WonHyoung Ryu

Mechanically-reinforced electrospun composite silk fibroin nanofibers containing hydroxyapatite nanoparticles

Electrospun silk fibroin (SF) scaffolds provide large surface area, high porosity, and interconnection for cell adhesion and proliferation and they may replace collagen for many tissue engineering applications. Despite such advantages, electrospun SF scaffolds are still limited as bone tissue replacement due to their low mechanical strengths. While enhancement of mechanical strengths by incorporating inorganic ceramics into polymers has been demonstrated, electrospinning of a mixture of SF and inorganic ceramics such as hydroxyapatite is challenging and less studied due to the aggregation of ceramic particles within SF. In this study, we aimed to enhance the mechanical properties of electrospun SF scaffolds by uniformly dispersing hydroxyapatite (HAp) nanoparticles within SF nanofibers. HAp nanoaprticles were modified by γ-glycidoxypropyltrimethoxysilane (GPTMS) for uniform dispersion and enhanced interfacial bonding between HAp and SF fibers. Optimal conditions for electrospinning of SF and GPTMS-modified HAp nanoparticles were identified to achieve beadless nanofibers without any aggregation of HAp nanoparticles. The MTT and SEM analysis of the osteoblasts-cultured scaffolds confirmed the biocompatibility of the composite scaffolds. The mechanical properties of the composite scaffolds were analyzed by tensile tests for the scaffolds with varying contents of HAp within SF fibers. The mechanical testing showed the peak strengths at the HAp content of 20 wt.%. The increase of HAp content up to 20 wt.% increased the mechanical properties of the composite scaffolds, while further increase above 20 wt.% disrupted the polymer chain networks within SF nanofibers and weakened the mechanical strengths.

Microfabrication Technology of Biodegradable Polymers for Interconnecting Microstructures

A microtechnology for synthetic biodegradable polymers has been developed to fabricate three-dimensionally (3-D) shaped and microstructured multilayer constructs for biomedical applications. A unique micromolding method is proposed to create deep and interconnecting microstructures which facilitate cellular scale interconnection across the layers for cell-to-cell communication and the supply of nutrients and oxygen. A geometrical condition and molding setup for interconnecting microstructuring are described and demonstrated experimentally. Furthermore, various types of synthetic biodegradable polymers are explored and compared with regard to the behavior during the proposed process. Considering the thermal instability of synthetic biodegradable polymers, it is also confirmed that the proposed process does not affect the property of the polymers. Finally, microfabricated 3-D multilayer constructs is presented to demonstrate the feasibility of the technology as a unique scaffolding method

Spatially discrete thermal drawing of biodegradable microneedles for vascular drug delivery

Spatially discrete thermal drawing is introduced as a novel method for the fabrication of biodegradable microneedles with ultra-sharp tip ends. This method provides the enhanced control of microneedle shapes by spatially controlling the temperature of drawn polymer as well as drawing steps and speeds. Particular focus is given on the formation of sharp tip ends of microneedles at the end of thermal drawing. Previous works relied on the fracture of polymer neck by fast drawing that often causes uncontrolled shapes of microneedle tips. Instead, this approach utilizes the surface energy of heated polymer to form ultra-sharp tip ends. We have investigated the effect of such temperature control, drawing speed, and drawing steps in thermal drawing process on the final shape of microneedles using biodegradable polymers. XRD analysis was performed to analyze the effect of thermal cycle on the biodegradable polymer. Load-displacement measurement also showed the dependency of mechanical strengths of microneedles on the microneedle shapes. Ex vivo vascular tissue insertion and drug delivery demonstrated microneedle insertion to tunica media layer of canine aorta and drug distribution in the tissue layer.

Impact insertion of transfer-molded microneedle for localized and minimally invasive ocular drug delivery.

