Freeze-drying Technique Research Articles

Preparation, characterization and bioactivities of nano anhydrous calcium phosphate added gelatin–chitosan scaffolds for bone tissue engineering

Gelatin, chitosan and nano calcium phosphate based composite scaffold with tailored architectures and properties has great potential for bone regeneration. Herein, we aimed to improve the physico chemical, mechanical and osteogenic properties of 3D porous scaffold by incorporation of dihydrogen calcium phosphate anhydrous (DCPA) nanoparticles into biopolymer matrix with variation in composition in the prepared scaffolds. Scaffolds were prepared from the slurry containing gelatin, chitosan and synthesized nano DCPA particle using lyophilization technique. DCPA nano particles were synthesized using calcium carbonate and phosphoric acid in water–ethanol medium. XRD pattern showed phase pure DCPA in synthesized nanopowder. Scaffolds were prepared by addition of DCPA nanoparticles to the extent of 5–10 wt% of total polymer into gelatin–chitosan solution with solid loading varying between 2.5 and 2.75 wt%. The prepared scaffold showed interconnected porosity with pore size varying between 110 and 200 micrometer. With addition of DCPA nanoparticles, average pore size of the prepared scaffolds decreased. With increase in nano ceramic phase content from 5 wt% to 10 wt% of total polymer, the compressive strength of the scaffold increased. Scaffold containing 10 wt% DCPA showed the highest average compressive strength of 2.2 MPa. Higher cellular activities were observed in DCPA containing scaffolds as compared to pure gelatin chitosan scaffold suggesting the fact that nano DCPA addition into the scaffold promoted better osteoblast adhesion and proliferation as evident from MTT assay and scanning electron microscopic (SEM) investigation of osteoblast cultured scaffolds. A higher degree of lamellopodia and filopodia extensions and better spreading behavior of osteoblasts were observed in FESEM micrographs of MG 63 cultured DCPA containing scaffold. The results demonstrated that both mechanical strength and osteogenic properties of gelatin–chitosan scaffold could be improved by addition of anhydrous dihydrogen calcium phosphate nanoparticles into it.

Effect of drying techniques on color and bioactive potential of two commercial edible Indonesian seaweed cultivars

Seaweeds (Kappaphycus alvarezii and Sargassum duplicatum) are potentially rich sources of bioactive compounds and functional constituents that are used in food applications. Sun, oven, vacuum, and freeze-drying techniques are commonly used seaweed drying techniques and could be classified as cheap (sun and oven) and expensive (vacuum and freeze) drying techniques based on ovens’ market price. Therefore, the study was designed to investigate the effect of cheap (sun and oven) and expensive (vacuum and freeze) drying techniques based on color and antioxidant potential of K. alvarezii and S. duplicatum using phenolic and antioxidant assays. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed to discriminate the drying techniques by instrumental color analysis. The results revealed that oven-dried seaweed extracts exhibited higher levels of phenolic (0.30 to 0.36 mg GAE g−1) and antioxidant potential (3.59 to 79.68%). PCA and HCA discriminated the drying techniques into two well-defined groups/clusters I (oven, vacuum, and freeze-drying) and II (sun drying) and revealed higher ΔE values of sun-dried seaweed samples, which was interpreted as color was preserved using drying techniques of cluster I. Overall, oven, vacuum, and freeze-drying techniques reported similarities in color characteristics and could be an alternate drying technique to preserve the color of seaweed cultivars. Therefore, oven drying technique is recommended due to low cost (compared with vacuum and freeze-drying) and an affordable alternative to sun drying, the preferred technique in fishing communities of low-and-middle-income countries, for the development of seaweed-enriched functional foods.

Esterified superhydrophobic nanofibrillated cellulose based aerogel for oil spill treatment

The present work aims towards the structuring of a physically cross-linked aerogels based on nanofibrillated cellulose (NFC) and polyvinyl alcohol (PVA), i.e., NFC/PVA aerogels for oil spillage cleanup. Highly porous (98%) NFC/PVA aerogels having both meso-micro pores were achieved by freeze drying technique. To impart super-hydrophobicity to the composite aerogel, a simple dip coating process was adopted using stearic acid chloride (SAC) solution. The SAC conjugated aerogels combined both superhydrophobic and oleophilic characteristics showed a contact angle of ∼159° and ∼0° with water and oil respectively. FESEM and X-ray microtomography images revealed a self-assembled 3D porous cellular structure of the aerogels. The prepared aerogels were found to be very efficient in separating a series of oil/water mixtures and various organic solvents with excellent selectivity and recyclability. Absorption capacity of the aerogels was at least 35 times higher than their dry weight. Simple mechanical squeezing method was adopted for repetitive uses.

