3D Bioprinting Technologies for Tissue Engineering Applications.

Three-dimensional (3D) printing (rapid prototyping or additive manufacturing) technologies have received significant attention in various fields over the past several decades. Tissue engineering applications of 3D bioprinting, in particular, have attracted the attention of many researchers. 3D scaffolds produced by the 3D bioprinting of biomaterials (bio-inks) enable the regeneration and restoration of various tissues and organs. These 3D bioprinting techniques are useful for fabricating scaffolds for biomedical and regenerative medicine and tissue engineering applications, permitting rapid manufacture with high-precision and control over size, porosity, and shape. In this review, we introduce a variety of tissue engineering applications to create bones, vascular, skin, cartilage, and neural structures using a variety of 3D bioprinting techniques.

Beneficial effect of aligned nanofiber scaffolds with electrical conductivity for the directional guide of cells.

Conducting polymer-based scaffolds receive biological and electrical signals from the extracellular matrix (ECM) or peripheral cells, thereby promoting cell growth and differentiation. Chitin, a natural polymer, is widely used as a scaffold because it is biocompatible, biodegradable, and nontoxic. In this study, we used an electrospinning technique to fabricate conductive scaffolds from aligned chitin/polyaniline (Chi/PANi) nanofibers for the directional guidance of cells. Pure chitin and random and aligned Chi/PANi nanofiber scaffolds were characterized using field emission scanning electron microscope (FE-SEM) and by assessing wettability, mechanical properties, and electrical conductivity. The diameters of aligned Chi/PANi nanofibers were confirmed to be smaller than those of pure chitin and random nanofibers owing to electrostatic forces and stretching produced by rotational forces of the drum collector. The electrical conductivity of aligned Chi/PANi nanofiber scaffolds was ~91% higher than that of random nanofibers. We also studied the viability of human dermal fibroblasts (HDFs) cultured on Chi/PANi nanofiber scaffolds in vitro using a CCK-8 assay, and found that cell viability on the aligned Chi/PANi nanofiber scaffolds was ~2.1-fold higher than that on random Chi/PANi nanofiber scaffolds after 7 days of culture. Moreover, cells on aligned nanofiber scaffolds spread in the direction of the aligned nanofibers (bipolar), whereas cells on the random nanofibers showed no spreading (6 h of culture) or multipolar patterns (7 days of culture). These results suggest that aligned Chi/PANi nanofiber scaffolds with conductivity exert effects that could improve survival and proliferation of cells with directionality.

Substance-P and transforming growth factor-ß in chitosan microparticle-pluronic hydrogel accelerates regenerative wound repair of skin injury by local ionizing radiation.

Clinical irradiation therapy for cancer could increase the risk of localized wound complications. This study was conducted to evaluate the potential use of a chitosan microparticle-pluronic F127 (CSMP-PF) hydrogel complex containing bioactive molecules, substance P and transforming growth factor-ß1, to regeneratively repair skin damaged by local ionizing radiation (IR). The BALB/c/bkl mice were locally irradiated to their limbs with a single 40 Gy dose of Co-60 γ rays to induce a skin injury. The morphological characteristics of the chitosan microparticles were analysed by scanning electron microscopy. The amounts of bioactive molecules taken up and released by the CSMP-PF hydrogel complex were measured. Haematoxylin and eosin staining of IR-damaged skin showed acanthosis and hyperkeratosis in the epidermis; and damage to hair follicles/skin appendages and adipose tissue, as well as panniculus carnosus, in the dermis. Injection of the CSMP-PF hydrogel complex into IR-damaged skin resulted in skin repair, suggesting that the complex has potential for use in the regenerative repair of IR-damaged skin.

3-dimensional bioprinting for tissue engineering applications.

The 3-dimensional (3D) printing technologies, referred to as additive manufacturing (AM) or rapid prototyping (RP), have acquired reputation over the past few years for art, architectural modeling, lightweight machines, and tissue engineering applications. Among these applications, tissue engineering field using 3D printing has attracted the attention from many researchers. 3D bioprinting has an advantage in the manufacture of a scaffold for tissue engineering applications, because of rapid-fabrication, high-precision, and customized-production, etc. In this review, we will introduce the principles and the current state of the 3D bioprinting methods. Focusing on some of studies that are being current application for biomedical and tissue engineering fields using printed 3D scaffolds.

Neuropeptide Substance-P-Conjugated Chitosan Nanofibers as an Active Modulator of Stem Cell Recruiting.

The goal to successful wound healing is essentially to immobilize and recruit appropriate numbers of host stem or progenitor cells to the wound area. In this study, we developed a chitosan nanofiber-immobilized neuropeptide substance-P (SP), which mediates stem cell mobilization and migration, onto the surfaces of nanofibers using a peptide-coupling agent, and evaluated its biological effects on stem cells. The amount of immobilized SP on chitosan nanofibers was modulated over the range of 5.89 ± 3.27 to 75.29 ± 24.31 ng when reacted with 10 to 500 ng SP. In vitro migration assays showed that SP-incorporated nanofibers induced more rapid migration of human mesenchymal stem cells on nanofibers compared to pristine samples. Finally, the conjugated SP evoked a minimal foreign body reaction and recruited a larger number of CD29- and CD44-positive stem cells into nanofibers in a mouse subcutaneous pocket model.

Gelatin blending and sonication of chitosan nanofiber mats produce synergistic effects on hemostatic functions.

