Intraoperative bioprinting of human adipose-derived stem cells and extra-cellular matrix induces hair follicle-like downgrowths and adipose tissue formation during full-thickness craniomaxillofacial skin reconstruction.

Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2 % or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo. Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.

Synergistic coupling between 3D bioprinting and vascularization strategies.

Three-dimensional (3D) bioprinting offers promising solutions to the complex challenge of vascularization in biofabrication, thereby enhancing the prospects for clinical translation of engineered tissues and organs. While existing reviews have touched upon 3D bioprinting in vascularized tissue contexts, the current review offers a more holistic perspective, encompassing recent technical advancements and spanning the entire multistage bioprinting process, with a particular emphasis on vascularization. The synergy between 3D bioprinting and vascularization strategies is crucial, as 3D bioprinting can enable the creation of personalized, tissue-specific vascular network while the vascularization enhances tissue viability and function. The review starts by providing a comprehensive overview of the entire bioprinting process, spanning from pre-bioprinting stages to post-printing processing, including perfusion and maturation. Next, recent advancements in vascularization strategies that can be seamlessly integrated with bioprinting are discussed. Further, tissue-specific examples illustrating how these vascularization approaches are customized for diverse anatomical tissues towards enhancing clinical relevance are discussed. Finally, the underexplored intraoperative bioprinting (IOB) was highlighted, which enables the direct reconstruction of tissues within defect sites, stressing on the possible synergy shaped by combining IOB with vascularization strategies for improved regeneration.

Intraoperative Bioprinting of Human Adipose-derived Stem cells and Extra-cellular Matrix Induces Hair Follicle-Like Downgrowths and Adipose Tissue Formation during Full-thickness Craniomaxillofacial Skin Reconstruction.

Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2% or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo . Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.

High-throughput bioprinting of the nasal epithelium using patient-derived nasal epithelial cells.

Progenitor human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models through biofabrication. However, this approach has limitations in terms of achieving the intricate three-dimensional (3D) structure of the natural nasal epithelium. 3D bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of progenitor hNECs ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4 weeks air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes, such as disease modeling, immunological studies, and drug screening.

High-Throughput Bioprinting of the Nasal Epithelium using Patient-derived Nasal Epithelial Cells.

Human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models. However, the manual approach is slow, low-throughput and has limitations in terms of achieving the intricate 3D structure of the natural nasal epithelium in a uniform manner. 3D Bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of nasal progenitor cells ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4-week air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes such as disease modeling, immunological studies, and drug screening.

Esophageal wound healing by aligned smooth muscle cell-laden nanofibrous patch.

The esophagus exhibits peristalsis via contraction of circularly and longitudinally aligned smooth muscles, and esophageal replacement is required if there is a critical-sized wound. In this study, we proposed to reconstruct esophageal tissues using cell electrospinning (CE), an advanced technique for encapsulating living cells into fibers that allows control of the direction of fiber deposition. After treatment with transforming growth factor-ß, mesenchymal stem cell-derived smooth muscle cells (SMCs) were utilized for cell electrospinning or three-dimensional bioprinting to compare the effects of aligned micropatterns on cell morphology. CE resulted in SMCs with uniaxially arranged and elongated cell morphology with upregulated expression levels of SMC-specific markers, including connexin 43, smooth muscle protein 22 alpha (SM22α), desmin, and smoothelin. When SMC-laden nanofibrous patches were transplanted into a rat esophageal defect model, the SMC patch promoted regeneration of esophageal wounds with an increased number of newly formed blood vessels and enhanced the SMC-specific markers of SM22α and vimentin. Taken together, CE with its advantages, such as guidance of highly elongated, aligned cell morphology and accelerated SMC differentiation, can be an efficient strategy to reconstruct smooth muscle tissues and treat esophageal perforation.

Comparison ofin-situversusex-situdelivery of polyethylenimine-BMP-2 polyplexes for rat calvarial defect repair via intraoperative bioprinting.

Gene therapeutic applications combined with bio- and nano-materials have been used to address current shortcomings in bone tissue engineering due to their feasibility, safety and potential capability for clinical translation. Delivery of non-viral vectors can be altered using gene-activated matrices to improve their efficacy to repair bone defects.Ex-situandin-situdelivery strategies are the most used methods for bone therapy, which have never been directly compared for their potency to repair critical-sized bone defects. In this regard, we first time explore the delivery of polyethylenimine (PEI) complexed plasmid DNA encoding bone morphogenetic protein-2 (PEI-pBMP-2) using the two delivery strategies,ex-situandin-situdelivery. To realize these gene delivery strategies, we employed intraoperative bioprinting (IOB), enabling us to 3D bioprint bone tissue constructs directly into defect sites in a surgical setting. Here, we demonstrated IOB of an osteogenic bioink loaded with PEI-pBMP-2 for thein-situdelivery approach, and PEI-pBMP-2 transfected rat bone marrow mesenchymal stem cells laden bioink for theex-situdelivery approach as alternative delivery strategies. We found thatin-situdelivery of PEI-pBMP-2 significantly improved bone tissue formation compared toex-situdelivery. Despite debates amongst individual advantages and disadvantages ofex-situandin-situdelivery strategies, our results ruled in favor of thein-situdelivery strategy, which could be desirable to use for future clinical applications.

