Substrates For Tissue Engineering Research Articles

Platelet-rich plasma-hyaluronic acid/chondrotin sulfate/carboxymethyl chitosan hydrogel for cartilage regeneration.

In this study, the chondrogenic potential of hyaluronic acid/chondrotin sulfate/carboxymethyl chitosan hydrogels with adipose-derived mesenchymal stem cells (ADMSCs) was evaluated. Here, hyaluronic acid, chondrotin sulfate, and carboxymethyl chitosan were used as the substrate for cartilage tissue engineering in which the hydrogel is formed due to electrostatic and hydrogen bonds through mixing the polymers. Because of the instability of this hydrogel in the biological environment, 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride/N-hydroxy-succinimide was used as a crosslinker to increase the hydrogel stability. The hydrogels showed reasonable stability due to the combined effect of self-crosslinking and chemical crosslinking. The cells were treated with the prepared hydrogel samples for 14 and 21 days in nondifferentiation medium for evaluation of the cellular behavior of ADMSCs. Gene expression evaluation was performed, and expression of specific genes involved in differentiation was shown in the crosslinked hydrogel with platelet-rich plasma (PRP) (H-EN-P) had increased the gene expression levels. Quantification of immunofluorescence intensity indicated the high level of expression of SOX9 in H-EN-P hydrogel. Based on the results, we confirmed that the presence of PRP and the similarity of the hydrogel constituents to the cartilage extracellular matrix could have positive effects on the differentiation of the cells, which is favorable for cartilage tissue engineering approaches.

Biomimetic 3D bacterial cellulose-graphene foam hybrid scaffold regulates neural stem cell proliferation and differentiation

Neural stem cell (NSC)-based therapy is a promising candidate for treating neurodegenerative diseases and the preclinical researches call an urgent need for regulating the growth and differentiation of such cells. The recognition that three-dimensional culture has the potential to be a biologically significant system has stimulated an extraordinary impetus for scientific researches in tissue engineering and regenerative medicine. Here, A novel scaffold for culturing NSCs, three-dimensional bacterial cellulose-graphene foam (3D-BC/G), which was prepared via in situ bacterial cellulose interfacial polymerization on the skeleton surface of porous graphene foam has been reported. 3D-BC/G not only supports NSC growth and adhesion, but also maintains NSC stemness and enhances their proliferative capacity. Further phenotypic analysis indicated that 3D-BC/G induces NSCs to selectively differentiate into neurons, forming a neural network in a short amount of time. The scaffold has good biocompatibility with primary cortical neurons enhancing the neuronal network activities. To explore the underlying mechanisms, RNA-Seq analysis to identify genes and signaling pathways was performed and it suggests that 3D-BC/G offers a more promising three-dimensional conductive substrate for NSC research and neural tissue engineering, and the repertoire of gene expression serves as a basis for further studies to better understand NSC biology.

Mechanical behaviour, hybridisation and osteoblast activities of novel baghdadite/ PCL-graphene nanocomposite scaffold: viability, cytotoxicity and calcium activity

ABSTRACT The research aimed to evaluate the mechanical behaviour, hybridisation and osteoblast activities of novel baghdadite/PCL-graphene nanocomposite scaffold. The mechanical behaviour was examined via measuring the compressive strength and Young’s module, the hybridisation was evaluated by Fourier transform infrared spectroscopy, Raman spectroscopy, and Brunauer Emmet Teller, and the osteoblast activities were assessed via MG-63 osteoblast cells. The results rendered PCL as a significant factor to enhance the mechanical strength of ceramic scaffolds. Due to the existence of σ and π covalent bonds in its structure, hydrophilicity and biocompatibility, graphene could be applied in scaffolds’ chemical compound to greatly enhance their mechanical and biological behaviours. This scaffold indicated compressive strength and Young’s module higher than 2 MPa and 0.05 GPa. Regarding cell behaviours, MG-63 osteoblast cells spread and attached well on the scaffolds confirming the viability, cytotoxicity, excellent cell attachment and proliferation. The results indicated that this scaffold possesses outstanding potential as a temporary substrate for bone tissue engineering.

