RESUMEN
Cartilage tissue damage and diseases are the most common clinical situation that occurs because of aging and injury, thereby causing pain and loss of mobility. The inability of cartilage tissue to self-repair is instrumental in developing tissue engineered substitutes. To this effect, the present study aims to engineer cartilage construct by culturing umbilical cord blood-derived human mesenchymal stem cells (hMSCs) on novel 3D porous scaffolds developed from natural biopolymers, silk fibroin (SF) and chitosan (CS), with addition of cartilage matrix components, glucosamine (Gl) and chondroitin sulfate (Ch). The presence of Gl and Ch is expected to enhance cartilage regeneration. The developed SF/CS-Gl-Ch scaffolds possess desired pore size in the range 56.55-168.15⯵m, 88-92% porosity, 44.7-46.8Ì contact angle, controlled swelling and biodegradability. Upon culturing under dynamic condition in a spinner flask bioreactor, the scaffold supported hMSCs attachment, proliferation, and further promoted chondrogenic differentiation. Cartilage-specific matrix and gene (Collagen II, Sox9 and aggrecan) expression analyses by histology, immunophenotype, immunofluorescence and quantitative PCR studies showed superiority of cell-scaffold construct generated in dynamic culture towards cartilage tissue generation as compared to cell aggregates formed by pellet culture. This study demonstrates the potentiality of SF/CS-Gl-Ch porous scaffold for the development of tissue construct for cartilage regeneration under dynamic culture condition.
Asunto(s)
Diferenciación Celular/fisiología , Fibroínas/metabolismo , Glicosaminoglicanos/metabolismo , Células Madre Mesenquimatosas/citología , Condrogénesis/fisiología , Colágeno/metabolismo , HumanosRESUMEN
Electrospinning technique converts polymeric solutions into nanoscale fibers using an electric field and can be used for various biomedical and clinical applications. Extracellular vesicles (EVs) are cell-derived small lipid vesicles enriched with biological cargo (proteins and nucleic acids) potential therapeutic applications. In this review, we discuss extending the scope of electrospinning by incorporating stem cell-derived EVs, particularly exosomes, into nanofibers for their effective delivery to target tissues. The parameters used during the electrospinning of biopolymers limit the stability and functional properties of cellular products. However, with careful consideration of process requirements, these can significantly improve stability, leading to longevity, effectiveness, and sustained and localized release. Electrospun nanofibers are known to encapsulate or surface-adsorb biological payloads such as therapeutic EVs, proteins, enzymes, and nucleic acids. Small EVs, specifically exosomes, have recently attracted the attention of researchers working on regeneration and tissue engineering because of their broad distribution and enormous potential as therapeutic agents. This review focuses on current developments in nanofibers for delivering therapeutic cargo molecules, with a special emphasis on exosomes. It also suggests prospective approaches that can be adapted to safely combine these two nanoscale systems and exponentially enhance their benefits in tissue engineering, medical device coating, and drug delivery applications.
Asunto(s)
Sistemas de Liberación de Medicamentos , Exosomas , Nanofibras , Regeneración , Células Madre , Ingeniería de Tejidos , Nanofibras/química , Humanos , Exosomas/metabolismo , Células Madre/citología , Ingeniería de Tejidos/métodos , Animales , Andamios del Tejido/químicaRESUMEN
Digital light processing (DLP) technology has gained significant attention for its ability to construct intricate structures for various applications in tissue modeling and regeneration. In this study, we aimed to design corneal lenticules using DLP bioprinting technology, utilizing dual network bioinks to mimic the characteristics of the human cornea. The bioink was prepared using methacrylated hyaluronic acid and methacrylated gelatin, where ruthenium salt and sodium persulfate were included for mediating photo-crosslinking while tartrazine was used as a photoabsorber. The bioprinted lenticules were optically transparent (85.45% ± 0.14%), exhibited adhesive strength (58.67 ± 17.5 kPa), and compressive modulus (535.42 ± 29.05 kPa) sufficient for supporting corneal tissue integration and regeneration. Puncture resistance tests and drag force analysis further confirmed the excellent mechanical performance of the lenticules enabling their application as potential corneal implants. Additionally, the lenticules demonstrated outstanding support for re-epithelialization and stromal regeneration when assessed with human corneal stromal cells. We generated implant ready corneal lenticules while optimizing bioink and bioprinting parameters, providing valuable solution for individuals suffering from various corneal defects and waiting for corneal transplants.
