram-negative bacteria. The chemical structures of the matrices were evaluated through ATR-FTIR and supported the intercalated composite formation. The thermal and mechanical stabilities of PLA matrices were also enhanced after OMMT and B incorporation. The lowest breaking strain value was recorded for PLA-OMMT/PBA composite compared to other B composites. The 100% and 50% extracts of the PLA-OMMT matrices showed modest cytotoxic effect on the human dermal fibroblasts (NHDF) on the second day culture that probably originated from TMOD. These results demonstrated that PLA-OMMT/B matrices, especially PBA including matrices, can be used as replaceable wound dressings that have limited interaction with cells but exhibit antibacterial activity and support the early stages of wound healing both morphologically and chemically.
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Antibacterianos , Nanofibras , Humanos , Antibacterianos/química , Nanofibras/química , Boro , Bacterias Gramnegativas , Bacterias Grampositivas , Poliésteres/química , Compuestos de BoroRESUMEN
In this study, a biological conduit, consisting of an adipocyte-derived mesenchymal stem cell (AdMSCs) sheet and amniotic membrane (AM), was designed for the reconstruction of peripheral nerve defects. To evaluate the effect of the produced conduit on neural regeneration, a 10-mm sciatic nerve defect was created in rats, and experiments were carried out on six groups, i.e., sham control group (SC), negative control group (NC), nerve autograft group (NG), the biological conduit (AdMSCs + AM) group, the commercial PGA tube conduit (PGA) group, and the conduit only consisting of AM (AM) group. The effects of different nerve repair methods on the peripheral nerve and gastrocnemius muscle were evaluated by functional, histological, and immunohistochemical tests. When the number of myelinated axons was compared between the groups of AdMSCs + AM and PGA, it was higher in the AdMSCs + AM group (p < 0.05). The percentage of gastrocnemius collagen bundle area of AdMSCs + AM group was found to be statistically lower than the PGA group (p < 0.05). The muscle fiber diameter of AdMSCs + AM group was lower than that of the NG group, but significantly higher than that of the PGA group and the AM group (p < 0.001). Muscle weight index was significantly higher in the AdMSCs + AM group compared to the PGA group (p < 0.05). It was observed that nerve regeneration was faster in the AdMSCs + AM group, and there was an earlier improvement in pin-prick score and sciatic functional index compared to the PGA group and the AM group. In conclusion, the biological conduit prepared from the AdMSCs sheet and AM is regarded as a new biological conduit that can be used as an alternative treatment method to nerve autograft in clinical applications.
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Células Madre Mesenquimatosas , Tejido Nervioso , Humanos , Ratas , Animales , Amnios , Nervio Ciático/cirugía , Nervio Ciático/trasplante , Modelos Animales de Enfermedad , Regeneración Nerviosa/fisiologíaRESUMEN
PURPOSE: To seek out the bone regeneration effect of human umbilical cord-mesenchymal stem cell (hUC-MSC)-derived exosomes of loaded chitosan/hydroxyapatite (CS/HA) scaffold in a rat calvarium bone regeneration model. MATERIALS AND METHODS: The hUC-MSC exosomes were purified and characterized. The scaffolds were prepared by a freeze-drying method. Animals were divided into five groups, and the CS/HA/exosome (CS/HA/Exo) scaffolds were transplanted to 5 × 2-mm critical-sized calvarial bone defects for repair in rats. All animals were sacrificed at the postoperative sixth week. Immunohistochemical and histologic analyses were performed. RESULTS: Scanning electron microscopy (SEM) images showed that the exosomes were round-shaped vesicles with bounded membrane, and the diameter of the exosomes was 83.728 ± 27.269 nm. Histologic analysis showed that mean new bone volumes were statistically significantly higher in the CS/HA/Exo group (1.83 ± 0.54, PCS/Exo-CS/HA/Exo = .000), and other new bone volumes in the other groups were statistically significant compared with the control (CS/Exo 1.50 ± 0.14 mm3; CS 1.20 ± 0.43 mm3; control 1.06 ± 0.10 mm3; and CS/HA 1.43 ± 0.66 mm3). CONCLUSION: The CS/HA/Exo combination is a novel treatment for bone defect repair to induce bone formation. The CS scaffold can significantly promote bone regeneration compared with the control. Moreover, the combination with HA and exosomes is promising for applications in bone tissue regeneration.