Fabrication and characterization of forcespun polycaprolactone microfiber scaffolds

Forcespinning technique was used to fabricate sub-micron size polycaprolactone (PCL) fibers. Forcespinning method uses centrifugal forces for the generation of fibers unlike the electrospinning method which uses electrostatic force. PCL has been extensively used as scaffolds for cell regeneration, substrates for tissue engineering and in drug delivery systems. The aim of this study is to qualitatively analyze the force spun fiber mats and investigate the effect of the spinneret rotational speed on the fiber morphology, thermal and mechanical properties. The extracted fibers were characterized by scanning electron microscopy differential scanning calorimetry, tensile testing and dynamic mechanical analysis. The results showed that higher rotational speeds produced uniform fibers with less number of beads. The crystallinity of the fibers decreased with increase in rotational speeds. The Young’s modulus of the forcespun fibers was found to be in the range of 3.5 to 6 MPa. Storage and loss moduli decreased with the increase in the fiber diameter. The fibers collected at farther distance from spinneret exhibited optimal mechanical properties compared to the fibers collected at shorter distances. This study will aid in extracting fibers with uniform geometries and lower beads to achieve the desired nanofiber drug release properties.

Fibroblast encapsulation in gelatin methacryloyl (GelMA) versus collagen hydrogel as substrates for oral mucosa tissue engineering

PurposeOver the past decades, a variety of biomaterials have been investigated in terms of their suitability for oral mucosa tissue engineering. The aim of this study was to compare collagen and GelMA hydrogels as connective tissue scaffolds for fibroblasts and as substrates for seeding and culture of oral epithelial keratinocyte cells. MethodsHuman primary oral fibroblast and keratinocyte cells were isolated from gingival biopsies. The mixture of fibroblasts with GelMA or collagen gel were aliquoted within six-well tissue culture plate inserts and cross-linked using visible light or reconstitution buffer/heat, respectively. The viability of fibroblasts in the hydrogels was investigated after one and three days of cultivation using the PrestoBlue assay. Following the addition and culture of oral keratinocytes onto the connective tissue constructs, the tissue-engineered oral mucosa was assessed histologically. ResultsThe tissue viability assay shows that collagen hydrogels encapsulating fibroblasts displayed significantly higher cell viability than cell-laden GelMA constructs after 24 and 72 h (p < 0.05). A stratified and differentiated epithelium has formed on the surface of cell-laden collagen hydrogel but not on the surface of the GelMA-based substrate. ConclusionCollagen-based scaffold offers superior biological properties compared to GelMA hydrogel in terms of oral fibroblast growth, as well as epithelial cell adhesion and differentiation. Therefore, collagen-based hydrogels remain the preferred choice for oral mucosa tissue engineering.

Polycaprolactone fumarate acts as an artificial neural network to promote the biological behavior of neural stem cells.

Herein, we investigated the effect of electrospun polycaprolactone fumarate (PCLF) nanofibers on neural stem cell (NSC) behavior in the in vitro setting. Murine NSCs were isolated from adult mice subventricular zone and immunophenotyped by flow cytometry assay and immunofluorescence staining. Cells were cultured on the plastic surface, laminin-coated surface, and electrospun PCLF nanofibers. Cell morphology, attachment, and spreading were evaluated by scanning electron microscopy analysis. Cell viability and proliferation rates were evaluated by MTT assay. The proliferation of plated cells was investigated by monitoring Ki-67-positive cells using flow cytometry analysis. The protein levels of Map-2 and GFAP were detected by using immunofluorescence staining to show neural and astrocyte differentiation capacity. Scanning electron microscopy images revealed an extensive distribution, morphological adaptation, and cell-to-cell connectivity in NSCs upon culture on the PCLF surface. MTT analysis showed that the NSCs had more survival rates on the PCLF surface compared to the laminin and control groups over time (p < 0.05). In contrast to the laminin group, Ki-67 analysis showed a decrease of proliferating cells in the PCLF group. Immunofluorescence staining revealed the prominent increase of Map-2 and GFAP reduction in NSCs from the PCLF group compared to the laminin and control groups, showing the stimulatory effect of PCLF on targeted maturation of NSCs (p < 0.05). In brief, PCLF based construct promotes NSCs morphological adaptation and neuronal differentiation, suggesting PCLF as an appropriate and applicable substrate in neural tissue engineering.