Strategies for 3D bioprinting of spheroids: A comprehensive review.

Biofabricated tissues have found numerous applications in tissue engineering and regenerative medicine in addition to the promotion of disease modeling and drug development and screening. Although three-dimensional (3D) printing strategies for designing and developing customized tissue constructs have made significant progress, the complexity of innate multicellular tissues hinders the accurate evaluation of physiological responses in vitro. Cellular aggregates, such as spheroids, are 3D structures where multiple types of cells are co-cultured and organized with endogenously secreted extracellular matrix and are designed to recapitulate the key features of native tissues more realistically. 3D Bioprinting has emerged as a crucial tool for positioning of these spheroids to assemble and organize them into physiologically- and histologically-relevant tissues, mimicking their native counterparts. This has triggered the convergence of spheroid fabrication and bioprinting, leading to the investigation of novel engineering methods for successful assembly of spheroids while simultaneously enhancing tissue repair. This review provides an overview of the current state-of-the-art in spheroid bioprinting methods and elucidates the involved technologies, intensively discusses the recent tissue fabrication applications, outlines the crucial properties that influence the bioprinting of these spheroids and bioprinted tissue characteristics, and finally details the current challenges and future perspectives of spheroid bioprinting efforts in the growing field of biofabrication.

miRNA induced 3D bioprinted-heterotypic osteochondral interface.

The engineering of osteochondral interfaces remains a challenge. MicroRNAs (miRs) have emerged as significant tools to regulate the differentiation and proliferation of osteogenic and chondrogenic formation in the human musculoskeletal system. Here, we describe a novel approach to osteochondral reconstruction based on the three-dimensional (3D) bioprinting of miR-transfected adipose-derived stem cell (ADSC) spheroids to produce a heterotypic interface that addresses the intrinsic limitations of the traditional approach to inducing zonal differentiation via the use of diffusible cytokines. We evaluated the delivery of miR-148b for osteogenic differentiation and the codelivery of miR-140 and miR-21 for the chondrogenic differentiation of ADSC spheroids. Our results demonstrated that miR-transfected ADSC spheroids exhibited upregulated expression of osteogenic and chondrogenic differentiation related gene and protein markers, and enhanced mineralization and cell proliferation compared to spheroids differentiated using a commercially-available differentiation medium. Upon confirmation of the osteogenic and chondrogenic potential of miR-transfected ADSC spheroids, using aspiration-assisted bioprinting, these spheroids were 3D bioprinted into a dual-layer heterotypic osteochondral interface with a stratified arrangement of distinct osteogenic and chondrogenic zones. The proposed approach holds great promise for the biofabrication of stratified tissues, not only for the osteochondral interfaces presented in this work, but also for other composite tissues and tissue interfaces, such as, but not limited to, the bone-tendon-muscle interface and craniofacial tissues.

Electrohydrodynamic-direct-printed cell-laden microfibrous structure using alginate-based bioink for effective myotube formation.

In this study, a fully aligned microfibrous structure fabricated using fibrin-assisted alginate bioink and electrohydrodynamic direct-printing was proposed for skeletal muscle tissue engineering. To safely construct the aligned alginate/fibrin microfibrous structure laden with myoblasts or endothelial cells, various printing conditions, such as an applied electric field, distance between the nozzle and target, and nozzle moving speed, were selected appropriately. Furthermore, to accelerate the formation of myotubes more efficiently, the alginate/fibrin bioink with vascular endothelial cells was co-printed into a spatially patterned structure within a myoblast-laden structure. The myoblast-laden structure co-cultured with endothelial cells presented fully aligned myotube formation and significantly greater myogenic differentiation compared to the myoblast-laden structure without the endothelial cells owing to the more abundant secretion of angiogenic cytokines. Also, when adipose stem cell- and endothelial cell-laden fibrous structure was implanted in a mouse volumetric muscle loss model, accelerated volumetric muscle repair was observed compared to the defect model. Based on the results, this study demonstrates an alginate-based bioink and new bio-fabricating method to obtain microfibrous cell-laden alginate/fibrin structures with mechanically stable and topographical cues. The proposed method can provide a myoblast/endothelial cell-laden fibrous alginate structure to efficiently induce engineering of skeletal muscle tissue, which could be used in muscle-on-a-chip or recovering structures of volumetric muscle defects.

