RESUMEN
BACKGROUND: Epidermal stem cells (EpSCs) play a vital role in wound healing and skin renewal. Although biomaterial scaffolds have been used for transplantation of EpSCs in wound healing, the ex vivo differentiation of EpSCs limits their application. METHODS: To inhibit the differentiation of EpSCs and maintain their stemness, we developed an electrospun polycaprolactone (PCL)+cellulose acetate (CA) micro/nanofiber for the culture and transplantation of EpSCs. The modulation effect on EpSCs of the scaffold and the underlying mechanism were explored. Liquid chromatography-tandem mass spectrometry for label-free quantitative proteomics was used to analyze proteomic changes in EpSCs cultured on scaffolds. In addition, the role of transplanted undifferentiated EpSCs in wound healing was also studied. RESULTS: In this study, we found that the PCL+CA micro/nanofiber scaffold can inhibit the differentiation of EpSCs through YAP activation-mediated inhibition of the Notch signaling pathway. Significantly differentially expressed proteomics was observed in EpSCs cultured on scaffolds and IV collagen-coated culture dishes. Importantly, differential expression levels of ribosome-related proteins and metabolic pathway-related proteins were detected. Moreover, undifferentiated EpSCs transplanted with the PCL+CA scaffold can promote wound healing through the activation of the Notch signaling pathway in rat full-thickness skin defect models. CONCLUSIONS: Overall, our study demonstrated the role of the PCL+CA micro-nanofiber scaffold in maintaining the stemness of EpSCs for wound healing, which can be helpful for the development of EpSCs maintaining scaffolds and exploration of interactions between biomaterials and EpSCs.
Asunto(s)
Nanofibras , Animales , Biomimética , Poliésteres , Proteómica , Ratas , Transducción de Señal , Células Madre , Andamios del Tejido , Cicatrización de HeridasRESUMEN
Developing basement membranes (BMs) substitute remains major problem for constructing functional tissue engineered skin because of its complex structure and multifunction of regulating cellular behavior. Herein, a stable electrospinning method was employed to generate a biomimetic model of natural BMs based on novel scaffold electrospun from Poly(É-caprolactone) (PCL) and cellulose acetate (CA) incorporated with chitosan (CS). The morphology, structure, surface hydrophilicity, roughness and mechanical tensile strength of prepared monolayer and tri-layered scaffold were comprehensively compared. Besides, co-culture system via seeding keratinocytes (Kcs) and fibroblasts (Fbs) on opposite side of tri-layered scaffold revealed more effective segregation of both cell types within the central nanofibrous barrier together with enhanced cell attachment and proliferation than that on the monolayer scaffold. Moreover, the deposition of type VII collagen and laminin-5 was examined in comparison with normal skin BMs. Furthermore, the histological studies revealed characteristics of reconstructing BM zone at the junction of dermis-epidermis after in vivo implantation for 2 weeks, and wound healing while the seeded cells interacted with the endogenous cells. Additionally, the expression of active integrin ß1 and phosphorylated focal adhesion kinase (FAK) was promoted with treatment of tri-layered scaffold. This study stressed that this tri-layer scaffold might provoke biomimetic responses of Kcs and Fbs and thus be applied for future development of BMs containing tissues.
Asunto(s)
Biomimética , Nanofibras , Membrana Basal , Proliferación Celular , Poliésteres , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Nerve growth factor (NGF) can promote the regeneration of peripheral nerve as well as contraction and reepithelization of wound. We constructed a bioengineered dermis containing microencapsulated NGF-expressing NIH-3T3 cells and study the effect of the microencapsule to the bioengineered dermis and seed cells. NGF gene was transfected into NIH-3T3 cells and enclosed into alginate-poly-L-lysine-alginate (APA) microencapsulation and cultivated in vitro. Content of NGF in microencapsules culturing supernatant was measured by enzyme linked immunosorbent assay (ELISA) method. These microencapsules were co-cultured with epidermic cells and fibroblast cells. Bioengineered dermis was constructed with NGF-expressing micorencapsules as seed cells using tissue engineering method. NIH-3T3 microencapsules, empty microencapsules, normal culture media were control groups. After one week culture, the characteristics of the dermis were described by MTT test, the content of hydroxyproline (HP), HE staining and ultrastructure photograph. We found the NGF-expressing microencapsulates can secret NGF steadly after cultured 8w in vitro, promot the proliferation of epidermic cells and secret collagen of fibroblast cells. These functions can maintaine in bioengineered dermis. So NGF-expressing NIH-3T3 microencapsulates can promote the quality of bioengineered dermis.