Crossing the hurdles of translation—a robust methodology for synthesis, characterization and GMP production of cross‐linked high molecular weight hyaluronic acid particles (cHA)

AbstractHyaluronic acid (HA) is one of the most widely used extracellular matrix substrate in tissue engineering, drug delivery, and other clinical applications, due to its unique physiochemical properties and ubiquitous biological presence. In the past decade, there has been a surge in research paradigms involving HA products to evaluate their commercial feasibility. Numerous papers and reviews have reported procedures for chemical modifications and cross‐linking of HA, but the intricacies of their scale‐up in the production processes are often not discussed. Protected by confidentiality agreements with industry partners, information on these procedures is exclusive and not accessible within an academic setting. Establishing translatable synthetic protocols of HA would address this significant gap in the field and facilitate their use in other applications. The current method details a reproducible and robust method for producing particles that are composed of high molecular weight hyaluronic acid (cHA) and cross‐linked via a 4‐arm polyethylene glycol amine linker using 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methyl‐morpholinium chloride chemistry. A critical analysis of previously reported procedures for their advantages and limitations (reaction parameters, molar equivalents, and reagents used for cross‐linking), forms the basis for this procedure and its subsequent adaptation to good manufacturing practices requirements. As a component of a Class III medical device, the reported cHA is in the form of non‐sized particles.

Selective laser sintered bio-inspired silicon-wollastonite scaffolds for bone tissue engineering.

The scaffolds, which morphologically and physiologically mimic natural features of the bone, are of high demand for regenerative medicine. To address this challenge, we have developed innovative bioactive porous silicon- wollastonite substrates for bone tissue engineering. Additive manufacturing through selective laser melting approach has been exploited to fabricate scaffolds of different architecture. Unique material combining osteoinductivity, osteoconductivity and bioactive elements allows flexibility in design. As the porous structure is required for the ingrowth of the bone tissue, the CAD designed scaffolds with pore size of 400μm and hierarchical gradient of pore size from 50μm to 350μm have been 3D printed and tested in vitro. The scaffolds have demonstrated not only the enhanced viability and differential patterning of human mesenchymal cells (hMSC) guided by the biomimetic design onto extra and intra scaffold space but also promoted the osteogenic differentiation in vitro. Both homogeneous and gradient scaffolds have shown the differential expression of primary transcription factors (RUNX2, OSX), anti-inflammatory factors and cytokines, which are important for the regulation of ossification. The effective elastic modulus and compressive strength of scaffolds have been calculated as 1.1±0.9GPa and 37±13.5MPa with progressive failure for homogeneous structured scaffold; and 1.8±0.9GPa and 71±9.5MPa for gradient-structured scaffold with saw-tooth fracture mode and sudden incognito failure zones. The finite element analysis reveals more bulk stress onto the gradient scaffolds when compared to the homogeneous counterpart. The findings demonstrate that as-produced composite ceramic scaffolds can pave the way for treating specific orthopaedic defects by tailoring the design through additive manufacturing.

Programmable release of 2-O-d-glucopyranosyl-l-ascorbic acid and heparin from PCL-based nanofiber scaffold for reduction of inflammation and thrombosis

Reduction of inflammation and thrombosis caused by implanted devices is critical for clinical success. To this end, the strategy based on programmable release of anti-inflammatory and anti-thrombotic agents from the widely-used polycaprolactone (PCL)/gelatin nanofiber scaffold is developed. The release of 2-O-d-Glucopyranosyl-l-ascorbic Acid (AA-2G) and heparin are controlled by reactive oxygen species (ROS)-responsive poly(ethylene glycol)-based β-thioether ester copolymer (PEGDA-EDT) and mesoporous silica nanoparticles (MSN) in the nanofiber, respectively. The in vitro assay demonstrate that the scaffolds are hemocompatible with the resistance of platelet adhesion; the control release of AA-2G prevents initial inflammation and oxidation of the blood cells, and the subsequent release of heparin entitles nanofibers with long-term anti-thrombotic capability. In addition, rapid endothelialization is obtained on the surface of nanofiber scaffolds for the further enhancement of the hemocompatibility. In vivo implant evaluation convinces that the nanofiber scaffolds possess high biocompatibility with the substantial resistance for inflammation and thrombosis. Hence, our work paves a new way to develop the anti-inflammatory and anti-thrombotic tissue-engineering substrates through programmable delivery of two or multiple drugs.