It has been challenging for microneedles to deliver drugs effectively to thin tissues with little background support such as the cornea. Herein, we designed a microneedle pen system, a single microneedle with a spring-loaded microneedle applicator to provide impact insertion. To firmly attach solid microneedles with 140 μm in height at the end of macro-scale applicators, a transfer molding process was employed. The fabricated microneedle pens were then applied to mouse corneas. The microneedle pens successfully delivered rhodamine dye deep enough to reach the stromal layer of the cornea with small entry only about 1000 μm(2). When compared with syringes or 30 G needle tips, microneedle pens could achieve more localized and minimally invasive delivery without any chances of perforation. To investigate the efficacy of microneedle pens as a way of drug delivery, sunitinib malate proven to inhibit in vitro angiogenesis, was delivered to suture-induced angiogenesis model. When compared with delivery by a 30 G needle tip dipped with sunitinib malate, only delivery by microneedle pens could effectively inhibit corneal neovascularization in vivo. Microneedle pens could effectively deliver drugs to thin tissues without impairing merits of using microneedles: localized and minimally invasive delivery.

Membrane-reinforced three-dimensional electrospun silk fibroin scaffolds for bone tissue engineering.

Electrospun silk fibroin (SF) scaffolds have drawn much attention because of their resemblance to natural tissue architecture such as extracellular matrix, and the biocompatibility of SF as a candidate material to replace collagen. However, electrospun scaffolds lack the physical integrity of bone tissue scaffolds, which require resistance to mechanical loadings. In this work, we propose membrane-reinforced electrospun SF scaffolds by a serial process of electrospinning and freeze-drying of SF solutions in two different solvents: formic acid and water, respectively. After wet electrospinning followed by replacement of methanol with water, SF nanofibers dispersed in water were mixed with aqueous SF solution. Freeze-drying of the mixed solution resulted in 3D membrane-connected SF nanofibrous scaffolds (SF scaffolds) with a thickness of a few centimeters. We demonstrated that the SF concentration of aqueous SF solution controlled the degree of membrane reinforcement between nanofibers. It was also shown that both increase in degree of membrane reinforcement and inclusion of hydroxyapatite (HAP) nanoparticles resulted in higher resistance to compressive loadings of the SF scaffolds. Culture of human osteoblasts on collagen, SF, and SF-HAP scaffolds showed that both SF and SF-HAP scaffolds had biocompatibility and cell proliferation superior to that of the collagen scaffolds. SF-HAP scaffolds with and without BMP-2 were used for in vivo studies for 4 and 8 weeks, and they showed enhanced bone tissue formation in rat calvarial defect models.

Perivascular biodegradable microneedle cuff for reduction of neointima formation after vascular injury

Restenosis often occurs at the site of vascular grafting and may become fatal for patients. Restenosis at anastomosis sites is due to neointimal hyperplasia (NH) and difficult to treat with conventional treatments. Such abnormal growth of smooth muscle cells in tunica media of vascular tissue can be reduced by delivering anti-proliferation drugs such as paclitaxel (PTX) to the inner vascular layer. Drug eluting stents (DES) or drug eluting balloon (DEB) have been developed to treat such vascular diseases. However, they are less efficient in drug delivery due to the drug loss to blood stream and inadequate to be applied to re-stenotic area in the presence of stent or anastomosis sites. Recently, we have introduced microneedle cuff (MNC) as perivascular delivery devices to achieve high delivery efficiency to tunica media. In this study, we investigated in vivo microneedle insertion and efficacy in treating NH using a rabbit balloon injury model. Microneedle shape was optimized for reliable insertion into tunica media layer. Uniform distribution of PTX in tunica media delivered by MNC devices was also confirmed. Animal study demonstrated significant NH reduction by MNC treatments and much higher delivery efficiency than flat type devices.

