SeungHyun Ahn

Designed hybrid scaffolds consisting of polycaprolactone microstrands and electrospun collagen-nanofibers for bone tissue regeneration

Biomedical scaffolds used in bone tissue engineering should have various properties including appropriate bioactivity, mechanical strength, and morphologically optimized pore structures. Collagen has been well known as a good biomaterial for various types of tissue regeneration, but its usage has been limited due to its low mechanical property and rapid degradation. In this work, a new hybrid scaffold consisting of polycaprolactone (PCL) and collagen is proposed for bone tissue regeneration. The PCL enhances the mechanical properties of the hybrid scaffold and controls the pore structure. Layered collagen nanofibers were used to enhance the initial cell attachment and proliferation. The results showed that the hybrid scaffold yielded better mechanical properties of pure PCL scaffold as well as enhanced biological activity than the pure PCL scaffold did. The effect of pore size on bone regeneration was investigated using two hybrid scaffolds with pore sizes of 200 ± 20 and 300 ± 27 μm. After post-seeding for 7 days, the cell proliferation with pore size, 200 ± 20 μm, was greater than that with pore size, 300 ± 27 μm, due to the high surface area of the scaffold.

Cells (MC3T3-E1)-Laden Alginate Scaffolds Fabricated by a Modified Solid-Freeform Fabrication Process Supplemented with an Aerosol Spraying

In this study, we propose a new cell encapsulation method consisting of a dispensing method and an aerosol-spraying method. The aerosol spray using a cross-linking agent, calcium chloride (CaCl(2)), was used to control the surface gelation of dispensed alginate struts during dispensing. To show the feasibility of the method, we used preosteoblast (MC3T3-E1) cells. By changing the relationship between the various dispensing/aerosol-spraying conditions and cell viability, we could determine the optimal cell-dispensing process: a nozzle size (240 μm) and an aerosol spray flow rate (0.93 ± 0.12 mL min(-1)), 10 mm s(-1) nozzle moving speed, a 10 wt % concentration of CaCl(2) in the aerosol solution, and 2 wt % concentration of CaCl(2) in the second cross-linking process. Based on these optimized process conditions, we successfully fabricated a three-dimensional, pore-structured, cell-laden alginate scaffold of 20 × 20 × 4.6 mm(3) and 84% cell viability. During long cell culture periods (16, 25, 33, and 45 days), the preosteoblasts in the alginate scaffold survived and proliferated well.

Coaxial structured collagen–alginate scaffolds: fabrication, physical properties, and biomedical application for skin tissue regeneration

Collagen is the most promising natural biomaterial and has been used in various tissue engineering applications for skin, bone, and cartilage because it provides good biocompatibility and low antigenicity. Although collagen is an excellent candidate material for various biomedical applications, its difficult processability and mechanical properties have remained important limitations. To overcome the problems, several methods including indirect printing combined with a sacrificing mold and low-temperature printing were suggested. However, it is difficult to fabricate precisely controlled 3D pore structure using the methods. In a previous study, we introduced a three-dimensional (3D) pore-structure-controlled collagen scaffold fabricated by a 3D dispensing system supplemented with a cryogenic and freeze-drying system. The fabricated scaffold had remarkably good cellular behaviour (cell migration and differentiation) but poor mechanical stability due to the highly porous structure consisting of micro-sized strands and poor mechanical nature of collagen. To overcome this deficiency, we designed a hybrid (core/shell) scaffold composed of an outer collagen and an inner alginate. The collagen/alginate scaffolds exhibited good structural stability (core–shell structure), increased Youngs modulus about seven times compared to pure collagen scaffold under a similar pore-structure, and resulted in good cell viability, similar to a pure collagen scaffold. In an in vivo test, the hybrid scaffold was used as a dermal substitute and provided good granulation tissue formation and rapid vascularisation.

Designed Three-Dimensional Collagen Scaffolds for Skin Tissue Regeneration

One of the challenges in tissue engineering is the development of a reproducible three-dimensional (3D) scaffold to support cell migration and infiltration. As a dermal substitute, 3D collagen scaffolds with precisely controlled pore structures were fabricated using an innovative cryogenic dispenser system. The scaffolds were composed of perpendicular, highly porous collagen strands in successive layers. The fabricated scaffolds were evaluated in an in vitro keratinocyte/fibroblast coculture test. Fibroblasts were well dispersed within the scaffold, and keratinocytes had completely migrated through the well-designed pore structure and differentiated on top of the scaffold surface. The differentiated keratinocytes generated a stratum corneum in the 3D dispensed scaffolds, similar to that in normal skin tissue.

Cell(MC3T3-E1)-printed poly(ϵ-caprolactone)/alginate hybrid scaffolds for tissue regeneration.

A new cell-printed scaffold consisting of poly(ϵ-caprolactone) (PCL) and cell-embedded alginate struts is designed. The PCL and alginate struts are stacked in an interdigitated pattern in successive layers to acquire a three-dimensional (3D) shape. The hybrid scaffold exhibits a two-phase structure consisting of cell (MC3T3-E1)-laden alginate struts able to support biological activity and PCL struts able to provide controllable mechanical support of the cell-laden alginate struts. The hybrid scaffolds exhibit an impressive increase in tensile modulus and maximum strength compared to pure alginate scaffolds. Laden cells are homogeneously distributed throughout the alginate struts and the entire scaffold, resulting in cell viability of approximately 84%.

