Biomimetic nanofibrous scaffolds: preparation and characterization of chitin/silk fibroin blend nanofibers

Electrospinning of chitin/silk fibroin (SF) blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was investigated to fabricate a biomimetic nanostructured scaffolds for tissue engineering. The morphology of the electrospun chitin/SF blend nanofibers was investigated with a field emission scanning electron microscope (FE-SEM). The average diameters of chitin/SF blend fibers decreased from 920 to 340 nm, with the increase of chitin content in blend compositions. The miscibility of chitin/SF blend fibers was examined by solution viscosity measurement. The chitin and SF were immiscible in the as-spun nanofibrous structure. The dimensional stability of chitin/SF blend nanofibers, with or without water vapor after-treatment, was conducted by immersing in water. As-spun SF-rich blend nanofibrous matrices were lost their fibrous structure after the water immersion for 24 h, and then changed into membrane-like structure. On the contrary, nanofibrous structures of water vapor-treated SF-rich blends were almost maintained. To assay the cytocompatibility and cell behavior on the chitin/SF blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal keratinocyte and fibroblasts seeded on the scaffolds were studied. Our results indicate that chitin/SF blend nanofibrous matrix, particularly the one that contained 75% chitin and 25% SF, could be a potential candidate for tissue engineering scaffolds because it has both biomimetic three-dimensional structure and an excellent cell attachment and spreading for NHEK and NHEF.     

Collagen-based biomimetic nanofibrous scaffolds: preparation and characterization of collagen/silk fibroin bicomponent nanofibrous structures

Electrospinning of collagen (COL)/silk fibroin (SF) blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol was investigated for fabrication of a biocompatible and biomimetic nanostructured scaffold for tissue engineering. The morphology of the electrospun COL/SF blend nanofibers was observed by scanning electron microscopy. The average diameters of COL/SF blend fibers ranged from 320 to 360 nm, irrespective of SF content in the blends. Both COL and SF components in the as-spun COL/SF blend matrices were stabilized by glutaraldehyde and water vapor, respectively, under the saturated glutaraldehyde aqueous solution at 25 degrees C. The glutaraldehyde vapor chemically stabilized the COL component via cross-linking, whereas the water vapor physically stabilized the SF component via crystallization to the beta-sheet structure. These structural changes of after-treated COL/SF blend matrices were examined using ATR-IR and CP/MAS (13)C NMR spectroscopy. To assay the cytocompatibility and cellular behavior of the COL/SF blend nanofibrous scaffolds, cell attachment and the spreading of normal human epidermal keratinocytes (NHEK) and fibroblasts (NHEF) seeded on the scaffolds were studied. In addition, both morphological changes and cellular responses of COL/SF blend nanofibrous matrices were also compared with COL/SF hybrid nanofibrous matrices. Generally similar levels of cell attachment and spreading of NHEF were shown in the COL/SF blend nanofibrous matrix compared with those of the pure COL and pure SF matrices; the cellular responses of NHEK were, however, markedly decreased in the COL/SF blend nanofibrous matrix as compared to the pure matrices. In contrast, cell attachment and spreading of NHEK on the COL/SF hybrid nanofibrous matrix were significantly higher than that of the COL/SF blend nanofibrous matrix. Our results indicate that a COL/SF hybrid nanofibrous matrix may be a better candidate than a COL/SF blend nanofibrous matrix for biomedical applications such as wound dressing and scaffolds for tissue engineering.     

Effect of chitin/silk fibroin nanofibrous bicomponent structures on interaction with human epidermal keratinocytes

To fabricate a biomimetic nanostructured bicomponent scaffolds, two types of chitin/silk fibroin (SF) nanofibrous scaffolds (blend scaffolds and hybrid scaffolds) were prepared by electrospinning or simultaneous electrospinning of chitin/SF solutions. The chitin/SF bicomponent scaffolds were after-treated with water vapor, and their nanofibrous structures were almost maintained. From the cytocompatibility and cell behavior on the chitin/SF blend or hybrid nanofibrous scaffolds, the hybrid matrix with 25% chitin and 75% SF as well as the chitin/SF blend nanofibers could be a potential candidate for tissue engineering scaffolds.     

