Hyung Woo Ju

3D electrospun silk fibroin nanofibers for fabrication of artificial skin

Tissue-engineered skin substitutes such as nanofibers from traditional electrospinning may offer an effective therapeutic option for the treatment of patients suffering from skin damages such as burns and diabetic ulcers. However, it is generally difficult for cells to infiltrate the nanofibers due to their small pore size and sheets-like appearance. In the present study, a facile and efficient strategy has successfully been introduced that can produce 3D silk fibroin nanofibers, obviating an intrinsic limitation of traditional and salt-leaching electrospinning by introducing cold-plate electrospinning. The cell attachment and infiltration studies indicated the use of 3D nanofiber scaffolds by cold-plate electrospinning as a potential candidate to overcome intrinsic barriers of electrospinning techniques. The 3D nanofiber scaffolds using this technique presented a high porosity with controlled thickness and an easy contouring of facial shape; these properties can contribute to the ideal candidate for artificial skin reconstruction. From the clinical editor: Electrospun nanofibers are considered as promising scaffolds for tissue engineering due to extracellular matrix mimicking factor resulting in a controllable 3D nanofibrous form. The cold-plate electrospinning technique can facilitate the fabrication of these biomaterials to create structures that could resemble the dermis.

Fabrication of 3D porous silk scaffolds by particulate (salt/sucrose) leaching for bone tissue reconstruction

Silk fibroin is a biomaterial being actively studied in the field of bone tissue engineering. In this study, we aimed to select the best strategy for bone reconstruction on scaffolds by changing various conditions. We compared the characteristics of each scaffold via structural analysis using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), the swelling ratio, water uptake, porosity, compressive strength, cell infiltration and cell viability (CCK-8). The scaffolds had high porosity with good inter pore connectivity and showed high compressive strength and modulus. In addition, to confirm bone reconstruction, animal studies were conducted in which samples were implanted in rat calvaria and investigated by micro-CT scans. In conclusion, the presented study indicates that using sucrose produces scaffolds showing better pore interconnectivity and cell infiltration than scaffolds made by using a salt process. In addition, in vivo experiments showed that hydroxyapatite accelerates bone reconstruction on implanted scaffolds. Accordingly, our scaffold will be expected to have a useful application in bone reconstruction.

Wound healing effect of electrospun silk fibroin nanomatrix in burn-model.

Silk fibroin has recently become an important biomaterial for tissue engineering application. In this study, silk fibroin nanomatrix was fabricated by electrospinning and evaluated as wound dressing material in a burn rat model. The wound size reduction, histological examination, and the quantification of transforming growth factor TGF-β1 and interleukin IL-1α, 6, and 10 were measured to evaluate the healing effects. The silk fibroin nanomatrix treatment exhibited effective performance in decreasing the wound size and epithelialization. Histological finding also revealed that the deposition of collagen in the dermis was organized by covering the wound area in the silk fibroin nanomatrix treated group. The expression level of pro-inflammatory cytokine (IL-1α) was significantly reduced in the injured skin following the silk fibroin nanomatrix treatment compared to the medical gauze (control) at 7 days after burn. Also, the expression level of TGF-β1 in the wound treated with silk fibroin nanomatrix peaked 21-days post-treatment whereas expression level of TGF-β1 was highest at day 7 in the gauze treated group. In conclusion, this data demonstrates that silk fibroin nanomatrix enhances the burn wound healing, suggesting it is a good candidate for burn wound treatment.

Three-dimensional electrospun silk-fibroin nanofiber for skin tissue engineering.

Tissue-engineered skin substitutes may offer an effective therapeutic option for the treatment of patients with skin damages. In this study, a novel three-dimensional (3D) scaffold composed of electrospun silk fibroin (SF) nanofiber was fabricated using electrospinning with the addition of NaCl crystals. It has well known that the electrospun SF nanofibers were excellent scaffold for tissue. However, it is generally difficult for cells to infiltrate the electrospun silk fibroin due to its small pore size. To resolve this problem, we dropped the NaCl crystals above the rotating collector, which become incorporated into the nanofibers. Three methods (freeze-drying, salt-leaching, and electrospinning with NaCl) for fabrication of SF scaffolds were compared to the difference of their characteristics using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), mechanical strength, porosity, swelling abilities, and cell proliferation. Additionally, using air-liquid culture system, keratinocytes were co-cultured with fibroblasts in each type of SF scaffolds to construct an artificial bilayer skin in vitro. In our experimental results, histologic findings in only electrospun SF scaffolds showed more proliferation of fibroblasts in deep layer and more differentiation of keratinocytes in superficial layer. The present study suggests that 3D electrospun SF scaffolds might be a suitable for skin tissue engineering.

