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.

A Microfluidic Device to Fabricate One-Step Cell Bead-Laden Hydrogel Struts for Tissue Engineering.

Cell-laden structures are widely applied for a variety of tissue engineering applications, including tissue restoration. Cell-to-cell interactions in bioprinted structures are important for successful tissue restoration, because cell-cell signaling pathways can regulate tissue development and stem cell fate. However, the low degree of cell-cell interaction in conventional cell-laden bioprinted structures is challenging for the therapeutic application of this modality. Herein, a microfluidic device with cell-laden methacrylated gelatin (GelMa) bioink and alginate as a matrix hydrogel is used to fabricate a functional hybrid structure laden with cell-aggregated microbeads. This approach effectively increases the degree of cell-to-cell interaction to a level comparable to cell spheroids. The hybrid structure is obtained using a one-step process without the exhausting procedure. It consists of cell bead fabrication and an extrusion process for the cell-bead laden structure. Different flow rates are appropriately selected to develop cell-laden struts with homogeneously distributed cell beads for each hydrogel in the process. The hybrid struts exhibit significantly higher cellular activities than those of conventional alginate/GelMa struts, which are bioprinted using similar cell densities and bioink formulations. Furthermore, hybrid struts with adipose stem cells are implanted into mice, resulting in significantly higher myogenesis in comparison to normally bioprinted struts.

A novel decellularized skeletal muscle-derived ECM scaffolding system for in situ muscle regeneration.

The cell-based tissue engineering strategies have gained attention in restoring normal tissue function after skeletal muscle injuries; however, these approaches require a donor tissue biopsy and extensive cell expansion process prior to implantation. In order to avoid this limitation, we developed a novel cell-free muscle-specific scaffolding system that consisted of a skeletal muscle-derived decellularized extracellular matrix (dECM) and a myogenic factor, insulin growth factor-1 (IGF-1). Rheological, morphological, and biological properties of this muscle-specific scaffold (IGF-1/dECM) as well as collagen and dECM scaffolds were examined. The cell viability in all scaffolds had over 90% at 1, 3, and 7 days in culture. The cell proliferation in the IGF-1/dECM was significantly increased when compared with other groups. More importantly, the IGF-1/dECM strongly supported the myogenic differentiation in the scaffold as confirmed by myosin heavy chain (MHC) immunofluorescence. We also investigated the feasibility in a rabbit tibialis anterior (TA) muscle defect model. The IGF-1/dECM had a significantly greater number of myofibers when compared to both collagen and dECM groups at 1 and 2 months after implantation. We demonstrated that this novel muscle-specific scaffolding system could effectively promote the muscle tissue regeneration in situ.

Preparation of functional human lysophosphatidic acid receptor 2 using a P9∗ expression system and an amphipathic polymer and investigation of its in vitro binding preference to Gα proteins.

Human lysophosphatidic acid receptor 2 (LPA2), a member of the G-protein coupled receptor family, mediates lysophosphatidic acid (LPA)-dependent signaling by recruiting various G proteins. Particularly, it is directly implicated in the progression of colorectal and ovarian cancer through G protein signaling cascades. To investigate the biochemical binding properties of LPA2 against various alpha subunits of G protein (Gα), a functional recombinant LPA2 was overexpressed in E. coli membrane with a P9∗ expression system, and the purified protein was stabilized with an amphipathic polymer that had been synthesized by coupling octylamine, glucosamine, and diethyl aminoproylamine at the carboxylic groups of poly-γ-glutamic acid. The purified LPA2 stabilized with the amphipathic polymer showed selective binding activity to the various Gα proteins as well as agonist-dependent dissociation from Gαi3. Understanding the binding properties of LPA2 against various Gα proteins advances the understanding of downstream signaling cascades of LPA2. The functional LPA2 prepared using a P9∗ expression system and an amphipathic polymer could also facilitate the development of LPA2-targeting drugs.

Macroscale pseudo-spheroids fabricated using methacrylated collagen-coated cells.

