Direct 3D cell-printing of human skin with functional transwell system.

Three-dimensional (3D) cell-printing has been emerging as a promising technology with which to build up human skin models by enabling rapid and versatile design. Despite the technological advances, challenges remain in the development of fully functional models that recapitulate complexities in the native tissue. Moreover, although several approaches have been explored for the development of biomimetic human skin models, the present skin models based on multistep fabrication methods using polydimethylsiloxane chips and commercial transwell inserts could be tackled by leveraging 3D cell-printing technology. In this paper, we present a new 3D cell-printing strategy for engineering a 3D human skin model with a functional transwell system in a single-step process. A hybrid 3D cell-printing system was developed, allowing for the use of extrusion and inkjet modules at the same time. We began by revealing the significance of each module in engineering human skin models; by using the extrusion-dispensing module, we engineered a collagen-based construct with polycaprolactone (PCL) mesh that prevented the contraction of collagen during tissue maturation; the inkjet-based dispensing module was used to uniformly distribute keratinocytes. Taking these features together, we engineered a human skin model with a functional transwell system; the transwell system and fibroblast-populated dermis were consecutively fabricated by using the extrusion modules. Following this process, keratinocytes were uniformly distributed onto the engineered dermis by the inkjet module. Our transwell system indicates a supportive 3D construct composed of PCL, enabling the maturation of a skin model without the aid of commercial transwell inserts. This skin model revealed favorable biological characteristics that included a stabilized fibroblast-stretched dermis and stratified epidermis layers after 14 days. It was also observed that a 50 times reduction in cost was achieved and 10 times less medium was used than in a conventional culture. Collectively, because this single-step process opens up chances for versatile designs, we envision that our cell-printing strategy could provide an attractive platform in engineering various human skin models.

Three-Dimensional Printing of Tissue/Organ Analogues Containing Living Cells.

The technical advances of three-dimensional (3D) printing in the field of tissue engineering have enabled the creation of 3D living tissue/organ analogues. Diverse 3D tissue/organ printing techniques with computer-aided systems have been developed and used to dispose living cells together with biomaterials and supporting biochemicals as pre-designed 3D tissue/organ models. Furthermore, recent advances in bio-inks, which are printable hydrogels with living cell encapsulation, have greatly enhanced the versatility of 3D tissue/organ printing. Here, we introduce 3D tissue/organ printing techniques and biomaterials that have been developed and widely used thus far. We also review a variety of applications in an attempt to repair or replace the damaged or defective tissue/organ, and develop the in vitro tissue/organ models. The potential challenges are finally discussed from the technical perspective of 3D tissue/organ printing.

Three-Dimensional Cell Printing of Large-Volume Tissues: Application to Ear Regeneration.

The three-dimensional (3D) printing of large-volume cells, printed in a clinically relevant size, is one of the most important challenges in the field of tissue engineering. However, few studies have reported the fabrication of large-volume cell-printed constructs (LCCs). To create LCCs, appropriate fabrication conditions should be established: Factors involved include fabrication time, residence time, and temperature control of the cell-laden hydrogel in the syringe to ensure high cell viability and functionality. The prolonged time required for 3D printing of LCCs can reduce cell viability and result in insufficient functionality of the construct, because the cells are exposed to a harsh environment during the printing process. In this regard, we present an advanced 3D cell-printing system composed of a clean air workstation, a humidifier, and a Peltier system, which provides a suitable printing environment for the production of LCCs with high cell viability. We confirmed that the advanced 3D cell-printing system was capable of providing enhanced printability of hydrogels and fabricating an ear-shaped LCC with high cell viability. In vivo results for the ear-shaped LCC also showed that printed chondrocytes proliferated sufficiently and differentiated into cartilage tissue. Thus, we conclude that the advanced 3D cell-printing system is a versatile tool to create cell-printed constructs for the generation of large-volume tissues.

Computer-aided multiple-head 3D printing system for printing of heterogeneous organ/tissue constructs.

Recently, much attention has focused on replacement or/and enhancement of biological tissues via the use of cell-laden hydrogel scaffolds with an architecture that mimics the tissue matrix, and with the desired three-dimensional (3D) external geometry. However, mimicking the heterogeneous tissues that most organs and tissues are formed of is challenging. Although multiple-head 3D printing systems have been proposed for fabricating heterogeneous cell-laden hydrogel scaffolds, to date only the simple exterior form has been realized. Here we describe a computer-aided design and manufacturing (CAD/CAM) system for this application. We aim to develop an algorithm to enable easy, intuitive design and fabrication of a heterogeneous cell-laden hydrogel scaffolds with a free-form 3D geometry. The printing paths of the scaffold are automatically generated from the 3D CAD model, and the scaffold is then printed by dispensing four materials; i.e., a frame, two kinds of cell-laden hydrogel and a support. We demonstrated printing of heterogeneous tissue models formed of hydrogel scaffolds using this approach, including the outer ear, kidney and tooth tissue. These results indicate that this approach is particularly promising for tissue engineering and 3D printing applications to regenerate heterogeneous organs and tissues with tailored geometries to treat specific defects or injuries.

