Three-Dimensional Artificial Skin Construct Bioprinted with a Marine-Based Biocomposite.

A variety of artificial skin scaffolds, including 3D-bioprinted constructs, have been widely studied for regenerating injured skin tissue. Here, we devised a new composite biomaterial ink using fish-skin-based decellularized extracellular matrices (dECM) from tilapia and cod fish. The composition of the biocomposite mixture was carefully selected to obtain a mechanically stable and highly bioactive artificial cell construct. In addition, the decellularized extracellular matrices were methacrylated, followed by exposure to UV light to initiate photo-cross-linking. Porcine-skin-based dECMMa (pdECMMa) and tilapia-skin-based dECMMa (tdECMMa) biomaterials were used as controls. Assessment of various biophysical parameters and in vitro cellular activities, including cytotoxicity, wound healing ability, and angiogenesis, showed that the biocomposite exhibited much higher cellular activities compared to the controls owing to the synergistic effect of the favorable biophysical properties of tdECMMa and bioactive components (collagen, glycosaminoglycans (GAGs), elastin, and free fatty acids) from the decellularized cod skin. Furthermore, the skin constructs bioprinted using the bioinks exhibited more than 90% cell viability, performed with 3 days of submerged culture and then 28 days of air-liquid culture. For all cell constructs, the expression of cytokeratin 10 (CK10) was observed on the top surface of the epidermal layer, and cytokeratin 14 (CK14) was detected in the lower section of the keratinocyte layer. However, more developed CK10 and CK14 antibodies were observed in the cell-laden biocomposite construct [tilapia-skin-based dECMMa with cod-skin-based dECM] than in the controls [porcine-skin-based dECMMa (pdECMMa) and tilapia-skin-based dECMMa (tdECMMa)]. Based on these results, we believe that the fish-skin-based biocomposite construct is a potential biomaterial ink for skin regeneration.

Fabrication of Mechanically Reinforced Gelatin/Hydroxyapatite Bio-Composite Scaffolds by Core/Shell Nozzle Printing for Bone Tissue Engineering.

In tissue engineering, biocompatible scaffolds are used as 3D cell niches to provide a similar environment to that of native tissue for seeded cells to regenerate the target tissue. When engineering bone tissue, high mechanical strength and calcium phosphate composition are essential factors to consider. In this study, we fabricated biocompatible composite scaffolds composed of synthetic polymers (polycaprolactone (PCL) and poly (vinyl alcohol) (PVA)), natural polymers (gelatin and collagen) and bioceramic (hydroxyapatite; HA) for bone tissue engineering. The synthetic polymers were used to enhance the mechanical properties of the composite scaffolds while the natural protein-based polymers were used to enhance various cellular activities, such as cell adhesion and proliferation. Meanwhile, the bioceramic was introduced to promote osteogenic differentiation. Composite scaffolds were evaluated for their physical characteristics, such as mechanical, swelling and protein absorbing properties as well as biological properties (cell proliferation, alkaline phosphatase (ALP) activities and calcium deposition) with human osteoblast-like cells (MG63). Consequently, incorporation of hydroxyapatite into the gelatin/PVA (C-GPH) scaffold showed 5-fold and 1.5-fold increase in calcium deposition and ALP activities, respectively compared to gelatin/PVA scaffold (C-GP). Moreover, compressive modulus also increased 1.8-fold. Integration of PCL core into gelatin/PVA/hydroxyapatite scaffold (C-PGPH) further amplified the compressive modulus 1.5-fold. In conclusion, the scaffold that is reinforced with HA particles and integrated with PCL core of the struts showed significant potential in field of bone tissue engineering.

Electrohydrodynamic jet process for pore-structure-controlled 3D fibrous architecture as a tissue regenerative material: fabrication and cellular activities.

In this study, we propose a new scaffold fabrication method, "direct electro-hydrodynamic jet process," using the initial jet of an electrospinning process and ethanol media as a target. The fabricated three-dimensional (3D) fibrous structure was configured with multilayered microsized struts consisting of randomly entangled micro/nanofibrous architecture, similar to that of native extracellular matrixes. The fabrication of the structure was highly dependent on various processing parameters, such as the surface tension of the target media, and the flow rate and weight fraction of the polymer solution. As a tissue regenerative material, the 3D fibrous scaffold was cultured with preosteoblasts to observe the initial cellular activities in comparison with a solid-freeform fabricated 3D scaffold sharing a similar structural geometry. The cell-culture results showed that the newly developed scaffold provided outstanding microcellular environmental conditions to the seeded cells (about 3.5-fold better initial cell attachment and 2.1-fold better cell proliferation).