Rationale: Cell spheroids have shown great promise as tools for creating effective three-dimensional (3D) tissue models, facilitating tissue reconstruction and organoid development, due to their high cell density and efficient cellular interactions. However, a significant challenge persists in creating large-scale tissue structures with a 3D geometrical architecture using spheroids, due to the continual condensation and reorganization of cells and their environments. Methods: The spherical cell aggregates (pseudo-cell spheroids) or macroscale cell aggregates were obtained by coating each adipose-derived stem cell (hASC) with methacrylated collagen (Col-Ma). Subsequently, the coated cells were printed into an alginate supporting bath and photocrosslinked through exposure to UV light. To assess the effectiveness of this procedure on regenerative potential, the generated cell aggregates were compared with conventional cell spheroids and bioprinted cell constructs using immunofluorescent staining and quantification of myogenic-related gene expressions. Moreover, the bioconstructs were implanted into a mouse model with volumetric muscle loss to further elucidate their regenerative and functional recovery properties. Results: The use of Col-Ma as a cell-coating material enables the rapid and physical aggregation of cells within several hours, regardless of the cell type. Furthermore, Col-Ma-coated cell aggregates can provide relatively lower hypoxic conditions than cell spheroids fabricated using the hanging drop method owing to the thin porous Col-Ma layer coated on the cells. In addition, the resulting structures maintain their geometrical architecture following cell fusion and possess the potential for efficient scale-up and 3D complex shape formation, making them more suitable for clinical applications than conventional cell spheroids. Finally, the feasibility of the Col-Ma-coated cylindrical human adipose-derived stem cells aggregates was assessed through implantation in a mouse volumetric muscle loss model, showing a significantly higher regenerative ability of muscle tissue than the normally bioprinted cell construct. Conclusion: Our newly proposed method has meaningful potential for various tissue engineering applications, supported by the improved cellular activities and efficient muscle regeneration observed in both in vitro and in vivo studies, and organ-chip models.

Fabrication of fully aligned self-assembled cell-laden collagen filaments for tissue engineering via a hybrid bioprinting process.

Cell-laden structures play a pivotal role in various tissue engineering applications, particularly in tissue restoration. Interactions between cells within bioprinted structures are crucial for successful tissue development and regulation of stem cell fate through intricate cell-to-cell signaling pathways. In this study, we developed a new technique that combines polyethylene glycol (PEG)-infused submerged bioprinting with a stretching procedure. This approach facilitated the generation of fully aligned collagen structures consisting of myoblasts and a low concentration (2 wt%) of collagen to efficiently encourage muscle tissue regeneration. By adjusting several processing parameters, we obtained biologically safe and mechanically stable cell-laden collagen filaments with uniaxial alignment. Notably, the cell filaments exhibited markedly elevated cellular activities compared to those exhibited by conventional bioprinted filaments, even at similar cell densities. Moreover, when we implanted structures containing adipose stem cells into mice, we observed a significantly increased level of myogenesis compared to that in normally bioprinted struts. Thus, this promising approach has the potential to revolutionize tissue engineering by fostering enhanced cellular interactions and promoting improved outcomes in regenerative medicine.

Enhancing Craniofacial Bone Reconstruction with Clinically Applicable 3D Bioprinted Constructs.

Medical imaging and 3D bioprinting can be used to create patient-specific bone scaffolds with complex shapes and controlled inner architectures. This study investigated the effectiveness of a biomimetic approach to scaffold design by employing geometric control. The biomimetic scaffold with a dense external layer showed improved bone regeneration compared to the control scaffold. New bone filled the defected region in the biomimetic scaffolds, while the control scaffolds only presented new bone at the boundary. Histological examination also shows effective bone regeneration in the biomimetic scaffolds, while fibrotic tissue ingrowth is observed in the control scaffolds. These findings suggest that the biomimetic bone scaffold, designed to minimize competition for fibrotic tissue formation in the bony defect, can enhance bone regeneration. This study underscores the notion that patient-specific anatomy can be accurately translated into a 3D bioprinting strategy through medical imaging, leading to the fabrication of constructs with significant clinical relevance.

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%.

Fabrication of self-assembled core-sheath microfibers via formulation of alginate-based bioinks.

Core-sheath microfibrous structures are widely used in various tissue engineering applications and drug delivery systems. However, the fabrication of the various core-sheath structures using a 3D printing process supplemented with a coaxial nozzle has been challenging due to the center positioning of the core nozzle enclosed in the bigger shell nozzle. In this work, we developed a new 3D printing process using an alginate-based bioink (a mixture of photo-crosslinkable hydrogel and alginate) and its in situ crosslinking process within a single glass nozzle of the 3D printer. By manipulating the alginate weight fraction, UV intensity, flow rate, and nozzle moving speed, we could fabricate various self-assembled core-sheath structures (straight, wavy, and crimped microfibers in the core region of the structure) in which the photocrosslinked hydrogel resided in the core, and alginate was positioned in the sheath region, like a virtual coaxial nozzle.