Current advances in three-dimensional tissue/organ printing.

Three-dimensional (3D) tissue/organ printing is a major aspect of recent innovation in the field of tissue engineering and regenerative medicine. 3D tissue/organ printing aims to create 3D living tissue/organ analogues, and have evolved along with advances in 3D printing techniques. A diverse range of computer-aided 3D printing techniques have been applied to dispose living cells together with biomaterials and supporting biochemical factors within pre-designed 3D tissue/organ analogues. Recent developments in printable biomaterials, such as decellularized extracellular matrix bio-inks have enabled improvements in the functionality of the resulting 3D tissue/organ analogues. Here, we provide an overview of the 3D printing techniques and biomaterials that have been used, including the development of 3D tissue/organ analogues. In addition, in vitro models are described, and future perspectives in 3D tissue/organ printing are identified.

Redefining the Septal L-Strut to Prevent Collapse.

During septorhinoplasty, septal cartilage is frequently resected for various purposes but the L-strut is preserved. Numerous materials are inserted into the nasal dorsum during dorsal augmenation rhinoplasty without considering nasal structural safety. This study used a finite element method (FEM) to redefine the septal L-strut, to prevent collapse as pressure moved from the rhinion to the supratip breakpoint on the nasal dorsum and as the contact percentage between the caudal L-strut and the maxillary crest changed. We designed a 1-cm-wide L-strut model based on computed tomography data. At least 45% of the width of the L-strut in the inferior portion of the caudal strut must be preserved during septoplasty to stabilize the septum. In augmentation rhinoplasty, the caudal L-strut must either be preserved perfectly or reinforced to prevent collapse or distortion of the L-strut. The dorsal augmentation material must be fixed in an augmentation pocket to prevent movement of graft material toward the supratip breakpoint, which can disrupt the L-strut. We conducted a numerical analysis using a FEM to predict tissue/organ behavior and to help clinicians understand the reasons for target tissue/organ collapse and deformation.

Redefining the septal L-strut in septal surgery.

In septal surgery, the surgeon preserves the L-strut, the portion anterior to a vertical line drawn from the rhinion to the anterior nasal spine (ANS) and at least a 1-cm width of the dorsal and caudal septal segment, to decrease the potential for loss of the tip and dorsal nasal support. However, nasal tip collapse and saddle deformities occur occasionally. We utilized a mechanical approach to determine the safe width size for the L-strut in contact with the maxillary crest. Five L-strut models were designed based on computed tomography data (80 patients) and previous studies (55 patients). All L-strut models connected the perpendicular plate of the ethmoid bone (PPE) and the maxillary crest and were assumed to be fixed to the PPE and maxillary crest. An approximated daily load was applied to the dorsal portion of the L-strut. Finite element analyses were performed to compare the stress, strain, and displacement distribution of all L-strut models. According to the differences in the contact area between the caudal L-strut and maxillary crest, there are significant differences in terms of the stress, strain, and displacement distribution in the L-strut. High stresses occurred at the inner corner of the L-strut when 60 - 100% of the strut was in contact with the maxillary crest. High stresses also occurred at the inferior portion of the caudal L-strut when 20 - 40% of the caudal strut was in contact with maxillary crest. We conclude that it is important to preserve the 1-cm width L-strut caudal segment, which corresponds to the portion posterior to a vertical line drawn from the rhinion to the ANS. In particular, we must maintain more than 40% of the contact area between the L-strut and the maxillary crest when the septal cartilage in the caudal portion of the L-strut is harvested.

A comparative study on collagen type I and hyaluronic acid dependent cell behavior for osteochondral tissue bioprinting.