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Quitosano , Exosomas , Células Madre Mesenquimatosas , Animales , Regeneración Ósea , Células Cultivadas , Quitosano/química , Quitosano/metabolismo , Quitosano/farmacología , Durapatita/química , Exosomas/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratas , Cráneo/patología , Cráneo/cirugía , Andamios del Tejido/química , Cordón UmbilicalRESUMEN
The development of a chitosan-based bioink that can provide a cell-friendly environment at relatively low concentration and moderate cross-linking conditions is still problematic. Here, we developed amorphous nanohydroxyapatite (nHAp) containing chitosan bioink formulations that can be gelled via the inclusion of glycerol phosphate (GP) and sodium hydrogen carbonate (SHC) into the polymer network under physiological conditions. Rheological analyses indicated that all the formulations showed shear-thinning characteristics compatible with the extrusion-based bioprinting. Also, the chitosan bioinks exhibited more gel-like structure as the weight fraction of nHAp increased from 10 % to 40 %. The printability of the chitosan-based bioinks was assessed and optimized by response surface methodology (RSM). These studies revealed that all the formulations can be successfully printed within the ranges of 50-70 kPa printing pressure and 4-11 mm/s printing speed. Multi-layered chitosan biomaterials with distinct pore structure were successfully fabricated with a high printability index. High cell viability was observed after bioprinting with pre-osteoblastic MC3T3-E1 cells. In conclusion, this study represents for the first time that chitosan biomaterials bearing suitable rheological properties and cellularity can be printed with controllable architecture for 3D bone scaffolds.
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Bioimpresión , Quitosano , Bioimpresión/métodos , Impresión Tridimensional , Materiales Biocompatibles , Ingeniería de Tejidos , Andamios del Tejido/químicaRESUMEN
Micro- or nano-surface topography of a biomaterial can improve various cellular activities for obtaining functional tissues. Electrospun fibers can gain further functionality when introduced topographic details to their surfaces. In this regard, we produced random and aligned polycaprolactone (PCL) micron/submicron fibers by the electrospinning method. Simultaneously, the surface structure of the fibers was altered by applying phase separation processes including non-solvent-induced phase separation (NIPS) and vapor-induced phase separation (VIPS) mechanisms. As a result, PCL fibers with porous, wrinkled, grooved, and crater-like morphology were obtained. Human dermal fibroblasts (BJ cells) and human keratinocytes (HS2) were cultured onto the fiber surfaces and the data were evaluated in terms of cell-material interactions. Results showed that not only the orientation of fibers but also fiber topography affected both cell-fiber and cell-cell interactions in different manners. It was observed that the wrinkled topography is the most suitable for both dermal fibroblasts and keratinocytes in terms of cell attachment and proliferation. We also concluded that cellular behavior was varied according to the morphology of the cells used. Morphological observations showed that HS2 cells proliferated more intensively on all surfaces compared to BJ cells. All these findings can be evaluated in terms of the design of tissue scaffolds, especially in skin tissue engineering.
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Poliésteres , Andamios del Tejido , Materiales Biocompatibles/química , Proliferación Celular , Fibroblastos , Humanos , Queratinocitos , Poliésteres/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Conventional wound healing treatments are insufficient for chronic wounds caused by factors such as senescence of fibroblasts, reduced growth factor synthesis, and poor angiogenesis. Recently, tissue engineering approaches have been investigated to develop effective therapies. In this study, a biochemical/biophysical stimulant-based 3D system was developed for the healing of chronic wounds. In this direction, genipin crosslinked chitosan (CHT)/gelatin (GEL) scaffolds were fabricated by freeze-drying and loaded with platelet-rich plasma (PRP). The scaffolds were seeded with human dermal fibroblasts and then, polychromatic light in near infrared region (NIR) was applied to the scaffolds for activating the platelets and stimulating the fibroblasts (photoactivation, PAC). Thus, fibroblasts were stimulated both chemically and physically by PRP and light, respectively. Cell migration, proliferation, morphology, gene expressions and reactive oxygen species (ROS) activity were evaluated in-vitro. Laminin and collagen 4 expressions that are important for extracellular matrix (ECM) formation, and PDGF (Platelet-derived growth factor) and VEGF (Vascular endothelial growth factor) expressions that are important for vascularization significantly increased in the presence of both PRP and light. Besides, PRP and light improved cell migration in 3D core-and shell model synergistically. Hydrogen peroxide content decreased in both PRP and light, indicating inhibition of ROS production. It was concluded that the stimulation of platelets with light in the NIR has a great potential to use for both platelets activation and stimulation of fibroblasts. As a result, an effective therapy can be developed for chronic wounds by using scaffold-based 3D systems together with PRP and photostimulation.