Biopolymers and Their Application in Bioprinting Processes for Dental Tissue Engineering.

Dental tissues are composed of multiple tissues with complex organization, such as dentin, gingiva, periodontal ligament, and alveolar bone. These tissues have different mechanical and biological properties that are essential for their functions. Therefore, dental diseases and injuries pose significant challenges for restorative dentistry, as they require innovative strategies to regenerate damaged or missing dental tissues. Biomimetic bioconstructs that can effectively integrate with native tissues and restore their functionalities are desirable for dental tissue regeneration. However, fabricating such bioconstructs is challenging due to the diversity and complexity of dental tissues. This review provides a comprehensive overview of the recent developments in polymer-based tissue engineering and three-dimensional (3D) printing technologies for dental tissue regeneration. It also discusses the current state-of-the-art, focusing on key techniques, such as polymeric biomaterials and 3D printing with or without cells, used in tissue engineering for dental tissues. Moreover, the final section of this paper identifies the challenges and future directions of this promising research field.

3D bioprinting using a new photo-crosslinking method for muscle tissue restoration.

Three-dimensional (3D) bioprinting is a highly effective technique for fabricating cell-loaded constructs in tissue engineering. However, the versatility of fabricating precise and complex cell-loaded hydrogels is limited owing to the poor crosslinking ability of cell-containing hydrogels. Herein, we propose an optic-fiber-assisted bioprinting (OAB) process to efficiently crosslink methacrylated hydrogels. By selecting appropriate processing conditions for the photo-crosslinking technique, we fabricated biofunctional cell-laden structures including methacrylated gelatin (Gelma), collagen, and decellularized extracellular matrix. To apply the method to skeletal muscle regeneration, cell-laden Gelma constructs were processed with a functional nozzle having a topographical cue and an OAB process that could induce a uniaxial alignment of C2C12 and human adipose stem cells (hASCs). Significantly higher degrees of cell alignment and myogenic activities in the cell-laden Gelma structure were observed compared with those in the cell construct that was printed using a conventional crosslinking method. Moreover, an in vivo regenerative potential was observed in volumetric muscle defects in a mouse model. The hASC-laden construct significantly induced greater muscle regeneration than the cell construct without topographical cues. Based on the results, the newly designed bioprinting process can prove to be highly effective in fabricating biofunctional cell-laden constructs for various tissue engineering applications.

The one-step fabrication of porous hASC-laden GelMa constructs using a handheld printing system.

The fabrication of highly porous cell-loaded structures in tissue engineering applications has been a challenging issue because non-porous cell-laden struts can cause severe cell necrosis in the middle region owing to poor transport of nutrients and oxygen. In this study, we propose a versatile handheld 3D printer for the effective fabrication of porous cell-laden methacrylated gelatin (GelMa) with high porosity (≈97%) by air injection and a bubble-making system using mesh filters through which a mixture of air/GelMa bioink is passed. In particular, the pore size and foamability of the cell constructs could be manipulated using various processing parameters (rheological properties of GelMa, filter size and number, and air-bioink volume ratio). To demonstrate the feasibility of the cell construct as a tissue engineering substitute for muscle regeneration, in vitro cellular activities and in vivo regeneration ability of human adipose stem cells were assessed. The in vitro results demonstrated that the human adipose stem cells (hASCs) fabricated using the handheld 3D printer were alive and well-proliferated. Furthermore, the in vivo results showed that the hASCs-constructs directly printed from the handheld 3D printer showed significant restoration of functionality and efficient muscle regeneration in the volumetric muscle loss model of mice. Based on these results, the fabrication method of the porous cell-laden construct could be a promising tool for regenerating muscle tissues.

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.

Correction: Mineralized biomimetic collagen/alginate/silica composite scaffolds fabricated by a low-temperature bio-plotting process for hard tissue regeneration: fabrication, characterisation and in vitro cellular activities.

Correction for 'Mineralized biomimetic collagen/alginate/silica composite scaffolds fabricated by a low-temperature bio-plotting process for hard tissue regeneration: fabrication, characterisation and in vitro cellular activities' by HyeongJin Lee et al., J. Mater. Chem. B, 2014, 2, 5785-5798, https://doi.org/10.1039/c4tb00931b.

Fabrication of bone-derived decellularized extracellular matrix/ceramic-based biocomposites and their osteo/odontogenic differentiation ability for dentin regeneration.