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%.

Bio-composites composed of a solid free-form fabricated polycaprolactone and alginate-releasing bone morphogenic protein and bone formation peptide for bone tissue regeneration.

Biomedical scaffolds should be designed with highly porous three-dimensional (3D) structures that have mechanical properties similar to the replaced tissue, biocompatible properties, and biodegradability. Here, we propose a new composite composed of solid free-form fabricated polycaprolactone (PCL), bone morphogenic protein (BMP-2) or bone formation peptide (BFP-1), and alginate for bone tissue regeneration. In this study, PCL was used as a mechanical supporting component to enhance the mechanical properties of the final biocomposite and alginate was used as the deterring material to control the release of BMP-2 and BFP-1. A release test revealed that alginate can act as a good release control material. The in vitro biocompatibilities of the composites were examined using osteoblast-like cells (MG63) and the alkaline phosphatase (ALP) activity and calcium deposition were assessed. The in vitro test results revealed that PCL/BFP-1/Alginate had significantly higher ALP activity and calcium deposition than the PCL/BMP-2/Alginate composite. Based on these findings, release-controlled BFP-1 could be a good growth factor for enhancement of bone tissue growth and the simple-alginate coating method will be a useful tool for fabrication of highly functional biomaterials through release-control supplementation.

Effects of a cell-imprinted poly(dimethylsiloxane) surface on the cellular activities of MG63 osteoblast-like cells: preparation of a patterned surface, surface characterization, and bone mineralization.

To understand the relationship between surface patterns and cellular activities, various types of pattern models have been investigated. In this study, we suggest a new surface pattern model, which replicates proliferated cells. We used osteoblast-like cells (MG63) as a target cell pattern and constructed various cell-imprinted surfaces using an electric field assisted casting method for different culturing times (4 h and 7 and 14 days). On the basis of scanning electron microscopy images and three-dimensional topographical optical images, we acquired the cells' unique patterns and used them for replicating patterned substrates. We then cultured MG63 cells in the patterned surfaces for 7 and 14 days to observe various cellular activities, cell viability, alkaline phosphatase (ALP) activity, and mineralization. Higher cellular activities were observed on the roughened surface as compared with the smooth surface. In particular, we obtained the most appropriate roughness value (R(a) = 702 ± 87 nm) from proliferated cells cultured over 14 days. On the basis of these findings, we demonstrate a new biomimical surface model that enhances cellular activities at the cell-substrate interface.

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.

Mechanically and biologically enhanced 3D-printed HA/PLLA/dECM biocomposites for bone tissue engineering.

Poly (L-lactic acid) (PLLA)-based biocomposites have been used in tissue engineering applications because of their reasonable biocompatibility and mechanical properties. However, the imperfect bioactive and mechanical properties of the composite make it difficult to be used in the region of bone defects that require high load-bearing. Therefore, this study introduced two fabricating strategies to induce mechanically and biologically enhanced hydroxyapatite (HA)/PLLA biocomposites. By introducing an in situ plasma treatment, which was simultaneously applied during the 3D-printing process, followed by the thermal annealing process, the flexural modulus of the composite was increased by 2.1-fold compared to the normal HA/PLLA composite. Furthermore, using the combinational process, efficient coating of bioactive material [decellularized extracellular matrix (dECM) derived from porcine bones] was possible. The fabricated biocomposite scaffold was assessed for various in vitro cellular activities such as cell proliferation and osteogenic activity. Based on the mechanical and biological studies, the HA/PLLA/dECM biocomposite scaffold is one of the promising scaffolds that can be applied in bone tissue regeneration.

Pressure/electric-field-assisted micro/nanocasting method for replicating a lotus leaf.

An electric field-aided process was introduced for a curable casting process. As a micro/nanosized pattern mask, a lotus leaf, which has a hierarchical structure, was used. The process consists of two steps: (1) applying an electric field to a liquid polymer and solidifying the polymer for use as a negative mold, and (2) using the negative polymer mold to fabricate a replicated poly(ethylene oxide) (PEO) surface in the original shape of the lotus leaf. In this process, the applied electric field induces unstable vibration of the liquid polymer, due to electrokinetic phenomena. The electrokinetic fluid motion resulted in well-replicated PEO surfaces. The quality of the fabricated surface was highly dependent on the applied field and pressure. We believe that this technique improves the quality of the standard nanocasting method and will be useful for fabricating micro/nanosized structures.