Effect of Non-Conventional Drying Methods on In Vitro Starch Digestibility Assessment of Cooked Potato Genotypes.

Potatoes (Solanum tuberosum L.) are a good dietary source of carbohydrates in the form of digestible starch (DS) and resistant starch (RS). As increased RS content consumption can be associated with decreased chronic disease risk, breeding efforts have focused on identifying potato varieties with higher RS content, which requires high-throughput analysis of starch profiles. For this purpose, freeze drying of potatoes has been used but this approach leads to inaccurate RS values. The present study objective was to assess the starch content (RS, DS and total starch (TS)) of three cooked potato genotypes that were dried using freeze drying and innovative drying techniques (microwave vacuum drying, instant controlled pressure drop drying and conductive hydro-drying) relative to freshly cooked potato samples. Depending on the genotype, all drying methods showed one or more starch measures that were significantly different from freshly cooked values. The combination of ultrasound and infrared assisted conductive hydro-drying was the only method identified to be associated with accurate assessment of DS and TS content relative to fresh samples. The drying treatments were all generally associated with highly variable RS content relative to fresh controls. We conclude that freshly cooked samples must be used for selecting varieties with a high proportion of RS starch as drying of cooked potatoes leads to unreliable RS measurements.

Development and Optimization of the Novel Fabrication Method of Highly Macroporous Chitosan/Agarose/Nanohydroxyapatite Bone Scaffold for Potential Regenerative Medicine Applications.

Bone scaffolds mimicking the three-dimensional bone structure are of essential importance for bone regeneration. The aim of this study was to develop and optimize the production method of highly macroporous bone scaffold composed of polysaccharide matrix (chitosan–agarose) reinforced with nanohydroxyapatite. The highly macroporous structure was obtained by the simultaneous application of a gas-foaming agent and freeze-drying technique. Fabricated variants of biomaterials (produced using different gas-foaming agent and solvent concentrations) were subjected to porosity evaluation and compression test in order to select the scaffold with the best properties. Then, bioactivity, cytotoxicity, and cell growth on the surface of the selected biomaterial were assessed. The obtained results showed that the simultaneous application of gas-foaming and freeze-drying methods allows for the production of biomaterials characterized by high total and open porosity. It was proved that the best porosity is obtained when solvent (CH3COOH) and foaming agent (NaHCO3) are applied at ratio 1:1. Nevertheless, the high porosity of novel biomaterial decreases its mechanical strength as determined by compression test. Importantly, novel scaffold is non-toxic to osteoblasts and favors cell attachment and growth on its surface. All mentioned properties make the novel biomaterial a promising candidate to be used in regenerative medicine in non-load bearing implantation sites.

A novel curcumin-loaded composite dressing facilitates wound healing due to its natural antioxidant effect.

PurposeTo prepare a novel wound dressing to facilitate cutaneous wound healing.MethodsCurcumin (Cur) was added to the ring-shaped β-cyclodextrin (CD) to form a β-CD–Cur inclusion complex (CD-Cur). CD-Cur was then integrated into a composite chitosan–alginate (CA) mix. Finally, CA-CD-Cur was generated with a freeze-drying technique. Water-uptake capacity, degradation rate, and drug-release kinetics of the newly formed dressing were investigated in vitro. In animal studies, cutaneous wounds in rats were created, treated with CA-CD-Cur, then compared to CA-Cur, CA, and gauze.ResultsCA-CD-Cur–treated wounds showed accelerated closure rates, improved histopathological results, and lower SOD, lipid peroxidation, pI3K, and pAktkt levels than other groups. On the contrary, catalase, IκBα, and TGFβ1 levels were higher than others.ConclusionCA-CD-Cur may facilitate cutaneous wound dressing that facilitate wound healing.