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Quitosano , Plasma Rico en Plaquetas , Proliferación Celular , Quitosano/química , Fibroblastos , Gelatina/química , Humanos , Especies Reactivas de Oxígeno/metabolismo , Andamios del Tejido/química , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
The blood-brain barrier (BBB) remains a major obstacle for the delivery of drugs in the treatment of many neurological diseases. In this study, we aimed to investigate the effects of radiofrequency electromagnetic fields (RF-EMFs) on the permeability of an in vitro BBB model under RF exposure alone, or in the presence of nanoparticles (NPs). For this purpose, an in vitro BBB model was established by seeding human umbilical vein endothelial cells (HUVECs) and human glioblastoma cell line (T98G) on the apical and basolateral sides of the transwell membrane, respectively. The integrity of the BBB model was confirmed by measuring transendothelial electrical resistance (TEER), and a fluorescein isothiocyanate (FITC)-dextran permeability assay was performed when the resistance reached 120 Ω cm2. After the RF-field exposure (13.56 MHz, 80 W, 10 min), we found that FITC-dextran transported across the in vitro BBB was increased 10-fold compared to FITC-dextran transported without an RF-field. This notable phenomenon, which can be called the burst permeability RF effect (BP-RF), has been proposed for the first time in the literature. Subsequently, the effect of the RF-field on BBB permeability was also investigated in the presence of superparamagnetic iron oxide nanoparticles (SPIONs) and magnetic poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-b-PEG) nanoparticles (m-PNPs). It was found that the amount of both transported NPs on the basolateral sides increased after exposure to the RF-field. As a result, the RF-field can be applied simultaneously during treatment with clinical agents or nanocarriers, improving the permeability of the BBB, which may contribute to therapeutic efficacy of many drugs that are used in neurological diseases.
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Bone morphogenetic proteins (BMPs), especially BMP-2, are being increasingly used in bone tissue engineering due to its osteo-inductive effects. Although recombinant human BMP-2 (rhBMP-2) was approved by Food and Drug Administration (FDA) to use for bone repair, its high doses cause undesired side effects. In order to reduce the BMP-2 dose for enhanced osteogenic differentiation, in this study we decided to suppress the synthesis of Noggin protein, the primary antagonist of BMP-2, on the MC3T3-E1 cells using Noggin targeted small interfering RNA (siRNA). Unlike other studies, Noggin siRNA (siNoggin) transfected cells were seeded on silk scaffolds, and osteogenic differentiation was investigated for a long-term period (21 days) with MTT, qPCR, SEM/EDS, and histological analysis. Besides, siNoggin transfected MC3T3-E1 cells were evaluated as a new cell source for tissue engineering studies. It was determined that Nog gene expression was suppressed in the siNoggin group and Ocn gene expression increased 5-fold compared to the control group (*p < 0.05). The osteogenic effect of BMP-2 was clearly observed in siNoggin transfected cells. According to the SEM/EDS analysis, the siNoggin group has mineral structures clustered on cells, which contain intense Ca and P elements. Histological staining showed that the siNoggin group has a more intense mineralized area than that of the control group. In conclusion, this study indicated that Noggin silencing by siRNA induces osteogenic differentiation in reduced BMP-2 doses for scaffold-based bone regeneration. This non-gene integration strategy has as a safe therapeutic potential to enhance tissue regeneration.