Poly(-caprolactone)/graphene oxide composite systems: A comparative study on hydrolytic degradation at extreme pH values

Polycaprolactone/Graphene oxide (PCL/GO) composites are shown to be promising substrates for tissue engineering as their degradation behavior is a key aspect in this type of application. The present paper studies the effect of different GO contents (0.1, 0.2 and 0.5 wt%) of PCL/GO composites on accelerated hydrolytic degradation at extreme pH values. Degradation kinetics at pH 13 is strongly affected by GO content, and speed up at higher percentages. The composite with 0.5 wt% of GO was completely degraded in 72 hours, while degradation at pH 1 presents a different profile and seems to have an induction period that lasts more than 1500 hours. Morphological changes, molecular weight distribution, weight loss, degree of swelling and calorimetric properties were investigated as a function of degradation time. According to the results obtained, the addition of small percentages of GO significantly influences the degradation behavior of the composites acting as degradation modulators.

Main-Chain Liquid Crystalline Hydrogels that Support 3D Stem Cell Culture.

Hydrogels are frequently utilized as three-dimensional matrices for the culture and regeneration of soft tissues, but one challenge with the existing hydrogels is that, though the natural extracellular matrix of tissues may be ordered, there are few biocompatible ways to incorporate anisotropy within hydrogels. Liquid crystalline (LC) polymers are well suited for this because of their combination of molecular ordering and polymer elasticity; however, the hydrophobic nature of LC monomers has hindered their polymerization into hydrogels under cytocompatible conditions. This work reports on the generation of main-chain LC hydrogels in aqueous media and the ability of LC phases to affect mesenchymal stem cell behavior. The synthesis results in high gel fraction materials, and calorimetry, thermomechanical analysis, and X-ray scattering show that the networks organize into LC phases in the dry and hydrogel states. Human mesenchymal stem cells (hMSCs) cultured within the hydrogels show excellent viability, and hMSC proliferation proceeds at a faster rate in LC hydrogels compared to non-LC hydrogels. TThe result is a new synthetic approach for calamitic liquid crystalline hydrogels, which support the encapsulation and culture of human stem cells and are expected to enable applications as anisotropic and responsive substrates for tissue engineering and regenerative medicine.

Characterization of novel calcium hydroxide-mediated highly porous chitosan-calcium scaffolds for potential application in dentin tissue engineering.

The aim of this study was to develop a highly porous calcium-containing chitosan scaffold suitable for dentin regeneration. A calcium hydroxide (Ca[OH]2 ) suspension was used to modulate the degree of porosity and chemical composition of chitosan scaffolds. The chitosan solution concentration and freezing protocol were adjusted to optimize the porous architecture using the phase-separation technique. Scanning electron microscopy/energy-dispersive spectroscopy demonstrated the fabrication of a highly porous calcium-linked chitosan scaffold (CH-Ca), with a well-organized and interconnected porous network. Scaffolds were cross-linked on glutaraldehyde (GA) vapor. Following a 28-day incubation in water, cross-linked CH scaffold had no changes on humid mass, and CH-Ca featured a controlled degradability profile since the significant humid mass loss was observed only after 21 (26.0%) and 28 days (42.2%). Fourier-transform infrared spectroscopy indicated the establishment of Schiff base on cross-linked scaffolds, along with calcium complexation for CH-Ca. Cross-linked CH-Ca scaffold featured a sustained Ca2+ release up to 21 days in a humid environment. This porous and stable architecture allowed for human dental pulp cells (HDPCs) to spread throughout the scaffold, with cells exhibiting a widely stretched cytoplasm; whereas, the cells seeded onto CH scaffold were organized in clusters. HDPCs seeded onto CH-Ca featured significantly higher ALP activity, and gene expressions for ALP, Col1, DMP-1, and DSPP in comparison to CH, leading to a significant 3.5 times increase in calcium-rich matrix deposition. In sum, our findings suggest that CH-Ca scaffolds are attractive candidates for creating a highly porous and bioactive substrate for dentin tissue engineering.

ECM Mimetic Electrospun Porous Poly (L-lactic acid) (PLLA) Scaffolds as Potential Substrates for Cardiac Tissue Engineering.