Microscale Simulations of Drying and Calendering Processes to Manufacture Porous Electrodes

Electrodes can suffer from inconsistencies in performance due to heterogeneity in particle arrangements. Manufacturing processes such as drying and calendering significantly alter the microstructure and introduce randomness, making it difficult to predict the final structure of the film, given process inputs. This has a crucial impact on the electrochemical and mechanical properties as well as the ultimate performance and lifetime of the battery [1]. The goal of this work is to analyze the behavior of electrode constituent materials during the drying and calendering steps using a new predictive model of microstructure. We have developed a model to simulate the drying and calendering processes using the open-source Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) [2]. Within LAMMPS, we developed a customization of smoothed particle hydrodynamics (SPH), which solves the Navier-Stokes equations on particles representing both solid and liquid materials present in the electrode film. We present here the development of the model and the resulting microstructure predictions for conventional Li-ion battery electrodes. Results are compared to experimental data where available, including microstructures determined from a new freeze-drying technique. Predicted properties such as effective conductivity and degree of heterogeneity can be used in more coarse-grained models to simulate cell performance.

Co nanoparticles decorated on N-doped rGO carbon aerogel as a highly efficient electrocatalyst for oxygen reduction

With sodium alginate and graphene oxide (GO) as raw materials, cobalt alginate/GO aerogel was prepared by ion-exchange reaction and subsequent freeze-drying technique. The aerogel was transported into a Co/N-rGO composite material through high-temperature carbonization at ammonia atmosphere. The Co/N-rGO composite material was then used for catalyzing oxygen reduction reaction in fuel cell and showed excellent catalytic activity and stability.

Transversely isotropic freeze-dried PVA hydrogels: Theoretical modelling and experimental characterization

Polyvinyl alcohol (PVA) hydrogels have recently emerged as suitable bio-mimetic materials for various applications such as scaffold engineering, controlled drug delivery and cell culture. In this paper, PVA hydrogels with different concentrations of PVA have been synthesized using uni-directional freeze-drying technique. Unidirectional freeze-dried hydrogels exhibit transverse isotropy. The microstructurally aligned porous architecture of the hydrogels has been confirmed from Scanning Electron Microscope (SEM) images. Experiments have been conducted to characterize their transversely isotropic and viscoplastic behavior. These materials have been observed to behave non-linearly when subjected to compressive mechanical loads. Theoretical models are proposed to predict the mechanical behavior of the transversely isotropic microstructurally aligned hydrogels. The first model is a visco-elasto-plastic model that incorporates the effect of non-Newtonian behavior effectively using a fractal dashpot. The second model is an anisotropic finite viscoplastic model. The predictions of both the models are found to be in close agreement with the experimental results.

Encapsulation and delivery of bioactive compounds using spray and freeze-drying techniques: A review

There are various bioactive components exist in plants, fruits, and vegetable origins that have many beneficial health effects (mainly antioxidant). However, they suffer from low-stability against the environmental condition. Thus, the encapsulation approach emerged to decrease their sensitivity and present a target delivery system. Generally, native carrier agents (polysaccharides and proteins) are being applied to embed the core materials. Accordingly, many encapsulation methods have been developed to protect vulnerable components by these carriers. Spray and freeze-drying are common encapsulation methods with the ability of powder production. Both feed emulsion production and drying process factors substantially influence the core embedded within the carrier agents. Spray-drying is well-known to higher applicable and scalable encapsulation procedure in the food and pharmaceutical industries. It is predominantly related to its lower process costs. Nevertheless, its application is limited for more sensitive bioactive compounds due to hot-air drying exertion. In contrast, freeze-drying has been mostly used for thermo-sensitive ingredients. Its application is restricted by economic drawbacks for which long process time (24–48 h) is required. However, freeze-drying is adequately taken into an advantage in the encapsulation of therapeutic compounds since high-added value products will be produced so that bioactive compounds with higher biological activity are needed.