To improve the hemostatic function of chitosan nanofiber mats, we studied the synergetic effects of gelatin blending and porosity control. Gelatin-blended-chitosan (Chi-Gel) nanofiber mats were evaluated with respect to surface morphology, mechanical properties and wettability, and functionally tested in a blood clotting study. The blood clotting efficiency of Chi-Gel nanofiber mats using rabbit whole blood in vitro was superior to that of chitosan nanofibers. Moreover, Chi-Gel nanofiber mats with enlarged porosity, produced by ultra-sonication, showed improved blood clotting efficiency, cell viability and cell infiltration compared with non-sonicated Chi-Gel nanofiber mats. Field-emission scanning electron microscopy revealed a richer density of platelets on sonicated nanofiber mats than on non-sonicated nanofiber mats after 3 min of blood clotting. The proliferation of human dermal fibroblast cells on sonicated Chi-Gel nanofiber mats using the DNA assay was higher than that on non-sonicated chitosan nanofiber mats after 7 days of culture. Confocal z-stack images showed that sonicated Chi-Gel nanofiber mats with high porosity supported active cell migration and infiltration into the 3-dimensional nanofiber mats. These results suggest that hydrophilic gelatin blending and sonication of chitosan nanofiber mats yields synergistic effects that not only improve hemostatic function but also promote wound repair.

Selectively micro-patterned fibronectin for regulating fate of human mesenchymal stem cell.

Microenvironment of the extracellular matrix can influence cellular responses through alternation of initial attachment and induce production of new tissue. To study the effect of such microenvironment on the relationship of cell cytoskeletal shape and its biological behaviors such as adhesion, proliferation and differentiation, we designed a patterned strip line of fibronectin on self assembled monolayers via microcontact printing. The physiological behavior of human mesenchymal stem cell (hMSC) on defined micro-patterns of fibronectin was evaluated after 4 h and 2 days of culture. Initial adhesion of hMSCs on a substrate with pattern spacing of 11 µm was stabilized faster than that on other substrates. Ratio of proliferating hMSC on 5 and 11 µm substrate constantly maintained a high rate. hMSCs on 5 and 11 µm substrate could adhere to substrate as spreading from fibronectin pattern line to several and lateral fibronectin pattern line. Their nucleus area could represent artificial increase by widely spreading on several fibronectin pattern lines. On the contrary to this, ratio of proliferating hMSC on 20 µm substrate constantly maintained a low rate less than even control and 0 µm substrate without fibronectin pattern. Tiny nucleus caused narrow and elongated hMSC morphology on 20 µm substrate gave the negative effect on the cell adhesion and proliferation. However, hMSCs on 20 µm substrate possessed not only slightly increased value of GO/G1 phase but also down regulation of CD marker expression compared with other groups. These results show initial adhesion and morphology of hMSC could regulate specific cellular behavior of hMSC.

Fabrication of conductive polymer-based nanofiber scaffolds for tissue engineering applications.

Natural and synthetic polymers, in particular those that are conductive, are of great interest in the field of tissue engineering and the pursuit of biomimetic extracellular matrix (ECM) structures for adhesion, proliferation, and differentiation of cells. In the present study, natural chitin and conductive polyaniline (PANi) blended solutions were electrospun to produce biodegradable and conductive biomimetic nanostructured scaffolds. The chitin/PANi (Chi-PANi) nanofibrous materials were characterized using field emission scanning electron microscopy, Fourier transform-infrared spectroscopy, wettability analysis, mechanical testing, and electrical conductivity measurements using a 4-point probe method. The calculated electrical conductivities of the PANi-containing nanofiber scaffolds significantly increased as the amount of PANi increased, reaching 5.21 ± 0.28 x 10(-3) S/cm for 0.3 wt% content of the conducting polymer. In addition, the viability of human mesenchymal stem cells (hMSCs) cultured on the Chi-PANi nanofiber scaffolds in vitro was found to be excellent. These results suggest that the Chi-PANi nanofiber scaffolds have great potential for use in tissue engineering applications that involve electrical stimulation.

Fabrication of sonicated chitosan nanofiber mat with enlarged porosity for use as hemostatic materials.

Electrospinning of pure chitosan was employed to obtain a nanofibrous hemostatic material. Owing to the water-solubility of the resulting acidic chitosan nanofibers, the optimum neutralization conditions were identified by testing various alkaline solutions, so that an insoluble material could be achieved. The pore size and thickness of the neutralized chitosan nanofibers mat could be controlled using ultra-sonication. The porosity of the chitosan mat was increased from 79.9% to 97.2% with ultra-sonication treatment for 1 min, and the water absorption time decreased from 110s to 9s. The blood clotting efficiency measured for the sonicated chitosan nanofiber mat was 1.35- and 3.41-fold better than the efficiencies of the Surgicel(®) and chitosan sponge, respectively. In addition, the proliferation of normal human dermal fibroblasts on the sonicated nanofiber mat was found to be 1.4-fold higher than that on the non-sonicated material after 7 days of culture.

Fabrications of nanofibers as crossed arrays by electrospinning.

We have developed a new method for obtaining nanofiber crossed arrays by exploiting an auxiliary electrode subjected to electrical frequencies, between the capillary tip and the grounded target in an electrospinning machine. The frequencies generated crossed arrays on a flat collector, used instead of a rotating wheel because of intersecting jets. We observed many straight and crossed structures. We determined the variation in morphology with changes in frequency, and characterized the samples using optical microscopy and a field emission scanning electron microscope. This paper reports on a simple, easy method for generating crossed array nanofibers on a flat substrate using electrical frequency in an auxiliary electrode.