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Osteogénesis , Seda , Proteína Morfogenética Ósea 2/genética , Diferenciación Celular , Humanos , Osteoblastos , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Chitosan has high biocompatibility, supports proliferation of many cells, and can be a good carrier for various growth factors. However, low attachment ratio and spheroid formation of several stem cell types on plain chitosan scaffolds/films is still a problem. In this study, it was aimed to obtain 3D scaffolds using medical grade chitosan (MC) with a high deacetylation degree (DD ≥ 92.6%) to overcome the spheroid formation of rat adipose tissue derived mesenchymal stem cells (rAdMSCs) on control chitosan (C, DD = 75%-85%) scaffolds. Genipin was used as a biological chemical crosslinker, and glycerol phosphate salt was used both as a pH adjusting agent and physical crosslinker. MTT and SEM analyses and live/dead staining indicated the increase in the attachment, cell viability, and proliferation of rAdMSCs on MC scaffolds with or without crosslinking when compared to the cells in spheroid formation on control scaffolds. Moreover, filamentous actin protein organization of rAdMSCs was found to be triggered on the crosslinked MC scaffolds. In conclusion, plain medical grade chitosan scaffolds with or without crosslinking prevented spheroid formation, supported the attachment, proliferation, and organization of rAdMSCs indicating that medical grade type of chitosan scaffolds with high DD can be a very good candidate as 3D carriers in stem cell cultivation.
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Reconstruction of bone defects is still a significant challenge. The aim of this study was to evaluate the effect of application of photobiomodulation (PBM) to enhance in vivo bone regeneration and osteogenic differentiation potential of adipose-derived stem cells (ADSCs) encapsulated in methacrylated gelatin (GEL-MA) hydrogels. Thirty-six Sprague-Dawley rats were randomly separated into 3 experimental groups (n = 12 each). The groups were control/blank defect (I), GEL-MA hydrogel (II), and ADSC-loaded GEL-MA (GEL-MA+ADSC) hydrogel (III). Biparietal critical sized bone defects (6 mm in size) are created in each animal. Half of the animals from each group (n = 6 each) were randomly selected for PBM application using polychromatic light in the near infrared region, 600-1200 nm. PBM was administered from 10 cm distance cranially in 48 h interval. The calvaria were harvested at the 20th week, and macroscopic, microtomographic, and histologic evaluation were performed for further analysis. Microtomographic evaluation demonstrated the highest result for mineralized matrix formation (MMF) in group III. PBM receiving samples of group III showed mean MMF of 79.93±3.41%, whereas the non-PBM receiving samples revealed mean MMF of 60.62±6.34 % (p=0.002). In terms of histologic evaluation of bone defect repair, the higher scores were obtained in the groups II and III when compared to the control group (2.0 for both PBM receiving and non-receiving specimens; p<0.001). ADSC-loaded microwave-induced GEL-MA hydrogels and periodic application of photobiomodulation with polychromatic light appear to have beneficial effect on bone regeneration and can stimulate ADSCs for osteogenic differentiation.
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Hidrogeles , Osteogénesis , Tejido Adiposo , Animales , Regeneración Ósea , Gelatina , Ratas , Ratas Sprague-Dawley , Células MadreRESUMEN
OBJECTIVES: Unpredictability with the final volume and viability of the graft are the major concerns in fat grafting. An experimental study was conducted to increase graft retention using photobiomodulation (PBM) with polychromatic light in near-infrared region (600-1200 nm) by utilizing its stimulatory effects on angiogenesis, neovascularization, adipocyte viability, and anti-inflammatory properties. METHODS: A total of 24 rats were divided into four groups (n = 6) according to the applied polychromatic light protocol to the recipient site (none, before fat transfer, after fat transfer, and combined). In all groups, inguinal fat pad was excised, measured for volume and weight, and transferred to the dorsum of the rat. At the end of the experiment, fat grafts were harvested from the recipient site for volume and weight measurements, histological, and immunohistochemical evaluation. RESULTS: Intergroup comparison revealed that fat graft retention regarding weight and volume, was significantly superior in Group IV (p = 0.049 and p = 0.043, respectively), which polychromatic light was applied both before and after transfer of the graft. Hematoxylin-eosin and Masson's trichrome stained sections showed absence of necrosis, fibrosis, inflammation, cyst formation, and increased vascularization of both inner and outer zones of the grafts in Group IV. Also, immunohistochemical staining scores for perilipin (indicator for adipocyte viability), CD31 and VEGF (indicators for angiogenesis and neovascularization) were significantly higher (p < 0.001). Ki67 scores were significantly lower in this group because of anti-inflammatory environment (p < 0.001). CONCLUSIONS: Application of PBM to the recipient site before and after fat transfer improved outcomes in rats at 56 day after fat grafting by means of volume retention, increased neovascularization and adipocyte viability and reduced necrosis, fibrosis and inflammation.