Hybrid scaffolds based on PLGA and silk for bone tissue engineering.

Porous silk scaffolds, which are considered to be natural polymers, cannot be used alone because they have a long degradation rate, which makes it difficult for them to be replaced by the surrounding tissue. Scaffolds composed of synthetic polymers, such as PLGA, have a short degradation rate, lack hydrophilicity and their release of toxic by‐products makes them difficult to use. The present investigations aimed to study hybrid scaffolds fabricated from PLGA, silk and hydroxyapatite nanoparticles (Hap NPs) for optimized bone tissue engineering. The results from variable‐pressure field emission scanning electron microscopy (VP–FE–SEM), equipped with EDS, confirmed that the fabricated scaffolds had a porous architecture, and the location of each component present in the scaffolds was examined. Contact angle measurements confirmed that the introduction of silk and HAp NPs helped to change the hydrophobic nature of PLGA to hydrophilic, which is the main constraint for PLGA used as a biomaterial. Thermo‐gravimetric analysis (TGA) and FT–IR spectroscopy confirmed thermal decomposition and different vibrations caused in functional groups of compounds used to fabricate the scaffolds, which reflected improvement in their mechanical properties. After culturing osteoblasts for 1, 7 and 14 days in the presence of scaffolds, their viability was checked by MTT assay. The fluorescent microscopy results revealed that the introduction of silk and HAp NPs had a favourable impact on the infiltration of osteoblasts. In vivo experiments were conducted by implanting scaffolds in rat calvariae for 4 weeks. Histological examinations and micro‐CT scans from these experiments revealed beneficial attributes offered by silk fibroin and HAp NPs to PLGA‐based scaffolds for bone induction. Copyright

3D silk fibroin scaffold incorporating titanium dioxide (TiO2) nanoparticle (NPs) for tissue engineering

The present study deals with fabrication of scaffolds composing of silk fibroin and TiO2 NPs fabricated using a salt-leaching process. At first instance, the TiO2 NPs were prepared by using sol-gel synthesis, affording to have average diameter of 77±21μm. Furthermore, the aqueous solutions of silk fibroin were mixed with 0.2%, 2.0% and 4.0% of TiO2 NPs and salt-leaching process was introduced which resulted in creation of porous scaffolds modified with TiO2 NPs. The presence of TiO2 NPs in scaffolds was confirmed by VP-FE-SEM-EDS, TGA and XRD. The presence of TiO2 NPs influenced in decrease in pore size and swelling behavior of composite scaffolds. The resultant mechanical property of scaffolds was improved upon the introduction of TiO2 NPs. Moreover, cell cytotoxicity results for 1, 3 and 7 days; revealed no toxic behavior to osteoblasts. However, a mild toxicity to NIH 3T3 fibroblasts was observed with the scaffolds containing 4.0% TiO2 NPs. The cell fixation results from 1 and 7 days of incubation indicated the attachment, spreading and subsequent proliferation of fibroblasts. However, these findings were independent to the amount of TiO2 NPs in scaffolds.

An experimental burn wound-healing study of non-thermal atmospheric pressure microplasma jet arrays.

In contrast with a thermal plasma surgical instrument based on coagulative and ablative properties, low‐temperature (non‐thermal) non‐equilibrium plasmas are known for novel medicinal effects on exposed tissue while minimizing undesirable tissue damage. In this study we demonstrated that arrays of non‐thermal microplasma jet devices fabricated from a transparent polymer can efficiently inactivate fungi (Candida albicans) as well as bacteria (Escherichia coli), both in vitro and in vivo, and that this leads to a significant wound‐healing effect. Microplasma jet arrays offer several advantages over conventional single‐jet devices, including superior packing density, inherent scalability for larger treatment areas, unprecedented material flexibility in a plasma jet device, and the selective generation of medically relevant reactive species at higher plasma densities. The therapeutic effects of our multi‐jet device were verified on second‐degree burns in animal rat models. Reduction of the wound area and the histology of the wound after treatment have been investigated, and expression of interleukin (IL)‐1α, ‐6 and ‐10 was verified to evaluate the healing effects. The consistent effectiveness of non‐thermal plasma treatment has been observed especially in decreasing wound size and promoting re‐epithelialization through collagen arrangement and the regulation of expression of inflammatory genes. Copyright