Fabrication of poly (lactic-co-glycolic acid)/decellularized articular cartilage extracellular matrix scaffold by three-dimensional printing technology and investigating its physicochemical properties

To manufacture a poly (lactic-co-glycolic acid) (PLGA) scaffold by low temperature deposition three-dimensional (3D) printing technology, prepare a PLGA/decellularized articular cartilage extracellular matrix (DACECM) cartilage tissue engineered scaffold by combining DACECM, and further investigate its physicochemical properties. PLGA scaffolds were prepared by low temperature deposition 3D printing technology, and DACECM suspensions was prepared by modified physical and chemical decellularization methods. DACECM oriented scaffolds were prepared by using freeze-drying and physicochemical cross-linking techniques. PLGA/DACECM oriented scaffolds were prepared by combining DACECM slurry with PLGA scaffolds. The macroscopic and microscopic structures of the three kinds of scaffolds were observed by general observation and scanning electron microscope. The chemical composition of DACECM oriented scaffold was analyzed by histological and immunohistochemical stainings. The compression modulus of the three kinds of scaffolds were measured by biomechanical test. Three kinds of scaffolds were embedded subcutaneously in Sprague Dawley rats, and HE staining was used to observe immune response. The chondrocytes of New Zealand white rabbits were isolated and cultured, and the three kinds of cell-scaffold complexes were prepared. The growth adhesion of the cells on the scaffolds was observed by scanning electron microscope. Three kinds of scaffold extracts were cultured with L-929 cells, the cells were cultured in DMEM culture medium as control group, and cell counting kit 8 (CCK-8) was used to detect cell proliferation. General observation and scanning electron microscope showed that the PLGA scaffold had a smooth surface and large pores; the surface of the DACECM oriented scaffold was rough, which was a 3D structure with loose pores and interconnected; and the PLGA/DACECM oriented scaffold had a rough surface, and the large hole and the small hole were connected to each other to construct a vertical 3D structure. Histological and immunohistochemical qualitative analysis demonstrated that DACECM was completely decellularized, retaining the glycosaminoglycans and collagen typeⅡ. Biomechanical examination showed that the compression modulus of DACECM oriented scaffold was significantly lower than those of the other two scaffolds ( P<0.05). There was no significant difference between PLGA scaffold and PLGA/DACECM oriented scaffold ( P>0.05). Subcutaneously embedded HE staining of the three scaffolds showed that the immunological rejections of DACECM and PLGA/DACECM oriented scaffolds were significantly weaker than that of the PLGA scaffold. Scanning electron microscope observation of the cell-scaffold complex showed that chondrocytes did not obviously adhere to PLGA scaffold, and a large number of chondrocytes adhered and grew on PLGA/DACECM oriented scaffold and DACECM oriented scaffold. CCK-8 assay showed that with the extension of culture time, the number of cells cultured in the three kinds of scaffold extracts and the control group increased. There was no significant difference in the absorbance ( A) value between the groups at each time point ( P>0.05). The PLGA/DACECM oriented scaffolds have no cytotoxicity, have excellent physicochemical properties, and may become a promising scaffold material of tissue engineered cartilage.

Revealing the pore characteristics and physicochemical properties of silica ionogels based on different sol-gel drying strategies

In this study, an exhaustive investigation about the effect of drying methods on the physical properties and microstructures of silica ionogels was conducted. Silica ionogels were synthesized by following one-step sol-gel process and dried either by supercritical, ambient pressure or freeze-drying techniques. After drying periods, characterization studies of the ionogels were carried out by performing FTIR, SEM, BET and TGA analyses. Results have shown that, although they all appeared in a monolithic structure, their microstructural pattern were completely different. Supercritically dried silica ionogel resulted in mesoporous structure with superior properties such as low density (ρ = 0.21 kg/m3), high porosity (ϕ = 90%) and high surface area (646 m2/g). On the other hand, silica ionogel dried in ambient conditions comprised both micro-meso pores and the pores are mainly closed form and freeze dried ionogel had non-homogeneous pore distribution with relatively larger pores.

Preparation, Characterization, and Properties of Inclusion Complexes of Balofloxacin with Cyclodextrins.

The study mainly aimed to improve the aqueous solubility of Balofloxacin (BLFX) by preparing the inclusion complexes (ICs) of BLFX with cyclodextrins (CDs). In this study, ICs in solid state were obtained by using beta-CD (β-CD), 2-hydroxypropyl-β-CD (HP-β-CD), 2, 6-dimethyl-β-CD (DM-β-CD) through a freeze-drying technique. The formation of ICs was confirmed through Fourier-transform infrared spectroscopy, differential scanning calorimetry, powder X-ray diffraction, nuclear magnetic resonance, and scanning electron microscopy. Results demonstrated that the water solubility and dissolution rates of three ICs were distinctly improved than that of parent BLFX. Bacteriostatic experiment manifested that the antibacterial effect of BLFX was not inhibited after encapsulation in CDs. The damage of BLFX to kidney and liver cells was reduced. Consequently, successful preparation of the ICs of BLFX with CDs provided possibility for devising new dosage form of BLFX, which held great promise for further applications in clinical fields.