Bioprinting is a promising technique for engineering composite tissues, such as osteochondral tissues. In this study, as a first step toward bioprinting-based osteochondral tissue regeneration, we systematically examined the behavior of chondrocytes and osteoblasts to hyaluronic acid (HA) and type I collagen (Col-1) hydrogels. First, we demonstrated that cells on hydrogels that were comprised of major native tissue extracellular matrix (ECM) components (i.e. chondrocytes on HA hydrogels and osteoblasts on Col-1 hydrogels) exhibited better proliferation and cell function than cells on non-native ECM hydrogels (i.e., chondrocytes on Col-1 hydrogels and osteoblasts on HA hydrogels). In addition, cells located near their native ECM hydrogels migrated towards them. Finally, we bioprinted three-dimensional (3D) osteochondral tissue-mimetic structures composed of two compartments, osteoblast-encapsulated Col-1 hydrogels and chondrocyte-encapsulated HA hydrogels, and found viability and functions of each cell type were well maintained within the 3D structures up to 14 days in vitro. These results suggest that with proper choice of hydrogel materials, bioprinting-based approaches can be successfully applied for osteochondral tissue regeneration.

3D printing of composite tissue with complex shape applied to ear regeneration.

In the ear reconstruction field, tissue engineering enabling the regeneration of the ear's own tissue has been considered to be a promising technology. However, the ear is known to be difficult to regenerate using traditional methods due to its complex shape and composition. In this study, we used three-dimensional (3D) printing technology including a sacrificial layer process to regenerate both the auricular cartilage and fat tissue. The main part was printed with poly-caprolactone (PCL) and cell-laden hydrogel. At the same time, poly-ethylene-glycol (PEG) was also deposited as a sacrificial layer to support the main structure. After complete fabrication, PEG can be easily removed in aqueous solutions, and the procedure for removing PEG has no effect on the cell viability. For fabricating composite tissue, chondrocytes and adipocytes differentiated from adipose-derived stromal cells were encapsulated in hydrogel to dispense into the cartilage and fat regions, respectively, of ear-shaped structures. Finally, we fabricated the composite structure for feasibility testing, satisfying expectations for both the geometry and anatomy of the native ear. We also carried out in vitro assays for evaluating the chondrogenesis and adipogenesis of the cell-printed structure. As a result, the possibility of ear regeneration using 3D printing technology which allowed tissue formation from the separately printed chondrocytes and adipocytes was demonstrated.

Effect of pore architecture and stacking direction on mechanical properties of solid freeform fabrication-based scaffold for bone tissue engineering.

Fabrication of a three-dimensional (3D) scaffold with increased mechanical strength may be an essential requirement for more advanced bone tissue engineering scaffolds. Various material- and chemical-based approaches have been explored to enhance the mechanical properties of engineered bone tissue scaffolds. In this study, the effects of pore architecture and stacking direction on the mechanical and cell proliferation properties of a scaffold were investigated. The 3D scaffold was prepared using solid freeform fabrication technology with a multihead deposition system. Various types of scaffolds with different pore architectures (lattice, stagger, and triangle types) and stacking directions (horizontal and vertical directions) were fabricated with a blend of polycaprolactone and poly lactic-co-glycolic acid. In compression tests, the triangle-type scaffold was the strongest among the experimental groups. Stacking direction affected the mechanical properties of scaffolds. An in vitro cell counting kit-8 assay showed no significant differences in optical density depending on the different pore architectures and stacking directions. In conclusion, mechanical properties of scaffolds can be enhanced by controlling pore architecture and stacking direction.

Development of a 3D bellows tracheal graft: mechanical behavior analysis, fabrication and an in vivo feasibility study.

Artificial tracheal grafts should have not only enough compressive strength to maintain an open tracheal lumen, but also sufficient flexibility for stable mechanical behavior, similar to the native trachea at the implant site. In this study, we developed a new 3D artificial tracheal graft using a bellows design for considering its mechanical behavior. To investigate the mechanical behavior of the bellows structure, finite element method (FEM) analysis in terms of longitudinal tension/compression, bending and radial compression was conducted. The bellows structure was then compared with the cylinder structure generally used for artificial tracheal grafts. The FEM analysis showed that the bellows had outstanding flexibility in longitudinal tension/compression and bending. Moreover, the bellows kept the lumen open without severe luminal deformation in comparison with the cylinder structure. A three-dimensional artificial tracheal graft with a bellows design was fabricated using indirect solid freeform fabrication technology, and the actual mechanical test was conducted to investigate the actual mechanical behavior of the bellows graft. The fabricated bellows graft was then applied to segmental tracheal reconstruction in a rabbit model to assess its applicability. The bellows graft was completely incorporated into newly regenerated connective tissue and no obstruction at the implanted site was observed for up to 8 weeks after implantation. The data suggested that the developed bellows tracheal graft could be a promising alternative for tracheal reconstruction.