Fabrication of duck’s feet collagen–silk hybrid biomaterial for tissue engineering

Collagen constituting the extracellular matrix has been widely used as biocompatible material for human use. In this study, we have selected ducks feet for extracting collagen. A simple method not utilizing harsh chemical had been employed to extract collagen from ducks feet. We fabricated ducks feet collagen/silk hybrid scaffold for the purpose of modifying the degradation rate of ducks feet collagen. This study suggests that extracted collagen from ducks feet is biocompatible and resembles collagen extracted from porcine which is commercially used. Ducks feet collagen is also economically feasible and it could therefore be a good candidate as a tissue engineering material. Further, addition of silk to fabricate a ducks feet collagen/silk hybrid scaffold could enhance the biostability of ducks feet collagen scaffold. Ducks feet collagen/silk scaffold increased the cell viability compared to silk alone. Animal studies also showed that ducks feet collagen/silk scaffold was more biocompatible than silk alone and more biostable than ducks feet or porcine collagen alone. Additionally, the results revealed that ducks feet collagen/silk hybrid scaffold had high porosity, cell infiltration and proliferation. We suggest that ducks feet collagen/silk hybrid scaffold could be used as a dermal substitution for full thickness skin defects.

Osteoinductive silk fibroin/titanium dioxide/hydroxyapatite hybrid scaffold for bone tissue engineering.

The present study demonstrated the fabrication that incorporation of titanium isopropoxide (TiO2) and hydroxyapatite (HA) nanoparticles into the silk fibroin (SF) scaffolds. In this process, we prepared TiO2 nanoparticles using sol-gel synthesis and the porous structure was developed by salt-leaching process. Homogeneous distribution of TiO2 and HA nanoparticles were confirmed by images of VP-FE-SEM and those equipped with energy dispersive X-ray spectrometer. Structural characteristics of the porous SF/TiO2/HA hybrid scaffold were also determined using FTIR analysis and X-ray diffractometer. In this study, the porous SF/TiO2/HA hybrid scaffold showed similar porosity, enhanced mechanical property, but decreased water binding abilities, compared with the porous SF scaffold. For evaluation of the osteogenic differentiation of rat bone marrow mesenchymal stem cells, alkaline phosphatase activity and osteogenic gene expression were employed. Our results revealed that the porous SF/TiO2/HA hybrid scaffold had improved osteoinductivity compared with the porous SF scaffold. These results suggest that the osteogenic property as well as mechanical property of the porous SF/TiO2/HA hybrid scaffold could be better than the porous SF scaffold. Therefore, the porous SF/TiO2/HA hybrid scaffold may be a good promising biomaterial for bone tissue engineering application.

Three-layered scaffolds for artificial esophagus using poly(ɛ-caprolactone) nanofibers and silk fibroin: An experimental study in a rat model.

The purpose of this study was to determine the feasibility of an artificial esophagus using a three-layered poly(ε-caprolactone) (PCL)-silk fibroin (SF) scaffold in a rat model. The artificial esophagus was a three-layered, hybrid-type prosthesis composed of an outer and inner layer of PCL with a middle layer of SF. After depositing the inner layer of the PCL scaffold by electrospinning, the lyophilized middle SF layer was created. The outer layer of PCL was produced following the same procedure used to make the inner PCL layer. Eleven rats were anesthetized using inhaled anesthesia. Circumferential defects of the cervical esophagus (n=11) were created and reconstructed. Groups of rats were sacrificed after the 1st and 2nd weeks. Three rats died of an esophageal fistula and wound infection. No gross evidence of a fistula, perforation, abscess formation, seroma accumulation, or surrounding soft-tissue necrosis was observed in the other rats sacrificed after the 1st and 2nd weeks. The artificial esophagus constructs produced complete healing of the circumferential defects by the 2nd week. The composition of the three-layered artificial esophagus was confirmed histologically to have an outer and inner layer of PCL and a middle layer of SF. The fusion of the PCL-SF scaffold and the regenerative tissue remained intact. Our study proposes a more practical experimental model for studying a three-layered PCL-SF scaffold in the esophagus. However, further studies on circumferential defect reconstruction in a rat model are still required.