Asunto(s)
Alginatos/química , Dermis/citología , Factor de Crecimiento Nervioso/genética , Polilisina/análogos & derivados , Fenómenos Fisiológicos de la Piel , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/química , Proliferación Celular , Regulación de la Expresión Génica , Ratones , Células 3T3 NIH , Polilisina/química , Transfección/métodosRESUMEN
Layer-by-layer coating technique is effective in modifying the surface of nanofibrous mats, but overmuch film-coating makes the mats less porous to hardly suit the condition for tissue engineering. We developed novel nanofibrous mats layer-by-layer coated by silk fibroin and lysozyme on the cellulose electrospun template via electrostatic interaction. The film-coating assembled on the mats was not excessive because the charge of the proteins varied in the coating process due to different pH value. In addition, pure nature materials made the mats nontoxic, biodegradable and low-cost. The morphology and composition variation during layer-by-layer coating process was investigated and the results showed that the structure and thickness of film-coatings could be well-controlled. The antibacterial assay and in vitro cell experiments indicated that the mats could actively inhibit bacteria and exhibit excellent biocompatibility. In vivo implant assay further verified the mats cultured with human epidermal cells could promote wound healing and avoid wound infection. Therefore, these mats showed promising prospects when performed for dermal reconstruction.
Asunto(s)
Fibroínas/química , Regeneración Tisular Dirigida/métodos , Membranas Artificiales , Muramidasa/química , Nanofibras/uso terapéutico , Piel/lesiones , Heridas Penetrantes/terapia , Administración Tópica , Animales , Materiales Biocompatibles Revestidos , Fibroínas/administración & dosificación , Regeneración Tisular Dirigida/instrumentación , Ensayo de Materiales , Muramidasa/administración & dosificación , Nanofibras/química , Nanofibras/ultraestructura , Ratas , Piel/patología , Piel Artificial , Propiedades de Superficie , Resultado del Tratamiento , Heridas Penetrantes/patologíaRESUMEN
Sequential interpenetrating polymer networks (IPN) hydrogels based on konjac glucomannan (KGM) and poly(methacrylic acid) (PMAA) were prepared by immersion of a solution of methacrylic acid (MAA) monomer with cross-linker N,N'-methylenebisacrylamide (MBAAm) and initiating into a pre-fabricated dried KGM gel. Polymerization and cross-linking of MAA inside the KGM network resulted in a novel biodegradable pH-sensitive IPN hydrogel. The studies on the swelling behavior of IPN hydrogels reveal their sensitive response to environment pH value. It was possible to modulate the degree of swelling of the IPN gels by changing the KGM/PMAA ratio and the cross-linking density of the PMAA component. The KGM component in the IPN can be degraded by ß-glycosidase Mannaway25L. In vitro drug release behavior of IPN hydrogels were investigated under different environments using model drugs 5-fluorouracil. The results suggested that such an IPN hydrogel can be exploited as carrier candidate for colon-specific drug delivery.
Asunto(s)
Preparaciones de Acción Retardada/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Mananos/química , Ácidos Polimetacrílicos/química , Tampones (Química) , Fluorouracilo/farmacología , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Soluciones , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Chitosan and pectin/organic rectorite (OREC) were initially deposited on the surface of cellulose acetate electrospun nanofibers by a layer-by-layer (LBL) technique to fabricate scaffolds for bacterial inhibition, and the cytotoxicity of the LBL structured scaffolds was also investigated. A couple of opposite charged material, pectin and OREC, were firstly used to fabricate the intercalated composites. The intercalated structure was determined by selected area electron diffraction. Field-emission scanning electron microscope, X-ray diffraction and X-ray photoelectron spectroscopy were applied for the characterization of LBL structured nanofibrous scaffolds. Antibacterial assay results showed that the diameters of the inhibition zone increased from 7.6 to 15.8 mm for Escherichia coli, as well as from 7.4 to 14.2 mm for Staphylococcus aureus. Finally, human epidermal (EP) cells grew well on the LBL films coating. These novel scaffolds could be an ideal candidate for wound dressings and food packaging.