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Adipocitos , Supervivencia de Injerto , Tejido Adiposo , Animales , Inflamación , Necrosis , Neovascularización Fisiológica , RatasRESUMEN
In skeletal muscle tissue engineering, success has not been achieved yet, since the properties of the tissue cannot be fully mimicked. The aim of this study is to investigate the potential use of poly-3-hydroxybutyrate (P3HB)/poly-ß-alanine (PBA) fibrous tissue scaffolds with piezoelectric properties for skeletal muscle regeneration. Random and aligned P3HB/PBA (5:1) fibrous matrices were prepared by electrospinning with average diameters of 951 ± 153 nm and 891 ± 247 nm, respectively. X-ray diffraction (XRD) analysis showed that PBA reinforcement and aligned orientation of fibers reduced the crystallinity and brittleness of P3HB matrix. While tensile strength and elastic modulus of random fibrous matrices were determined as 3.9 ± 1.0 MPa and 86.2 ± 10.6 MPa, respectively, in the case of aligned fibers they increased to 8.5 ± 1.8 MPa and 378.2 ± 4.2 MPa, respectively. Aligned matrices exhibited a soft and an elastic behaviour with ~70% elongation in similar to the natural tissue. For the first time, d33 piezoelectric modulus of P3HB/PBA matrices were measured as 5 pC/N and 5.3 pC/N, for random and aligned matrices, respectively. Cell culture studies were performed with C2C12 myoblastic cell line. Both of random and aligned P3HB/PBA fibrous matrices supported attachment and proliferation of myoblasts, but cells cultured on aligned fibers formed regular and thick myofibril structures similar to the native muscle tissue. Reverse transcription polymerase chain reaction (RT-qPCR) analysis indicated that MyoD gene was expressed in the cells cultured on both fiber orientation, however, on the aligned fibers significant increase was determined in Myogenin and Myosin Heavy Chain (MHC) gene expressions, which indicate functional tubular structures. The results of RT-qPCR analysis were also supported with immunohistochemistry for myogenic markers. These in vitro studies have shown that piezoelectric P3HB/PBA aligned fibrous scaffolds can successfully mimic skeletal muscle tissue with its superior chemical, morphological, mechanical, and electroactive properties.
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Poliésteres , Ingeniería de Tejidos , Ácido 3-Hidroxibutírico , Proliferación Celular , Hidroxibutiratos , Músculo Esquelético , Regeneración , Andamios del Tejido , beta-AlaninaRESUMEN
In this work we investigated tenogenic differentiation of adipose-derived mesenchymal stem cells (AdMSCs), which were seeded onto silk fibroin/poly-3-hydroxybutyrate (SF/P3HB) scaffolds with aligned topography, and high mechanical strength. The electrospinning process was optimized by using the response surface method (RSM) and SF/P3HB nanofibrous matrices with a total polymer concentration of 5% (SF: PHB = 3: 1), flow rate 1 mL/h, collector rotation speed 2000 rpm, applied voltage 14 kV, and collector distance 25 cm were obtained. The average fiber diameter was 699 ± 203 nm and 80% of the nanofibers were aligned within the ±15o range. SF reinforcement reduced the crystallinity of P3HB, and the elastic modulus was found to be 197.0 ± 7.7 MPa. The scaffolds showed bacteriostatic effect. A 21-day of cell culture study was performed with rat rAdMSCs in the absence and presence of tenogenic differentiation factor-5 (GDF-5). The results demonstrated that SF/P3HB scaffolds allow the cells to proliferate and differentiate to the tenocytes. However, no significant effect of GDF-5 on the differentiation of cells was observed. These findings indicated that our aligned SF/P3HB scaffolds have a significant potential to be used for tendon tissue engineering.