Three-dimensional hierarchical composite scaffolds consisting of polycaprolactone, ß-tricalcium phosphate, and collagen nanofibers: fabrication, physical properties, and in vitro cell activity for bone tissue regeneration.

ß-Tricalcium phosphate (ß-TCP) and collagen have been widely used to regenerate various hard tissues, but although Bioceramics and collagen have various biological advantages with respect to cellular activity, their usage has been limited due to ß-TCP's inherent brittleness and low mechanical properties, along with the low shape-ability of the three-dimensional collagen. To overcome these material deficiencies, we fabricated a new hierarchical scaffold that consisted of a melt-plotted polycaprolactone (PCL)/ß-TCP composite and embedded collagen nanofibers. The fabrication process was combined with general melt-plotting methods and electrospinning. To evaluate the capability of this hierarchical scaffold to act as a biomaterial for bone tissue regeneration, physical and biological assessments were performed. Scanning electron microscope (SEM) micrographs of the fabricated scaffolds indicated that the ß-TCP particles were uniformly embedded in PCL struts and that electrospun collagen nanofibers (diameter = 160 nm) were well layered between the composite struts. By accommodating the ß-TCP and collagen nanofibers, the hierarchical composite scaffolds showed dramatic water-absorption ability (100% increase), increased hydrophilic properties (20%), and good mechanical properties similar to PCL/ß-TCP composite. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffold was 2.2 times higher than that on the PCL/ß-TCP composite scaffold. Additionally, the proliferation rate of the cells was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we conclude that the collagen nanofibers and ß-TCP particles in the scaffold provide good synergistic effects for cell activity.

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(ethylene oxide) 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.

Micro/nano-hierarchical scaffold fabricated using a cell electrospinning/3D printing process for co-culturing myoblasts and HUVECs to induce myoblast alignment and differentiation.

Human skeletal muscle is composed of intricate anatomical structures, including uniaxially arranged myotubes and widely distributed blood capillaries. In this regard, vascularization is an essential part of the successful development of an engineered skeletal muscle tissue to restore its function and physiological activities. In this paper, we propose a method to obtain a platform for co-culturing human umbilical vein endothelial cells (HUVECs) and C2C12 cells using cell electrospinning and 3D bioprinting. To elaborate, on the surface of mechanical supporters (polycaprolactone and collagen struts) with a topographical cue, HUVECs-laden alginate bioink was uniaxially electrospun. The electrospun HUVECs showed high cell viability (90%), homogeneous cell distribution, and efficient HUVEC growth. Furthermore, the myoblasts (C2C12 cells), which were seeded on the vascularized structure (HUVECs-laden fibers), were co-cultured to facilitate myoblast regeneration. As a result, the scaffold that included myoblasts and HUVECs represented a high degree of the myosin heavy chain (MHC) with striated patterns and enhanced myogenic-specific gene expressions (MyoD, troponin T, MHC and myogenin) as compared to the scaffold that included only myoblasts. STATEMENT OF SIGNIFICANCE: Cell electrospinning is an advanced electrospinning method that improves cell-matrix interactions by embedding cells directly into micro/nanofibers. Here, cell electrospinning was employed to achieve not only the homogeneous human umbilical vein endothelial cells (HUVECs) distribution with a high cell-viability (~90%), but also highly aligned topographical cue. Moreover, the uniaxially micropatterned PCL/collagen struts as a physical support were generated using three-dimensional (3D) printing, and was covered with HUVEC-laden micro/nanofibers. This hierarchical structure provided meaningful mechanical stability, homogeneous cell distribution, and HUVEC transformation into a narrow, elongated structure. Furthermore, the myoblasts (C2C12 cells) were seeded on the HUVECs-laden fibers and cocultured to facilitate myogenesis. In brief, a myosin heavy chain with striated patterns and enhanced myogenic specific gene expressions were represented.

ASC/chondrocyte-laden alginate hydrogel/PCL hybrid scaffold fabricated using 3D printing for auricle regeneration.