Hydroxyapatite-Collagen Composite Made from Coral and Chicken Claws for Bone Implant Application

Synthesis of porous hydroxyapatite-collagen composites for bone implant applications has been carried out. Hydroxyapatite synthesized from coral by the precipitation method, while Collagen synthesized from chicken claws. Collagen formation was carried out by freeze-dry technique with variations in freezing time of 2, 4 and 6 hours at -80 ° C. The next process was by drying in a lyophilizer. Characterization of samples was carried out using Fourier Transform Infra Red (FTIR), Scanning Electron Microscopy (SEM), compressive strength test and cytotoxicity test with Microtetrazolium (MTT) assay. FTIR results proved that collagen uptake and hydroxyapatite combine chemically. This is indicated by the absorption of functional groups that did not coincide between collagen and hydroxyapatite functional groups with composites. SEM observations showed that the largest pore size was obtained at freezing for 2 hours which was 774 μm and the smallest in freezing for 6 hours was 640 μm. This pore size was an important parameter of the bone implant because it played a role in the osteoinductive process. The composite compressive strength test results for freezing 2 hours, 4 hours and 6 hours respectively was 737 KPa, 842 KPa and 707.7 KPa. The results of the cytotoxicity test with MTT showed the percentage of cell viability above 100%. This means that the Hydroxyapatite-collagen composite is non-toxic. So, the sample formed has qualified as a bone implant candidate.

Bioavailability and Bioactivity of Encapsulated Phenolics and Carotenoids Isolated from Red Pepper Waste.

In order to deactivate the health properties of bioactive compounds, they need to withstand the effects of food processing, their potential release from the food matrix, and remain bio-accessible in the gastrointestinal tract. Bio-actives from different plants are prone to oxidative degradation, and encapsulation is an effective method in improving their stability. In the present study, red pepper waste (RPW), a by-product of vegetable processing industry, was encapsulated in whey protein using spray and freeze-drying techniques. The aim was to evaluate the effects of in vitro gastrointestinal digestion on the release and bioactivity of encapsulated bio-actives, after each digestion step. The results showed that the release of phenolics and carotenoids, as well as antioxidants, anti-hyperglycemic, and anti-inflammatory activities are influenced by pH and intestinal fluid, with pH 7.5 exhibited at higher levels. There was a rapid initial release of carotenoids from whey protein matrices, while a more gradual increase of phenolics was observed, reaching around 50% for both encapsulates first at 6 h and 37 °C, and small intestine conditions. The encapsulation of RPW demonstrated a protective effect against pH changes and enzymatic activities along digestion, and contributed to the increase in bio-accessibility in the gut. Also, the results suggest that encapsulation is an efficient method for valorization of bio-actives from RPW, with improvements in nutrition, color, and bioactive properties.

Nanocomposite scaffold seeded with mesenchymal stem cells for bone repair.

The mechanical property of bone tissue scaffolds is one of the most important aspects in bone tissue engineering that has remained problematic. In our previous study, we fabricated a three-dimensional scaffold from nano-hydroxyapatite/gelatin (nHA/Gel) and investigated its efficiency in promoting bone regeneration both in vitro and in vivo. In the present study, the effect of adding silicon carbide (SiC) on the mechanical and biological behaviors of the nHA/Gel/SiC and bone regeneration in vivo were determined. nHA and SiC were synthesized and characterized by the X-ray diffraction pattern and transmission electron microscope image. Layer solvent casting, freeze drying, and lamination techniques were applied to prepare these scaffolds. Then, the biocompatibility and cell adhesion behavior of the synthesized nHA/Gel/SiC scaffolds were investigated. For in vivo studies, rats were categorized into three groups: blank defect, blank scaffold, and rat bone marrow mesenchymal stem cells (rBM-MSCs)/scaffold. After 1, 4, and 12 weeks post-injury, the rats were sacrificed and the calvaria were harvested. Sections with a thickness of 5 µm thickness were prepared and stained with hematoxylin-eosin and Masson's Trichrome, and immunohistochemistry was performed. Our results showed that SiC effectively increased the mechanical properties of the nHA/Gel/SiC scaffold. No significant differences were observed in biocompatibility, cell adhesion, and cytotoxicity of the nHA/Gel/SiC in comparison with the nHA/Gel nanocomposite. Based on histological and immunohistochemical studies, both osteogenesis and collagenization were significantly higher in the rBM-MSCs/scaffold group, quantitatively and qualitatively. The present study strongly suggests the potential of SiC as an alternative strategy to improve the mechanical and biological properties of bone tissue engineering scaffolds, and shows that the pre-seeded nHA/Gel/SiC scaffold with rBM-MSCs improves osteogenesis in the engineered bone implant.