Cardiac tissue engineering (CTE) aims to generate potential scaffolds to mimic extracellular matrix (ECM) for recreating the injured myocardium. Highly porous scaffolds with properties that aid cell adhesion, migration and proliferation are critical in CTE. In this study, electrospun porous poly (l-lactic acid) (PLLA) porous scaffolds were fabricated and modified with different ECM derived proteins such as collagen, gelatin, fibronectin and poly-L-lysine. Subsequently, adult human cardiac fibroblasts (AHCF) were cultured on the protein modified and unmodified fibers to study the cell behavior and guidance. Further, the cytotoxicity and reactive oxygen species (ROS) assessments of the respective fibers were performed to determine their biocompatibility. Excellent cell adhesion and proliferation of the cardiac fibroblasts was observed on the PLLA porous fibers regardless of the surface modifications. The metabolic rate of cells was on par with the conventional cell culture ware while the proliferation rate surpassed the latter by nearly two-folds. Proteome profiling revealed that apart from being an anchorage platform for cells, the surface topography has modulated significant expression of the cellular proteome with many crucial proteins responsible for cardiac fibroblast growth and proliferation.

Cell growth on electrospun nanofiber mats from polyacrylonitrile (PAN) blends

Nanofiber mats can be produced by electrospinning from diverse polymers and polymer blends as well as with embedded ceramics, metals, etc. The large surface-to-volume ratio makes such nanofiber mats a well-suited substrate for tissue engineering and other cell growth experiments. Cell growth, however, is not only influenced by the substrate morphology, but also by the sterilization process applied before the experiment as well as by the chemical composition of the fibers. A former study showed that cell growth and adhesion are supported by polyacrylonitrile/gelatin nanofiber mats, while both factors are strongly reduced on pure polyacrylonitrile (PAN) nanofibers. Here we report on the influence of different PAN blends on cell growth and adhesion. Our study shows that adding ZnO to the PAN spinning solution impedes cell growth, while addition of maltodextrin/pea protein or casein/gelatin supports cell growth and adhesion.

Intact extracellular matrix component promotes maintenance of liver-specific functions and larger aggregates formation of primary rat hepatocytes.

The extracellular matrix (ECM) in a liver-specific extracellular matrix (L-ECM) scaffold facilitates hepatocyte viability and maintains hepatocyte functions in vitro. However, whether an intact composition of ECM is required for an efficient ECM-based substrate design remains to be clarified. In this study, two L-ECM hydrogels, namely L-ECM I and L-ECM II, were prepared by pepsin solubilization at 4 °C and 25 °C, respectively. The solubility at 4 °C was 50% whereas that at 25 °C was 95%, thus indicating well-preserved L-ECM. Analysis confirmed higher ECM protein components (especially collagen) in L-ECM II, along with denser fiber network and larger fiber diameter. L-ECM II gel exhibited high compression strength and suitable viscoelastic properties. Furthermore, hepatocytes in L-ECM II showed higher expression of liver-specific functions in 3D culture and wider spread while maintaining the cell-cell contacts in 2D culture. Therefore, an intact L-ECM is important to realize effective substrates for liver tissue engineering.

The Background and Scope of Polyphosphazenes as Biomedical Materials

Although many traditional organic polymers have been evaluated for uses in biology and medicine, relatively few have proved to be satisfactory for crucial uses such as surgical sutures or mesh, tissue engineering substrates, controlled drug delivery, or soft matter. A reason for this is that most large-volume commercial polymers are optimized for some mechanical engineering purpose, and biomedical compatibility or bioerodibility is not one of the target properties. Thus, biomedical scientists and engineers have been forced to improvise and compromise by using widely available non-biological polymers, mainly because those materials are already available in commercial quantities. Polyphosphazenes offer an opportunity to solve many of these problems. A crucial need exists for new polymers that can be utilized in orthopedics, cardiovascular, dental, neural, or drug delivery applications, yet very few long-existing polymers have properties that are ideal for these medical uses. Our research seeks to design and find methods to synthesize polymeric materials that are specifically designed to solve a range of medical challenges. In our program, we make use of an unusual polymer backbone comprised of alternating phosphorus and nitrogen atoms to which are attached a wide range of organic side groups. This system is unique in the wide range of different property combinations that can be generated, and many of these combinations can be matched precisely to the needs of medical materials.

Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications.

This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS-PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yielding long-term stability and adequate stiffness for 3D cell culture, as confirmed with compression testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types.

Versatile Biomaterial Platform Enriched with Graphene Oxide and Carbon Nanotubes for Multiple Tissue Engineering Applications.