Tissue engineering using adipose derived stem cells (ASCs) has become one of the most promising treatments for defective articular cartilage owing to the stability and dynamic differentiation of ASCs. In this study, we fabricated a 3D hybrid scaffold using poly(ε-caprolactone) (PCL) to support the mechanical properties of the regenerating auricle cartilage, and injected a cell-laden alginate hydrogel, containing a mixture of ASCs and chondrocytes, into the PCL scaffold. Using the cell-laden 3D auricle structure, the in vitro chondrogenesis of the ASCs with and without the presence of chondrocytes was examined. Additionally, the feasibility of utilizing the 3D cell-laden auricle structure for cartilage tissue engineering was evaluated in a rat model. In our in vitro and in vivo experiments, we observed that as the ASCs were co-cultured with the chondrocytes, chondrogenic differentiation was encouraged, and the regeneration of cartilage was significantly increased.

Nano/microscale topographically designed alginate/PCL scaffolds for inducing myoblast alignment and myogenic differentiation.

For regenerating skeletal muscle tissue, cell alignment and myotube formation in a scaffold are required. To achieve this goal, various studies have focused on controlling the myoblast orientation by manipulating the topographical structures of scaffolds. In the present study, a combined process involving electrospinning and three-dimensional (3D) printing was used to obtain a hierarchical structure consisting of microscale and nanoscale topographical structures by using alginate nanofibers and a polycaprolactone (PCL)-fibrillated micro-strut. In the structure, a micropatterned PCL strut, which was obtained using 3D printing and a leaching process supplemented with a sacrificial material, was employed for not only enhancing the mechanical stability, but also inducing myotube formation, while highly aligned alginate nanofibers fabricated using a modified electrospinning process facilitated myoblast attachment and alignment. The cell orientation and myotube formation of C2C12 cells cultured in the 3D hierarchical structure were significantly better than those of two controls (alginate-coated PCL strut and alginate nanofiber-deposited PCL strut, not fibrillated). These results confirm that the hierarchical scaffold has immense potential as a biomaterial for muscle-tissue regeneration.

Three-dimensional gelatin/PVA scaffold with nanofibrillated collagen surface for applications in hard-tissue regeneration.

The surface topography of a tissue-engineered scaffold is widely known to play an essential role in bone tissue engineering applications. Therefore, the cell-to-material interaction should be considered when developing scaffolds for bone tissue regeneration. Bone is a dynamic tissue with a distinct hierarchical structure composed of mostly collagen and bioceramics. In this study, the surface of gelatin/PVA scaffold (CF-G5P5) coated with fibrillated collagen was fabricated to enhance cell proliferation and osteogenic differentiation for bone tissue regeneration. The physical and biological properties of the fabricated scaffolds were investigated. As a result, the CF-G5P5 scaffold increased surface roughness and increased protein absorption compared to a gelatin/PVA scaffold (G5P5) by 1.6 times from OD value 0.43 to 0.71 after 12 h, cell proliferation increased 1.7 times from OD value 0.57 to 0.96, and differentiation increased by 1.5 times from 100 to 151%. Based on the results, the CF-G5P5 scaffold developed can be considered as a highly potential bone tissue regenerative material.

The fabrication of uniaxially aligned micro-textured polycaprolactone struts and application for skeletal muscle tissue regeneration.

One of the most important factors in skeletal muscle tissue regeneration is the alignment of muscle cells to mimic the native tissue. In this study, we developed a PCL-based scaffold with uniaxially aligned surface topography by stretching a 3D-printed scaffold. We examined the formation of aligned patterns by stretching the samples at different temperatures and stretching rates. This was possible through the effects of crystalline and amorphous regions on micro-textured deformation during the stretching process. We characterized the physical and biological properties of unstretched and stretched PCL struts. The stretched PCL showed greater surface roughness, protein absorption ability, and wettability. Moreover, myoblasts were cultured on the stretched and unstretched samples to analyze cellular activity. The cells cultured on the stretched samples were aligned along the pattern and showed a more elongated morphology. Furthermore, proliferation and differentiation were increased on the stretched samples resulting in a greater number of myotubes. We also discuss the possible alternative applications of this developed scaffold in other tissues.

Skeletal myotube formation enhanced through fibrillated collagen nanofibers coated on a 3D-printed polycaprolactone surface.