A cryogenic direct-plotting system for fabrication of 3D collagen scaffolds for tissue engineering

The goal of tissue engineering is to repair or regenerate damaged tissue using a combination of cellular biology and materials engineering techniques. One of the challenging problems in tissue engineering is the development of a reproducible three-dimensional (3D) scaffold to support cell migration and infiltration. Although natural polymers, such as dissolved collagen or alginate, are considered ideal for this purpose, their hydrophilic properties have hindered the fabrication of designed 3D scaffold structures. To overcome this problem, we developed a novel system for the cryogenic plotting of 3D scaffolds. Using this technique, we created various 3D collagen scaffolds with designed pore structures that exhibited desired properties. The diameter of the individual collagen strands, which varied from 250 µm to 500 µm, was reproducibly dependent on processing parameters, and the final collagen scaffold showed little shrinkage (less than 12%) relative to the initial design. To evaluate the fabricated scaffold, we adapted the scaffold to regenerate skin tissue. Immunohistochemical analysis demonstrated that co-cultured keratinocytes and fibroblasts completely migrated throughout the 3D collagen scaffold and keratinocytes were well differentiated on the surface of scaffold like a human skin.

A three-dimensional hierarchical collagen scaffold fabricated by a combined solid freeform fabrication (SFF) and electrospinning process to enhance mesenchymal stem cell (MSC) proliferation

Collagen has the advantage of being very similar to macromolecular substances that can be recognized and metabolized in the biological environment. Although the natural material has superior property for this purpose, its use to fabricate reproducible and pore-structure-controlled 3D structures, which are designed to allow the entry of sufficient cells and the easy diffusion of nutrients, has been limited due to its low processability. Here, we propose a hybrid technology that combines a cryogenic plotting system with an electrospinning process. Using this technique, an easily pore-size-controllable hierarchical 3D scaffold consisting of micro-sized highly porous collagen strands and micro/nano-sized collagen fibers was fabricated. The pore structure of the collagen scaffold was controlled by the collagen micro/nanofibers, which were layered in the scaffold. The hierarchical scaffolds were characterized with respect to initial cell attachment and proliferation of bone marrow-derived mesenchymal stem cells within the scaffolds. The hierarchical scaffold exhibited incredibly enhanced initial cell attachment and cell compactness between pores of the plotted scaffold relative to the normally designed 3D collagen scaffold.

A new hybrid scaffold constructed of solid freeform-fabricated PCL struts and collagen struts for bone tissue regeneration: fabrication, mechanical properties, and cellular activity

We propose a new technique for the fabrication of hybrid scaffolds using melt-plotting and a low temperature plate. This method is useful for the fabrication of a scaffold composed of heterogeneous biomaterials. We applied the new technique to collagen and polycaprolactone (PCL), which are stacked in interdigitated struts in successive layers to acquire a three-dimensional (3D) shape. The fabricated scaffolds exhibited a two-phase structure consisting of collagen struts to enhance the biological activity and PCL struts to increase the mechanical stability. They also exhibited a pore size under 100% pore interconnectivity appropriate for bone tissue regeneration. The fabricated hybrid scaffolds were assessed not only for mechanical properties, but also for biological capabilities by culturing osteoblast-like cells (MG63) on pure PCL, collagen, and hybrid scaffolds. Compared with the pure PCL scaffold, the hybrid scaffold exhibited higher biological activity, such as cell viability (an increase of about 27% at 7 days), alkaline phosphatase (ALP) activity (an increase of about 36% at 14 days), and calcium deposition. The pure collagen exhibited the highest value for most biological activities studied. In addition, the hybrid scaffolds exhibited a dramatic increase of Youngs modulus compared to those of pure collagen scaffolds. These results suggest that this hybrid scaffold is potentially useful as a biomedical scaffold for bone tissue regeneration.

Fabrication of cell-laden three-dimensional alginate-scaffolds with an aerosol cross-linking process

A modified dispensing method for fabricating three-dimensional (3D) cell-laden alginate scaffolds is presented that utilizes aerosolized calcium chloride solutions for crosslinking. To evaluate the process, preosteoblasts (MC3T3-E1) mixed with 3.5 wt% alginate were fabricated into a 3D matrix (20 × 20 × 4.5 mm3). The fabricated cell-laden structure was highly porous and had uniformly designed pore size and shape. We compared two different dispensing nozzle sizes (310 and 610 μm) by observing the cell distribution in the struts and determining the cell viability within the scaffolds. The scaffold-embedded cells were 85% viable compared to the initial cell viability, and the cell distribution was homogeneous. This innovative cell dispensing technique is likely to be a promising fabrication tool for obtaining functional 3D scaffolds for tissue regeneration.

Fabrication of three-dimensional collagen scaffold using an inverse mould-leaching process

Natural biopolymers, such as collagen or chitosan, are considered ideal for biomedical scaffolds. However, low processability of the materials has hindered the fabrication of designed pore structures controlled by various solid freeform-fabrication methods. A new technique to fabricate a biomedical three-dimensional collagen scaffold, supplemented with a sacrificial poly(ethylenexa0oxide) mould is proposed. The fabricated collagen scaffold shows a highly porous surface and a three-dimensional structure with high porosity as well as mechanically stable structure. To show its feasibility for biomedical applications, fibroblasts/keratinocytes were co-cultured on the scaffold, and the cell proliferation and cell migration of the scaffold was more favorable than that obtained with a spongy-type collagen scaffold.