Carbon-based nanomaterials, such as graphene oxide (GO) or carbon nanotubes (CNTs) are currently used in various medical applications due to their positive influence on biocompatibility, adhesion, proliferation, and differentiation, as well as their contribution to modulating cell behavior in response to nanomaterial substrates. In this context, in this study, novel flexible membranes based on cellulose acetate (CA) enriched with CNT and GO in different percentages were tested for their versatility to be used as substrates for soft or hard tissue engineering (TE), namely, for their ability to support human adipose-derived stem cells (hASCs) adhesion during adipogenic or osteogenic differentiation. For this purpose, differentiation markers were assessed both at gene and protein levels, while histological staining was performed to show the evolution of the processes in response to CA-CNT-GO substrates. Micro-CT analysis indicated porous morphologies with open and interconnected voids. A slightly lower total porosity was obtained for the samples filled with the highest amount of GO and CNTs, but thicker walls, larger and more uniform pores were obtained, providing beneficial effects on cell behavior and increased mechanical stability. The addition of 1 wt% GO and CNT to the biocomposites enhanced hASCs adhesion and cytoskeleton formation. The evolution of both adipogenic and osteogenic differentiation processes was found to be augmented proportionally to the GO-CNT concentration. In conclusion, CA-CNT-GO biomaterials displayed good properties and versatility as platforms for cell differentiation with potential as future implantable materials in TE applications.

Enhanced Osteogenic Differentiation of Human Fetal Cartilage Rudiment Cells on Graphene Oxide-PLGA Hybrid Microparticles.

Poly(d,l–lactide–co–glycolide) (PLGA) has been extensively explored for bone regeneration applications; however, its clinical use is limited by low osteointegration. Therefore, approaches that incorporate osteoconductive molecules are of great interest. Graphene oxide (GO) is gaining popularity for biomedical applications due to its ability to bind biological molecules and present them for enhanced bioactivity. This study reports the preparation of PLGA microparticles via Pickering emulsification using GO as the sole surfactant, which resulted in hybrid microparticles in the size range of 1.1 to 2.4 µm based on the ratio of GO to PLGA in the reaction. Furthermore, this study demonstrated that the hybrid GO-PLGA microparticles were not cytotoxic to either primary human fetal cartilage rudiment cells or the human osteoblast-like cell line, Saos-2. Additionally, the GO-PLGA microparticles promoted the osteogenic differentiation of the human fetal cartilage rudiment cells in the absence of exogenous growth factors to a greater extent than PLGA alone. These findings demonstrate that GO-PLGA microparticles are cytocompatible, osteoinductive and have potential as substrates for bone tissue engineering.

Design and characterization of biodegradable multi layered electrospun nanofibers for corneal tissue engineering applications.

Tissue engineering is one of the most promising areas for treatment of various ophthalmic diseases particularly for patients who suffer from limbal stem cell deficiency and this is due to the lack of existence of appropriate matrix for stem cell regeneration. The aim of this research project is to design and fabricate triple layered electrospun nanofibers as a suitable corneal tissue engineering scaffold and the objective is to investigate and perform various in vitro tests to find the most optimum and suitable scaffold for this purpose. Electrospun scaffolds were prepared in three layers. Poly(d, l-lactide-co-glycolide; PLGA, 50:50) nanofibers were electrospun as outer and inner layers of the scaffold and aligned type I collagen nanofibers were electrospun in the middle layer. Furthermore, the scaffolds were cross-linked by 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride and glutaraldehyde. Structural, physical, and mechanical properties of scaffolds were investigated by using N2 adsorption/desorption isotherms, Fourier transform infrared spectroscopy, contact angle measurement, tensile test, degradation, shrinkage analysis, and scanning electron microscopy (SEM). In addition, capability to support cell attachment and viability were characterized by SEM, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and 4',6-diamidino-2-phenylindole staining. According to the result of Brunauer-Emmett-Teller analysis, specific surface area of electrospun scaffold was about 23.7 m2 g-1 . Tensile tests on cross-linked scaffolds represented more suitable hydrophilicity and tensile behavior. In addition, degradation rate analysis indicated that noncross-linked scaffolds degraded faster than cross-linked one and cross-linking led to controlled shrinkage in the scaffold. The SEM analysis depicted nano-sized fibers in good shape. Also, the in vitro study represented an improved cell attachment and proliferation in the presence of human endometrial stem cells for both cross-linked and noncross-linked samples. The current study suggests the possibility of producing an appropriate substrate for successful cornea tissue engineering with a novel design.