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Fibroínas , Hidroxibutiratos , Poliésteres , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Fibroínas/química , Fibroínas/farmacología , Hidroxibutiratos/química , Hidroxibutiratos/farmacología , Células Madre Mesenquimatosas , Ratones , Poliésteres/química , Poliésteres/farmacología , RatasRESUMEN
In osteochondral tissue engineering, while the biochemical and mechanical properties of hydrogels guide stem cell proliferation and differentiation, physical and chemical stimulators also affect the differentiation of stem cells. Herein, we presented a patient and tissue-specific strategy for the development of biomimetic osteochondral constructs with gradient compositions. Osteochondral constructs were fabricated by gradually printing of bio-inks consisting of therapeutic platelet-rich plasma (PRP), adipose tissue-derived mesenchymal stem cells (AdMSCs), and extracellular matrix (ECM) mimetic hydrogel, microwave-assisted methacrylated gelatin (Gel-MA). Periodic application of light in the near infrared region (600-1200 nm wavelength) was used to induce platelet activation and also AdMSCs' differentiation. Gel-MA has the same structure as type I collagen and PRP has cartilage tissue-specific bioactive components, so they provide the appropriate environment for the differentiation of AdMSCs to osteochondral tissue. Histology, immunocytochemistry, and biochemical analyses indicated enhanced glycosaminoglycan (GAG) and calcium content, mineralization, and ECM production. Furthermore, RT-PCR results indicated the expressions of bone- and cartilage-specific genes. In conclusion, the periodically photoactivated hydrogels with relatively low degradation rate and high mechanical strength, and tissue-specific biomimetic structure promoted in-vitro osteochondral tissue formation including hyaline and hypertrophic cartilage and bone phases.
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Gelatina , Plasma Rico en Plaquetas , Cartílago , Humanos , Hidrogeles , Tinta , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
The growing safety problems about the use of bone morphogenetic protein 2 (BMP-2) is one of the recent issues that was improved by using low doses of BMP-2 with the support of other osteoinductive agents and/or using appropriate carriers. The aim of the present study is to investigate the effect of scaffold-based dual release system including melatonin (MEL) and BMP-2 loaded polylactic-co-glycolic acid (PLGA) microparticles on the osteogenic activity of pre-osteoblastic MC3T3-E1 cells. MEL and BMP-2 loaded microparticles were prepared by double emulsion solvent evaporation method in the average diameters of ~2 µm and ~11 µm, respectively and loaded into chitosan/hydroxyapatite (HAp) scaffolds. In vitro MC3T3-E1 culture studies were carried out comparatively with blank scaffolds, single (BMP-2 or MEL) releasing groups and dual (BMP-2 and MEL) releasing group. Microscopic observations and hematoxylin/eosin staining showed enhanced number of cells and dense ECM in dual release group. The expressions of differentiation markers, Runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) and also mineralization were higher in dual release group than that of the other groups. Our findings showed that BMP-2 at low doses (~20 ng per scaffold) was sufficient in terms of osteogenic activity with controlled release systems where it was used in combination with MEL (~10 µg per scaffold).
Asunto(s)
Proteína Morfogenética Ósea 2 , Melatonina , Diferenciación Celular , Glicoles , Osteogénesis , Andamios del TejidoRESUMEN
Bone morphogenetic protein two (BMP-2) has been widely used as an osteoinductive agent in the treatment of bone diseases. However, some side effects, such as osteoclast activation have emerged when it was used at high doses. In this study, by considering the osteoclast-suppressing capability of melatonin (MEL), its effect on osteoclast differentiation induced by BMP-2 was investigated. These two factors, MEL and BMP-2, were embedded into chitosan/hydroxyapatite (HAp) scaffolds that were characterized morphologically by scanning electron microscopy (SEM) and micro-computed tomography (µ-CT). Release profiles of MEL and BMP-2 from scaffolds were determined in vitro and then, the differentiation of RAW 264.7 cells to osteoclasts was investigated on the scaffolds. Results of tartrate-resistant acid phosphatase (TRAP) staining, SEM imaging and expression of cathepsin K gene showed that, in the presence of BMP-2, osteoclast differentiation increased, whereas it decreased in MEL and MEL/BMP-2 embedded scaffolds suggesting that melatonin successfully attenuated osteoclast differentiation induced by BMP-2. Thus, the MEL/BMP-2 loaded chitosan/HAp scaffolds that have dual function in enhancing bone formation and inhibiting osteoclast activity are recommended biomaterials in the field of bone regeneration.