Electrospinning of carboxymethyl chitin/poly(vinyl alcohol) nanofibrous scaffolds for tissue engineering applications

A novel fibrous membrane of carboxymethyl chitin (CMC)/poly(vinyl alcohol) (PVA) blend was successfully prepared by electrospinning technique. The concentration of CMC (7%) with PVA (8%) was optimized, blended in different ratios (0–100%) and electrospun to get nanofibers. Fibers were made water insoluble by chemical followed by thermal cross-linking. In vitro mineralization studies identified the ability of formation of hydroxyapatite deposits on the nanofibrous surfaces. Cytotoxicity of the nanofibrous scaffold was evaluated using human mesenchymal stem cells (hMSCs) by the MTT assays. The cell viability was not altered when these nanofibrous scaffolds were pre-washed with phosphate buffer containing saline (PBS) before seeding the cells. The SEM images also revealed that cells were able to attach and spread in the nanofibrous scaffolds. Thus our results indicate that the nanofibrous CMC/PVA scaffold supports cell adhesion/attachment and proliferation and hence this scaffold will be a promising candidate for tissue engineering applications.     

Fabrication,Characterization and Cellular Compatibility of Poly(Hydroxy Alkanoate) Composite Nanofibrous Scaffolds for Nerve Tissue Engineering

Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm⁻¹ can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.     

Electrospun poly (ɛ-caprolactone)/silk fibroin core-sheath nanofibers and their potential applications in tissue engineering and drug release

One of the key tenets of tissue engineering is to develop scaffold materials with favorable biodegradability, surface properties, outstanding mechanical strength and controlled drug release property. In this study, we generated core-sheath nanofibers composed of poly (?-caprolactone) (PCL) and silk fibroin (SF) blends via emulsion electrospinning. Nanofibrous scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), contact angle and tensile measurements. An in vitro FITC release study was conducted to evaluate sustained release potential of the core-sheath structured nanofibers. We found that the conformation of SF contained in PCL/SF composite nanofibers was transformed from random coil to β-sheet when treated with methanol, leading to improved crystallinity and tensile strength of nanofibrous scaffolds. The hydrophobicity and diameter of nanofibers decreased when we increased the content of SF in PCL/SF composite nanofibers. Furthermore, we evaluated the potential of fabricated PCL/SF composite nanofibers as scaffold in vitro. The results confirmed that fabricated PCL/SF scaffolds improved cell attachment and proliferation. Our results demonstrated the feasibility to generate core-sheath nanofibers composed of PCL and SF using a single-nozzle technique. The produced nanofibrous scaffolds with sustained drug release have potential application in tissue engineering.     

Improved infiltration of stem cells on electrospun nanofibers

Nanofibrous scaffolds have been recently used in the field of tissue engineering because of their nano-size structure which promotes cell attachment, function, proliferation and infiltration. In this study, nanofibrous polyethersulfone (PES) scaffolds was prepared via electrospinning. The scaffolds were surface modified by plasma treatment and collagen grafting. The surface changes then investigated by contact angle measurements and FTIR-ATR. The results proved grafting of the collagen on nanofibers surface and increased hydrophilicity after plasma treatment and collagen grafting. The cell interaction study was done using stem cells because of their ability to differentiate to different kinds of cell lines. The cells had normal morphology on nanofibers and showed very high infiltration through collagen grafted PES nanofibers. This infiltration capability is very useful and needed to make 3D scaffolds in tissue engineering.     

Silk–PVA Hybrid Nanofibrous Scaffolds for Enhanced Primary Human Meniscal Cell Proliferation

In this study, silk fibroin nanofibrous scaffolds were developed to investigate the attachment and proliferation of primary human meniscal cells. Silk fibroin (SF)–polyvinyl alcohol (PVA) blended electrospun nanofibrous scaffolds with different blend ratios (2:1, 3:1, and 4:1) were prepared. Morphology of the scaffolds was characterized using atomic force microscopy (AFM). The hybrid nanofibrous mats were crosslinked using 25 % (v/v) glutaraldehyde vapor. In degradation study, the crosslinked nanofiber showed slow degradation of 20 % on weight after 35 days of incubation in simulated body fluid (SBF). The scaffolds were characterized with suitable techniques for its functional groups, porosity, and swelling ratio. Among the nanofibers, 3:1 SF:PVA blend showed uniform morphology and fiber diameter. The blended scaffolds had fluid uptake and swelling ratio of 80 % and 458 ± 21 %, respectively. Primary meniscal cells isolated from surgical debris after meniscectomy were subcultured and seeded onto these hybrid nanofibrous scaffolds. Meniscal cell attachment studies confirmed that 3:1 SF:PVA nanofibrous scaffolds supported better cell attachment and growth. The DNA and collagen content increased significantly with 3:1 SF:PVA. These results clearly indicate that a blend of SF:PVA at 3:1 ratio is suitable for meniscus cell proliferation when compared to pure SF-PVA nanofibers.     