An in vitro model using spheroids-laden nanofibrous structures for attaining high degree of myoblast alignment and differentiation.

A spheroid is an aggregation of single cells with structural and functional characteristics similar to those of 3D native tissues, and it has been utilized as one of the typical in vitro three-dimensional (3D) cell models. Scaffold-free spheroids provide outstanding reflection of tissue complexity in a 3D in vivo-like environment, but they can neither fabricate realistic macroscale 3D complex structures without avoiding necrosis nor receive direct external stimuli (i.e., stimuli from mechanical or topographical cues). Here, we propose a spheroid-laden electrospinning process to obtain in vitro model achieved using the synergistic effect of the unique bioactive components provided by the spheroids and stimulating effects provided by the aligned nanofibers. Methods: To show the functional activity of the spheroid-laden structures, we used myoblast-spheroids to obtain skeletal muscle, comprising highly aligned myotubes, utilizing an uniaxially arranged topographical cue. The spheroid-electrospinning was used to align spheroids directly by embedding them in aligned alginate nanofibers, which were controlled with various materials and processing parameters. Results: The spheroids laden in the alginate nanofibers showed high cell viability (>90%) and was compared with that of a cell-laden alginate nanofiber that was electrospun with single cells. Consequently, the spheroids laden in the aligned nanofibers showed a significantly higher degree of myotube formation and maturation. Conclusion: Results suggested that the in vitro model using electrospun spheroids could potentially be employed to understand myogenic responses for various in vitro drug tests.

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.

Cell-Electrospinning and Its Application for Tissue Engineering.

Electrospinning has gained great interest in the field of regenerative medicine, due to its fabrication of a native extracellular matrix-mimicking environment. The micro/nanofibers generated through this process provide cell-friendly surroundings which promote cellular activities. Despite these benefits of electrospinning, a process was introduced to overcome the limitations of electrospinning. Cell-electrospinning is based on the basic process of electrospinning for producing viable cells encapsulated in the micro/nanofibers. In this review, the process of cell-electrospinning and the materials used in this process will be discussed. This review will also discuss the applications of cell-electrospun structures in tissue engineering. Finally, the advantages, limitations, and future perspectives will be discussed.

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.

4D Bioprinting: Technological Advances in Biofabrication.

The development of the three-dimensional (3D) printer has resulted in significant advances in a number of fields, including rapid prototyping and biomedical devices. For 3D structures, the inclusion of dynamic responses to stimuli is added to develop the concept of four-dimensional (4D) printing. Typically, 4D printing is useful for biofabrication by reproducing a stimulus-responsive dynamic environment corresponding to physiological activities. Such a dynamic environment can be precisely designed with an understanding of shape-morphing effects (SMEs), which enables mimicking the functionality or intricate geometry of tissues. Here, 4D bioprinting is investigated for clinical use, for example, in drug delivery systems, tissue engineering, and surgery in vivo. This review presents the concept of 4D bioprinting and smart materials defined by SMEs and stimulus-responsive mechanisms. Then, biomedical smart materials and applications are discussed along with future perspectives.

Anisotropically Aligned Cell-Laden Nanofibrous Bundle Fabricated via Cell Electrospinning to Regenerate Skeletal Muscle Tissue.

For muscle regeneration, a uniaxially arranged micropattern is important to mimic the structure of the natural extracellular matrix. Recently, cell electrospinning (CE) has been tested to fabricate cell-laden fibrous structures by embedding cells directly into micro/nanofibers. Although homogenous cell distribution and a reasonable cell viability of the cell-laden fibrous structure fabricated using the CE process are achieved, unique topographical cues formed by an aligned fibrous structure have not been applied. In this study, a CE process to achieve not only homogeneous cell distribution with a high cell viability, but also highly aligned cells, which are guided by aligned alginate fibers is employed. To attain the aligned cell-laden fibrous structure, various processing conditions are examined. The selected condition is applied using C2C12 myoblast cells to ensure the biocompatibility and guidance of cell elongation and alignment. As a control, a cell-printed scaffold using a 3D bioprinter is used to compare the efficiency of cell alignment and differentiation of myoblasts. Highly arranged, multinucleated cell morphology is confirmed in the CE scaffold, which successively facilitates myogenic differentiation. It is believed that this study will be a new platform for obtaining cell alignment and will significantly benefit the efforts on muscle regeneration.