This work focused on considering the cellular responses of the growth and differentiation of myoblasts, C2C12, on fibrillated collagen-coated poly(ε-caprolactone) (PCL) surfaces. Through a fibrillation processing window using NaCl and collagen weight fractions, collagen fibril coating density can be controlled. Three different collagen-fibril densities coated on PCL strut were used to investigate the effects of the collagen fibril on the myoblast activities. After physical and cellular analyses of the scaffolds, such as surface morphology, fibronectin absorption, wettability, and mechanical properties, the rate of cell growth and the proficiency of the myoblasts to develop skeletal myotubes were evaluated. Based on the results, although the coated collagen nanofibers were randomly distributed, the fibrillated collagen layer with the appropriate density on the PCL surface promoted a greater myotube formation than that of the control, which had no fibrillated collagen. In particular, relatively higher densities of collagen fibril showed significantly greater myotube formation than those of the control (not-fibrillated collagen-coated on the PCL surface) and lower density of collagen fibril.

Three-Dimensional Hierarchical Nanofibrous Collagen Scaffold Fabricated Using Fibrillated Collagen and Pluronic F-127 for Regenerating Bone Tissue.

It is well known that a nanoscale fibrous structure can provide a unique stage for encouraging reasonable cell activities including attachment and proliferation owing to its similar topological structure to the extracellular matrix. Hence, the structure has been widely applied in tissue regeneration. Type-I collagen has been typically used as a typical tissue regenerative material owing to its biocompatibility and abundance, although it has potential for antigenicity. In particular, collagen has been fabricated in two different forms, porous spongy and nanofibers. However, although the structures provided outstanding cellular activities, they exhibit disadvantages such as low cell migration capabilities in a spongy scaffold owing to the low degree of interconnected macropores and low processability in fabricating three-dimensional (3D) structures in an electrospun collagen scaffold. Hence, the fabrication of 3D nanofibrous collagen structures with interconnected macropores can be extremely challenging. In this work, we developed a 3D collagen scaffold consisting of multilayered nanofibrous struts fabricated using a 3D printing process and pluronic F-127 (PF-127), which is a thermoreversible polymer. After optimizing various processing conditions, we successfully achieved the 3D nanofibrous collagen mesh structure with fully interconnected macropores. A 3D-printed collagen scaffold that was fabricated using a low-temperature printing process was applied as a control. Through various analyses using physical properties (surface morphology, fibronectin absorption, mechanical properties, etc.) and cell activities using preosteoblasts (MC3T3-E1), we are convinced that the newly designed 3D nanofibrous collagen scaffold can be a new promising scaffold for bone tissue engineering.

Gelatin/PVA scaffolds fabricated using a 3D-printing process employed with a low-temperature plate for hard tissue regeneration: Fabrication and characterizations.

Tissue engineering aims to repair or replace damaged tissues or organs using biomedical scaffolds cultured with cells. The scaffolds composed of biomaterials should guide the cells to mature into functional tissues or organs. An ideal scaffold to regenerate hard tissues should have mechanical stability as well as biocompatibilities. It has been well known that gelatin can provide outstanding biological activities, but its low mechanical stability can be one of obstacles to be used in hard tissue regeneration. To overcome the issue, we used PVA, which can reinforce the low mechanical stability of the gelatin. The gelatin/PVA scaffolds have been fabricated using a low temperature 3D-printing process. By manipulating various weight fractions of PVA/gelatin, we can obtain the optimal mixture ratio in aspect of the physical and biological properties of the scaffolds. As a result, a weight fraction of 5:5 showed appropriate mechanical strength and enhanced cell activities, such as cell proliferation and differentiation. The gelatin/PVA scaffold showed potential for future application as biomedical scaffold in soft and hard tissue regeneration.

Fabrication of micro/nanoporous collagen/dECM/silk-fibroin biocomposite scaffolds using a low temperature 3D printing process for bone tissue regeneration.

Biomaterials must be biocompatible, biodegradable, and mechanically stable to be used for tissue engineering applications. Among various biomaterials, a natural-based biopolymer, collagen, has been widely applied in tissue engineering because of its outstanding biocompatibility. However, due to its low mechanical properties, collagen has been a challenge to build a desired/complex 3D porous structure with appropriate mechanical strength. To overcome this problem, in this study, we used a low temperature printing process to create a 3D porous scaffold consisting of collagen, decellularized extracellular matrix (dECM) to induce high cellular activities, and silk-fibroin (SF) to attain the proper mechanical strength. To show the feasibility of the scaffold, pre-osteoblast (MC3T3-E1) cells were grown on the fabricated scaffold. Various in vitro cellular activities (cell-viability, MTT assay, and osteogenic activity) for pure collagen, collagen/dECM, and collagen/SF/dECM scaffolds were compared.