Electrospun sodium alginate/poly(ethylene oxide) core-shell nanofibers scaffolds potential for tissue engineering applications

Core-shell structure nanofibers of sodium alginate/poly(ethylene oxide) were prepared via electrospinning their dispersions in water solution. The core-shell structure morphology of the obtained nanofibers was viewed under scanning electron microscope (SEM) and transmission electron microscope (TEM), and X-ray photoelectron spectroscopy (XPS) analysis was used to further quantify the chemical composition of the core-shell composite SA/PEO nanofibers surface in detail. Furthermore, one-step cross-linking method through being immersed in CaCl₂ solution was investigated to improve the anti-water property of the electrospun nanofibers mats in order to facilitate their practical applications as tissue engineering scaffolds, and the changes of the structural of nanofibers before and after cross-linking was characterized by Fourier transform infrared (FT-IR). Indirect cytotoxicity assessment indicated that SA/PEO nanofibers membrane was nontoxic to the fibroblasts cells, and cell culture suggested that SA/PEO nanofibers tended to promote fibroblasts cells attachment and proliferation. It was assumed that the nanofibers membrane of electrospun SA/PEO could be used for tissue engineering scaffolds.     

Coaxial electrospinning of (fluorescein isothiocyanate-conjugated bovine serum albumin)-encapsulated poly(epsilon-caprolactone) nanofibers for sustained release

As an aim toward developing biologically mimetic and functional nanofiber-based tissue engineering scaffolds, we demonstrated the encapsulation of a model protein, fluorescein isothiocyanate-conjugated bovine serum albumin (fitcBSA), along with a water-soluble polymer, poly(ethylene glycol) (PEG), within the biodegradable poly(epsilon-caprolactone) (PCL) nanofibers using a coaxial electrospinning technique. By variation of the inner flow rates from 0.2 to 0.6 mL/h with a constant outer flow rate of 1.8 mL/h, fitcBSA loadings of 0.85-2.17 mg/g of nanofibrous membranes were prepared. Variation of flow rates also resulted in increases of fiber sizes from ca. 270 nm to 380 nm. The encapsulation of fitcBSA/PEG within PCL was subsequently characterized by laser confocal scanning microscopy, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) analysis. In vitro release studies were conducted to evaluate sustained release potential of the core-sheath-structured composite nanofiber PCL-r-fitcBSA/PEG. As a negative control, composite nanofiber PCL/fitcBSA/PEG blend was prepared from a normal electrospinning method. It was found that core-sheath nanofibers PCL-r-fitcBSA/PEG pronouncedly alleviated the initial burst release for higher protein loading and gave better sustainability compared to that of PCL/fitcBSA/PEG nanofibers. The present study would provide a basis for further design and optimization of processing conditions to control the nanostructure of core-sheath composite nanofibers and ultimately achieve desired release kinetics of bioactive proteins (e.g., growth factors) for practical tissue engineering applications.     

In vitro study of smooth muscle cells on polycaprolactone and collagen nanofibrous matrices

Biodegradable polycaprolactone and collagen nanofibers were produced by electrospinning, with fiber diameters of around 300-700nm and features similar to the extracellular matrix of natural tissue. Human coronary artery smooth muscle cells (SMCs) seeded on nanofibrous matrices tend to maintain normal phenotypic shape and growth tends to be guided by the nanofiber orientation. The SMC and nanofibrous matrix interaction was observed by SEM, MTS assay, trypan blue exclusion method and laser scanning confocal microscopy. The results showed that the proliferation and growth rate of SMCs were not different on polycaprolactone (PCL) nanofibrous matrices coated with collagen or tissue culture plates. PCL nanofibrous matrices coated with collagen showed that the SMCs migrated towards inside the nanofibrous matrices and formed smooth muscle tissue. This approach may be useful for engineering a variety of tissues in various structures and shapes, and also to demonstrate the importance of matching both the initial mechanical properties and degradation rate of nanofibrous matrices to the specific tissue engineering.     

Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration

The physical and spatial architectural geometries of electrospun scaffolds are important to their application in tissue engineering strategies. In this work, poly(epsilon-caprolactone) microfiber scaffolds with average fiber diameters ranging from 2 to 10 microm were individually electrospun to determine the parameters required for reproducibly fabricating scaffolds. As fiber diameter increased, the average pore size of the scaffolds, as measured by mercury porosimetry, increased (values ranging from 20 to 45 microm), while a constant porosity was observed. To capitalize on both the larger pore sizes of the microfiber layers and the nanoscale dimensions of the nanofiber layers, layered scaffolds were fabricated by sequential electrospinning. These scaffolds consisted of alternating layers of poly(epsilon-caprolactone) microfibers and poly(epsilon-caprolactone) nanofibers. By electrospinning the nanofiber layers for different lengths of time, the thickness of the nanofiber layers could be modulated. Bilayered constructs consisting of microfiber scaffolds with varying thicknesses of nanofibers on top were generated and evaluated for their potential to affect rat marrow stromal cell attachment, spreading, and infiltration. Cell attachment after 24 h did not increase with increasing number of nanofibers, but the presence of nanofibers enhanced cell spreading as evidenced by stronger F-actin staining. Additionally, increasing the thickness of the nanofiber layer reduced the infiltration of cells into the scaffolds under both static and flow perfusion culture for the specific conditions tested. The scaffold design presented in this study allows for cellular infiltration into the scaffolds while at the same time providing nanofibers as a physical mimicry of extracellular matrix.     

Electrospun chitosan-graft-poly (ε -caprolactone)/poly (ε-caprolactone) cationic nanofibrous mats as potential scaffolds for skin tissue engineering

This research is aimed to develop cationic nanofibrous mats with improved cellular adhesion profiles and stability of three-dimensional fibrous structure as potential scaffolds for skin tissue engineering. Firstly, amino-remained chitosan-graft-poly (?-caprolactone) (CS-g-PCL) was synthesized with a facile one-step manner by grafting ?-caprolactone oligomers onto the hydroxyl groups of CS via ring-opening polymerization by using methanesulfonic acid as solvent and catalyst. And then, CS-g-PCL/PCL nanofibrous mats were obtained by electrospinning of CS-g-PCL/PCL mixed solution. Scanning electron microscopy (SEM) images showed that the morphologies and diameters of the nanofibers were mainly affected by the weight ratio of CS-g-PCL to PCL. The enrichment of amino groups on the nanofiber surface was confirmed by X-ray photoelectron spectroscopy (XPS). With the increase of CS-g-PCL in CS-g-PCL/PCL nanofiber, the content of amino groups on the nanofiber surface increased, which resulted in the increase of zeta-potential of nanofibers. Studies on cell-scaffold interaction were carried out by culturing mouse fibroblast cells (L929) on CS-g-PCL/PCL nanofibrous mats with various contents of CS-g-PCL by assessing the growth, proliferation and morphologies of cells. The results of MTS assay and SEM observation showed that CS-g-PCL/PCL (2/8) mats with a moderate surface zeta-potential (ζ=3mV) were the best in promoting the cell attachment and proliferation. Toluidine blue staining further confirmed that L929 cells grew well and exhibited a normal morphology on the CS-g-PCL/PCL (2/8) mats. These results suggested the potential utilization of CS-g-PCL/PCL (2/8) nanofibrous mats for skin tissue engineering.     

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications.     

Zinc oxide-doped poly(urethane) spider web nanofibrous scaffold via one-step electrospinning: a novel matrix for tissue engineering

Zinc oxide (ZnO) nanostructures have been commonly studied for electronic purposes due to their unique piezoelectric and catalytic properties; however, recently, they have been also exploited for biomedical applications. The purpose of this study was to fabricate ZnO-doped poly(urethane) (PU) nanocomposite via one-step electrospinning technique. The utilized nanocomposite was prepared by using colloidal gel composed of ZnO and PU, and the obtained mats were vacuum dried at 60 °C overnight. The physicochemical characterization of as-spun composite nanofibers was carried out by X-ray diffraction pattern, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, electron probe microanalysis, and transmission electron microscopy, whereas the thermal behavior was analyzed by thermogravimetric analysis. The viability, attachment, and proliferation of NIH 3T3 mouse fibroblast cells on the ZnO/PU composite nanofibers were analyzed by in vitro cell compatibility test. The morphological features of the cells attached on nanofibers were examined by Bio-SEM. We conclude that the electrospun nanofibrous scaffolds with unique spider nets had good biocompatibility. Cytotoxicity experiments indicated that the mouse fibroblasts could attach to the nanocomposite after being cultured. Thus, the current work demonstrates that the as-synthesized ZnO/PU hybrid nanofibers represent a promising biomaterial to be exploited for various tissue engineering applications.     