Three-Dimensional Microfibrous Bundle Structure Fabricated Using an Electric Field-Assisted/Cell Printing Process for Muscle Tissue Regeneration.

In tissue engineering, biomimetic scaffolds are developed to provide cells with a microenvironment that promotes cellular activities. In this study, we present a three-dimensional (3D) fibrous bundle structure fabricated using an electrohydrodynamic process and a cell printing process using myoblast-laden collagen bioink. An anisotropic topographical cue in a 3D structure is an important factor for muscle tissue regeneration, and therefore, the fibrous bundle structure was uniaxially stretched using optimized conditions for fiber alignment. In addition, for stable cell attachment to facilitate the effect of topological cues, the myoblasts were efficiently released from the collagen bioink. We observed that the 3D fibrous bundle structure was an effective in vitro platform that induced cell proliferation and the formation of myotubes. The synergistic combination of the aligned topological cues and high biocompatibility of collagen enhanced the formation of myotubes, which was represented by the relative expression of myogenic genes (Myf5, Myh2, MyoD, and Myogenin). Therefore, we could confirm the feasibility of the 3D fibrous bundle structure for the regeneration of skeletal muscle tissues.

Biomimetic cellulose/calcium-deficient-hydroxyapatite composite scaffolds fabricated using an electric field for bone tissue engineering.

Cellulose has been widely used as micro/nanofibers in various applications of tissue regeneration, but has certain limitations for bone regeneration, e.g., low biocompatibility in inducing osteogenesis. In addition, the low processability from the decomposition property before melting can be a significant obstacle to fabricating a required complex structure through a 3D-printing process. Herein, to overcome the low osteogenic activity of pure cellulose, we suggest a new cellulose-based composite scaffold consisting of cellulose and a high weight fraction (70 wt%) of calcium-deficient-hydroxyapatite (CDHA), which was obtained from the hydrolysis of α-tricalcium phosphate. Using biocompatible components, we fabricated a 3D pore-structure controllable composite scaffold consisting of microfibrous bundles through an electrohydrodynamic printing (EHDP) process supplemented with an ethanol bath. To obtain a mechanically stable and repeatable 3D mesh structure, various process parameters (nozzle-to-target distance, electric field strength, flow rate, and nozzle moving speed) were considered. As a control, a mesh structure fabricated using a normal EHDP process and with a similar pore geometry was used. A variety of cellular responses using preosteoblasts (MC3T3-E1) indicate that a CDHA/cellulose composite scaffold provides an efficient platform for inducing significantly high bone mineralization.

Preparation and characterization of gelatin/α-TCP/SF biocomposite scaffold for bone tissue regeneration.

In this study, we suggest a new biocomposite scaffold composed of gelatin/α-TCP (tricalcium phosphate)/SF (silk-fibroin) (GTS) which has enhanced mechanical strength and high level of cellular activity. To fabricate GTS scaffold, a temperature-controlled 3D printing process was used and appropriate printing conditions were selected based on rheological data. To show the feasibility as a biomedical scaffold for bone tissue regeneration, the various physical and biological results, using MG63 (osteoblast-like cells), of the GTS scaffold were compared with those of a pure gelatin (G) and gelatin/α-TCP (GT) composite scaffold. GTS scaffolds showed enhanced mechanical properties in dry and wet state compared to those of the G and GT scaffolds. Also, significantly high cell-proliferation and differentiation of MG63 cells were observed in the GTS scaffold. Therefore, the GTS composite scaffold will be one of highly potential biomaterials to be used in bone regeneration.

Development of a tannic acid cross-linking process for obtaining 3D porous cell-laden collagen structure.

Cell-printing is an emerging technique that enables to build a customized structure using biomaterials and living cells for various biomedical applications. In many biomaterials, alginate has been widely used for rapid gelation, low cost, and relatively high processability. However, biocompatibilities enhancing cell adhesion and proliferation were limited, so that, to overcome this problem, an outstanding alternative, collagen, has been extensively investigated. Many factors remain to be proven for cell-printing applications, such as printability, physical sustainability after printing, and applicability of in vitro cell culture. This study proposes a cell-laden collagen scaffold fabricated via cell-printing and tannic acid (TA) crosslinking process. The effects of the crosslinking agent (0-3wt% TA) in the cell-laden collagen scaffolds on physical properties and cellular activities using preosteoblasts (MC3T3-E1) were presented. Compared to the cell-laden collagen scaffold without TA crosslinking, the scaffold with TA crosslinking was significantly enhanced in mechanical properties, while reasonable cellular activities were observed. Concisely, this study introduces the possibility of a cell-printing process using collagen and TA crosslinking and in vitro cell culture for tissue regeneration.