Rapid and repeatable fabrication of high A/R silk fibroin microneedles using thermally-drawn micromolds

Thermal drawing is a versatile rapid prototyping method that can freely form microneedle (MN) structures with ultra-high aspect ratio without relying on any complex and expensive process. However, it is still challenging to repeatedly produce MNs with identical shapes using this thermal drawing due to small fluctuations in processing conditions such as temperatures, drawing speeds, drawing heights, or parallelism in the drawing setup. In addition, thermal drawing is only applicable to thermoplastic materials and most natural biomaterials are incompatible with this method. Thus, we propose use of thermal drawing to fabricate master molds with high aspect ratios and replicate the shape by micromolding. In this work, high A/R MNs with various body profiles were fabricated by thermal drawing and replicated to silk fibroin (SF) MNs multiple times using micromolding. The original MN shape was precisely copied to the SF MNs. Methanol treatment enhanced the mechanical strength of SF MNs up to about 113% more depending on the treatment duration. We also demonstrated that methanol exposure time could effectively control drug release rates from SF MNs.

In vivo O2 measurement inside single photosynthetic cells.

The oxygen evolution of single cells was investigated using a nano-probe with an ultra-micro electrode (UME) in a submicron sized system in combination with a micro-fluidic system. A single cell was immobilized in the micro-fluidic system and a nano-probe was inserted into the cytosolic space of the single cell. Then, the UME was used for an in vivo amperometric experiment at a fixed potential and electrochemical impedance spectroscopy to detect oxygen evolution of the single cell under various light intensities.

Open micro-fluidic system for atomic force microscopy-guided in situ electrochemical probing of a single cell

Ultra-sharp nano-probes and customized atomic force microscopy (AFM) have previously been developed in our laboratory for in situ sub-cellular probing of electrochemical phenomena in living plant cells during their photosynthesis. However, this AFM-based electrochemical probing still has numerous engineering challenges such as immobilization of the live cells, compatibility of the immobilization procedure with AFM manipulation of the probe, maintenance of biological activity of the cells for an extended time while performing the measurements, and minimization of electrochemical noise. Thus, we have developed an open micro-fluidic channel system (OMFC) in which individual cells can be immobilized in micro-traps by capillary flow. This system affords easy AFM access and allows for maintenance of the cells in a well-defined chemical environment, which sustains their biological activity. The use of micro-channels for making the electrochemical measurements significantly reduces parasitic electrical capacitances and allows for current detection in the sub-pico-ampere range at high signal bandwidths. The OMFC was further studied using simulation packages for optimal design conditions. This system was successfully used to measure light-dependent oxidation currents of a few pico-amperes from the green alga Chlamydomonas reinhardtii.

Electrospun Silk Fibroin Nanofibrous Scaffolds with Two-Stage Hydroxyapatite Functionalization for Enhancing the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

The development of functional scaffolds with improved osteogenic potential is important for successful bone formation and mineralization in bone tissue engineering. In this study, we developed a functional electrospun silk fibroin (SF) nanofibrous scaffold functionalized with two-stage hydroxyapatite (HAp) particles, using mussel adhesive-inspired polydopamine (PDA) chemistry. HAp particles were first incorporated into SF scaffolds during the electrospinning process, and then immobilized onto the electrospun SF nanofibrous scaffolds containing HAp via PDA-mediated adhesive chemistry. We obtained two-stage HAp-functionalized SF nanofibrous scaffolds with improved mechanical properties and capable of providing a bone-specific physiological microenvironment. The developed scaffolds were tested for their ability to enhance the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and repair bone defect in vivo. To boost their ability for bone repair, we genetically modified hADMSCs with the transcriptional coactivator with PDZ-binding motif (TAZ) via polymer nanoparticle-mediated gene delivery. TAZ is a well-known transcriptional modulator that activates the osteogenic differentiation of mesenchymal stem cells (MSCs). Two-stage HAp-functionalized SF scaffolds significantly promoted the osteogenic differentiation of TAZ-transfected hADMSCs in vitro and enhanced mineralized bone formation in a critical-sized calvarial bone defect model. Our study shows the potential utility of SF scaffolds with nanofibrous structures and enriched inorganic components in bone tissue engineering.