Electrospun poly(lactic acid)/chitosan core-shell structure nanofibers from homogeneous solution

The core-shell structure nanofibers of poly(lactic acid)/chitosan with different weight ratios were successfully electrospun from homogeneous solution. The preparation process was more simple and effective than double-needle electrospinning. The nanofibers were obtained with chitosan in shell while poly(lactic acid) in core attributing to phase separation, which were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and energy dispersive spectrometer (EDS). The electrospun nanofibrous membrane was evaluated in vitro by using mouse fibroblasts (L929) as reference cell lines. Cell culture results indicated that these materials were good in promoting cell growth and attachment, thus they could be used for tissue engineering and wound healing dressing.     

Improved osteogenic differentiation of human induced pluripotent stem cells cultured on polyvinylidene fluoride/collagen/platelet-rich plasma composite nanofibers

Blood transfusion or blood products, such as plasma, have a long history in improving health, but today, platelet-rich plasma (PRP) is used in various medical areas such as surgery, orthopedics, and rheumatology in many ways. Considering the high efficiency of tissue engineering in repairing bone defects, in this study, we investigated the combined effect of nanofibrous scaffolds in combination with PRP on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). Electrospinning was used for fabricating nanofibrous scaffolds by polyvinylidene fluoride/collagen (PVDF/col) with and without PRP. After scaffold characterization, the osteoinductivity of the fabricated scaffolds was studied by culturing human iPSCs under osteogenic medium. The results showed that PRP has a considerable positive effect on the biocompatibility of the PVDF/col nanofibrous scaffold when examined by protein adsorption, cell attachment, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In addition, the results obtained from alkaline phosphatase activity and calcium content assays demonstrated that nanofibers have higher osteoinductivity while grown on PRP-incorporated PVDF/col nanofibers. These results were also confirmed while the osteogenic differentiation of the iPSCs was more investigated by evaluating the most important bone-related genes expression level. According to the results, it can be concluded that PVDF/col/PRP has much more osteoinductivity while compared with the PVDF/col, and it can be introduced as a promising bone bio-implant for use in bone tissue engineering applications.     

Carboxymethyl chitin/organic rectorite composites based nanofibrous mats and their cell compatibility

In this study, carboxymethyl chitin (CMC) - organic rectorite (OREC)/poly (vinyl alcohol) (PVA) composite nanofibrous mats were successfully prepared via electrospinning. SAXRD pattern showed that the interlayer distance of OREC was increased from 3.68 to 4.08nm, which verified that polymer chains were intercalated into the interlayer of OREC. Field emission scanning electron microscopy, Fourier transform infrared spectra and energy-dispersive X-ray spectroscopy were used to characterize the morphology and microcosmic structure of nanofibrous mats. Thermal properties of mats were determined by differential scanning calorimetry. To evaluate the cell compatibility of mats, mouse lung fibroblast (L929) was chosen for cell attachment and spreading assay. The results shows that nanofibrous mats contained OREC have better thermal properties. Besides, the addition of OREC has little effect on the cell compatibility of nanofibrous mats.     

Formation of collagen-glycosaminoglycan blended nanofibrous scaffolds and their biological properties

The development of blended collagen and glycosaminoglycan (GAG) scaffolds can potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native extracellular matrix (ECM). In this study, we were able to obtain novel nanofibrous collagen-GAG scaffolds by electrospinning collagen blended with chondroitin sulfate (CS), a widely used GAG, in a mixed solvent of trifluoroethanol and water. The electrospun collagen-GAG scaffold with 4% CS (COLL-CS-04) exhibited a uniform fiber structure with nanoscale diameters. A second collagen-GAG scaffold with 10% CS consisted of smaller diameter fibers but exhibited a broader diameter distribution due to the different solution properties in comparison with COLL-CS-04. After cross-linking with glutaraldehyde vapor, the collagen-GAG scaffolds became more biostable and were resistant to collagenase degradation. This is evidently a more favorable environment allowing increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without cross-linking did not increase the biostability but still promoted cell growth. The potential of applying the nanoscale collagen-GAG scaffold in tissue engineering is significant since the nanodimension fibers made of natural ECM mimic closely the native ECM found in the human body. The high surface area characteristic of this scaffold may maximize cell-ECM interaction and promote tissue regeneration faster than other conventional scaffolds.