Asunto(s)
Bioimpresión , Trasplante de Córnea , Humanos , Ingeniería de Tejidos , Andamios del Tejido/química , Córnea , Impresión Tridimensional , HidrogelesRESUMEN
Cornea-related injuries are the most common cause of blindness worldwide. Transplantation remains the primary approach for addressing corneal blindness, though the demand for donor corneas outmatches the supply by millions. Tissue adhesives employed to seal corneal wounds have shown inefficient healing and incomplete vision restoration. We have developed a biodegradable hydrogel - Kuragel, with the ability to promote corneal regeneration. Functionalized gelatin and hyaluronic acid form photo-crosslinkable hydrogel with transparency and compressive modulus similar to healthy human cornea. Kuragel composition was tuned to achieve sufficient adhesive strength for sutureless integration to host tissue, with minimal swelling post-administration. Studies in the New Zealand rabbit mechanical injury model affecting corneal epithelium and stroma demonstrate that Kuragel efficiently promotes re-epithelialization within 1 month of administration, while stroma and sub-basal nerve plexus regenerate within 3 months. We propose Kuragel as a regenerative treatment for patients suffering from corneal defects including thinning, by restoration of transparency and thickness.
RESUMEN
Biological materials derived from extracellular matrix (ECM) proteins have garnered interest as their composition is very similar to that of native tissue. Herein, we report the use of human cornea derived decellularized ECM (dECM) microparticles dispersed in human fibrin sealant as an accessible therapeutic alternative for corneal anterior stromal reconstruction. dECM microparticles had good particle size distribution (≤10 µm) and retained the majority of corneal ECM components found in native tissue. Fibrin-dECM hydrogels exhibited compressive modulus of 70.83 ± 9.17 kPa matching that of native tissue, maximum burst pressure of 34.3 ± 3.7 kPa, and demonstrated a short crosslinking time of ~17 min. The fibrin-dECM hydrogels were found to be biodegradable, cytocompatible, non-mutagenic, non-sensitive, non-irritant, and supported the growth and maintained the phenotype of encapsulated human corneal stem cells (hCSCs) in vitro. In a rabbit model of anterior lamellar keratectomy, fibrin-dECM bio-adhesives promoted corneal re-epithelialization within 14 days, induced stromal tissue repair, and displayed integration with corneal tissues in vivo. Overall, our results suggest that the incorporation of cornea tissue-derived ECM microparticles in fibrin hydrogels is non-toxic, safe, and shows tremendous promise as a minimally invasive therapeutic approach for the treatment of superficial corneal epithelial wounds and anterior stromal injuries.
Asunto(s)
Córnea/citología , Matriz Extracelular/metabolismo , Cicatrización de Heridas , Animales , Cadáver , Proliferación Celular , Córnea/patología , Córnea/fisiología , Enfermedades de la Córnea/patología , Enfermedades de la Córnea/terapia , Matriz Extracelular/química , Fibrina/química , Humanos , Hidrogeles/química , Conejos , Trasplante de Células Madre , Células Madre/citología , Células Madre/metabolismo , Ingeniería de TejidosRESUMEN
AIM: Cartilage damage is a common age-related problem that leads to progressive proteoglycan loss. Glucosamine stimulates proteoglycan synthesis and, therefore, its effect on the cartilage extracellular matrix synthesis over silk fibroin:chitosan (SF:CS) tissue-engineered scaffold was investigated for cartilage construct generation. MATERIALS & METHODS: Human mesenchymal stem cells (hMSCs) were cultured and differentiated over SF:CS-glucosamine porous scaffold, under dynamic culture condition in spinner flask bioreactor. RESULTS: hMSCs-seeded scaffold in dynamic culture exhibited homogenous cell distribution, proliferation and higher cell density at the core than static culture. Glucosamine in scaffold promoted proteoglycan and collagenous matrix synthesis as revealed by histological and immunofluorescence studies. Quantitative-PCR analysis showed upregulation of cartilage-specific genes, thereby confirming the chondrogenic differentiation. CONCLUSION: The chondrogenic differentiation of hMSCs was enhanced by the synergistic effect of glucosamine incorporated in SF:CS scaffold and influence of 3D dynamic culture environment, thereby resulting in chondrogenic phenotype of the cells that promoted cartilage regeneration.