Using Chitosan Besides Nano Hydroxyapatite and Fluorohydroxyapatite Boost Dental Pulp Stem Cell Proliferation

The dental tissue scaffold must provide a favorable surface for dental pulp stem cell attachment and proliferation. Employing nanohydroxyapatite (HA) and nanofluorohydroxyapatite (FHA) beside synthetic and organic polymer in favor of scaffolds would be used in bone and dental tissue engineering. In this research, nanoHA and FHA/chitosan scaffolds were synthesized by freeze-drying technique. Surface morphology, chemical composition and hydrophilicity have a great impact on initial cell attachment which will further affect the cell viability and proliferation which evaluated by SEM, XRD and contact angle measurement. Bioactivity of scaffolds was investigated by immersion in simulated body fluid (SBF) and cell proliferation assay. In freeze-drying technique percentage usage of hydroxyapatite could be risen up to 40% and shown better macro-mechanical and physical properties and bioactivity. According to obtained results by adding chitosan, contact angle was decreased by %54 and %37 for polycaprolactone (PCL)/HA and PCL/FHA scaffolds. In addition, addition of chitosan causes significant increase in the cell proliferation for PCL/HA and PCL/FHA up to 81% and 164%, respectively. These results indicate that PCL/FHA/chitosan scaffold represent a big potential for dental tissue engineering.

Corrigendum to “Synthesis and characterization of bioscaffolds using freeze drying technique for bone regeneration” [Biocatal. Agric. Biotechnol. 20 (2019) 101184

Corrigendum to “Synthesis and characterization of bioscaffolds using freeze drying technique for bone regeneration” [Biocatal. Agric. Biotechnol. 20 (2019) 101184

Raking Effects on Aggregate Densification and Channelling in Thickening

Thickening performance is greatly influenced by aggregate densification and channelling development; however, the microstructures of aggregates and channelling are unable to directly observe because of high turbidity mud bed during thickening. Computed tomography scan imaging technique has been introduced to investigate the effects of aggregate densification and channelling in the mud bed samples during unclassified tailings thickening. The samples were prepared through deep-freezing and freeze-drying techniques, immediately after sampling from the operation of a pilot thickener. Based on the information of void ratio, pore-size distribution, and permeability, obtained from 3D reconstruction images of the aggregates structure, aggregate densification and channelling development have been characterized. Channelling patterns have been classified by the size and shape of the connection throat of the pore between aggregates. As the aggregate structure broke and densified, the void ratio and pore size of the mud bed were decreased. Thickening performance enhancement was predicted based on solid flux and permeability of mud bed, and the result indicated raking on aggregate densification and channelling apparently improves the unclassified tailings dewatering extent.

Fabrication and characterization of novel bilayer scaffold from nanocellulose based aerogel for skin tissue engineering applications

The aim of this study was to fabricate a novel bilayer scaffold containing cellulose nanofiber/poly (vinyl) alcohol (CNF/PVA) to evaluate its potential use in skin tissue engineering. Here, the scaffolds were fabricated using a novel one-step freeze-drying technique with two different concentrations of the aforementioned polymers. FE-SEM analysis indicated that the fabricated scaffolds had interconnected pores with two defined pore size in each layer of the bilayer scaffolds that can recapitulate the two layers of the dermis and epidermis of the skin. Lower concentration of polymers causes higher porosity with larger pore size and increased water uptake and decreased mechanical strength. FTIR proved the presence of functional groups and strong hydrogen bonding between the molecules of CNF/PVA and the efficient crosslinking. The MTT assay showed that these nanofibrous scaffolds meet the requirement as a biocompatible material for skin repair. Here, for the first time, we fabricated bilayer scaffold using a novel one-step freeze-drying technique only by controlling the polymer concentration with spending less time and energy.