Asunto(s)
Diferenciación Celular , Quitosano/química , Condrogénesis , Fibroínas/química , Glucosamina/química , Células Madre Mesenquimatosas/metabolismo , Andamios del Tejido/química , Técnicas de Cultivo de Célula/métodos , Humanos , Células Madre Mesenquimatosas/citologíaRESUMEN
Cartilage construct generation includes a scaffold with appropriate composition to mimic matrix of the damaged tissue on which the stem cells grow and differentiate. In this study, umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) were seeded on freeze dried porous silk-fibroin (SF)/chitosan (CS) scaffolds. Influence of static and dynamic (spinner flask bioreactor) culture conditions on the developing cartilage construct were studied by in-vitro characterization for viability, proliferation, distribution, and chondrogenic differentiation of hMSCs over the scaffold. Constructs developed in spinner flask consisted of 62% live cells, and exhibited 543% more cell density at the core than constructs cultured in static system. Quantification of DNA and glycosaminoglycans accumulation after 21 days showed the progression of chondrogenic differentiation of hMSCs was higher in dynamic culture compared to static one. In constructs generated under dynamic condition, histology staining for proteoglycan matrix, and fluorescence staining for collagen-II and aggrecan showed positive correlation between early and late stage chondrogenic markers, which was further confirmed by quantitative PCR analysis, showing low collagen-I expression and highly expressed Sox9, collagen-II and aggrecan. The present study demonstrated that construct generated by combining 3D SF/CS scaffold with UCB-hMSCs under dynamic condition using spinner flask bioreactor can be used for cartilage tissue regeneration for future medical treatments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 397-407, 2018.
Asunto(s)
Cartílago/fisiología , Técnicas de Cultivo de Célula/métodos , Sangre Fetal/citología , Fibroínas/farmacología , Células Madre Mesenquimatosas/citología , Andamios del Tejido/química , Animales , Bombyx , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrogénesis/efectos de los fármacos , ADN/metabolismo , Fluorescencia , Glicosaminoglicanos/metabolismo , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/ultraestructura , PorosidadRESUMEN
Chondroitin sulfate (Ch) is one of the main structural components of cartilage tissue, therefore, its presence in tissue engineered scaffold is expected to enhance cartilage regeneration. Previously, silk fibroin/chitosan (SF/CS) blend was proven to be a potential biomaterial for tissue development. In this study, the effect of Ch on physicochemical and biological properties of SF/CS blend was investigated and scaffolds with 0.8 wt% Ch was found to be favorable. The scaffolds possess pore size of 37-212 µm, contact angle 46.2-50.3°, showed controlled swelling and biodegradation. The biocompatibility of scaffold was confirmed by subcutaneous implantation in mouse. Human mesenchymal stem cells (hMSCs) seeded scaffolds cultured under spinner flask bioreactor promoted cell attachment, proliferation, distribution, and metabolic activity in vitro. The histology and immunofluorescence studies revealed that combined effect of Ch and dynamic condition resulted in higher glycosaminoglycan secretion and native cartilage type matrix synthesis in comparison to SF/CS scaffolds used as control. Higher expression of collagen-II, Sox9, aggrecan and decrease in collagen-I expression represented by quantitative polymerase chain reaction study confirmed the progression of chondrogenic differentiation. This study successfully demonstrates the potentiality of SF/CS-Ch scaffold for hMSCs recruitment and redirecting cartilage tissue regeneration with enhanced chondrogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2576-2587, 2018.
Asunto(s)
Técnicas de Cultivo de Célula , Condrogénesis , Sulfatos de Condroitina/química , Fibroínas/química , Células Madre Mesenquimatosas/metabolismo , Andamios del Tejido/química , Animales , Humanos , Células Madre Mesenquimatosas/citología , RatonesRESUMEN
Deformities in tissues and organs can be treated by using tissue engineering approach offering the development of biologically functionalized scaffolds from a variety of polymer blends which mimic the extracellular matrix and allow adjusting the material properties to meet the defect architecture. In recent years, research interest has been shown towards the development of chitosan (CS) based biomaterials for tissue engineering applications, because of its minimal foreign body reactions, intrinsic antibacterial property, biocompatibility, biodegradability and ability to be molded into various geometries and forms thereby making it suitable for cell ingrowth and conduction. The present work involves the fabrication of nanofibrous scaffold from CS and poly(vinyl alcohol) blends by free-surface electrospinning method. The morphology and functional characteristics of the developed scaffolds were assessed by field emission scanning electron microscopy and fourier transformed infra-red spectra analysis. The morphological analysis showed the average fiber diameter was 269 nm and thickness of the mat was 200-300 µm. X-ray diffraction study confirmed the crystalline nature of the prepared scaffolds, whereas hydrophilic characteristic of the prepared scaffolds was confirmed by measured contact angle. The scaffolds possess an adequate biodegradable, swelling and mechanical property that is found desirable for tissue engineering applications. The cell study using umbilical cord blood-derived mesenchymal stem cells has confirmed the in vitro biocompatibility and cell supportive property of the scaffold thereby depicting their potentiality for future clinical applications.