The goal of this study was to fabricate bioactive cell-laden biocomposites supplemented with bone-derived decellularized extracellular matrix (dECM) with calcium phosphate ceramic, and to assess the effect of the biocomponents on the osteogenic and odontogenic differentiation of human dental pulp stem cells (hDPSCs). By evaluating the rheological properties and selecting printing parameters, mechanically stable cell-laden 3D biocomposites with high initial cell-viability (>90%) and reasonable printability (≈0.9) were manufactured. The cytotoxicity of the biocomposites was evaluated via MTT assay and nuclei/F-actin fluorescent analyses, while the osteo/odontogenic differentiation of the hDPSCs was assessed using histological and immunofluorescent analyses and various gene expressions. Alkaline phosphate activity and alizarin red staining results indicate that the dECM-based biocomposites exhibit significantly higher osteogenic activities, including calcification, compared to the collagen-based biocomposites. Furthermore, immunofluorescence images and gene expressions demonstrated upregulation of dentin matrix acidic phosphoprotein-1 and dentin sialophosphoprotein in the dECM-based biocomposites, indicating acceleration of the odontogenic differentiation of hDPSCs in the printed biocomposites. The hDPSC-laden biocomposite was implanted into the subcutaneous region of mice, and the biocomposite afforded clear induction of osteo/odontogenic ectopic hard tissue formation 8 weeks post-transplantation. From these results, we suggest that the hDPSC-laden biocomposite is a promising biomaterial for dental tissue engineering.

Highly porous multiple-cell-laden collagen/hydroxyapatite scaffolds for bone tissue engineering.

Efficient vascularization within a scaffold is an essential criterion for evaluating the success of volumetric bone formation. Various strategies using angiogenic growth factors and cell-based approaches to induce effective osteogenic and angiogenic activities have been investigated. In this study, we propose a new highly porous multiple-cell-laden collagen/hydroxyapatite scaffold fabricated using a whipped bioink. After in vitro culturing of cells in the porous scaffolds for an extended culture period, osteogenic and angiogenic activities were significantly enhanced owing to the well-developed microporous cell-supporting matrix inducing efficient crosstalk between the adipose stem cells and endothelial cells compared to those of the normally bioprinted cell-constructs. Furthermore, the in vitro results were thoroughly evaluated by in vivo experiments using a posterolateral lumbar spinal fusion model of an ovariectomized mouse. Based on these results, the porous multiple-cell-laden scaffolds enhanced spine fusion in the event of osteoporosis.

Bio-printing of aligned GelMa-based cell-laden structure for muscle tissue regeneration.

Volumetric muscle loss (VML) is associated with a severe loss of muscle tissue that overwhelms the regenerative potential of skeletal muscles. Tissue engineering has shown promise for the treatment of VML injuries, as evidenced by various preclinical trials. The present study describes the fabrication of a cell-laden GelMa muscle construct using an in situ crosslinking (ISC) strategy to improve muscle functionality. To obtain optimal biophysical properties of the muscle construct, two UV exposure sources, UV exposure dose, and wall shear stress were evaluated using C2C12 myoblasts. Additionally, the ISC system showed a significantly higher degree of uniaxial alignment and myogenesis compared to the conventional crosslinking strategy (post-crosslinking). To evaluate the in vivo regenerative potential, muscle constructs laden with human adipose stem cells were used. The VML defect group implanted with the bio-printed muscle construct showed significant restoration of functionality and muscular volume. The data presented in this study suggest that stem cell-based therapies combined with the modified bioprinting process could potentially be effective against VML injuries.

Wound healing ability of acellular fish skin and bovine collagen grafts for split-thickness donor sites in burn patients: Characterization of acellular grafts and clinical application.

Due to its high polyunsaturated fatty acid content, acellular fish skin has emerged as a dermal substitute for the promotion of wound healing as it decreases scar formation while providing pain relief. However, various systematic studies on acellular fish skin, such as its biophysical analysis, in vitro activities, and clinical application, have not been sufficiently investigated. In this study, we conducted a comparative study to evaluate the wound-healing ability of acellular fish skin graft (Kerecis®) with that of the widely used bovine collagen skin graft (ProHeal®). The skin grafts were evaluated not only in terms of their biophysical properties, but also their in vitro cellular activities, using fibroblasts, keratinocytes, and human endothelial cells. The clinical study evaluated wound healing in 52 patients with acute burns who underwent skin grafting on donor sites from January 2019 to December 2020. The study was conducted with two groups; while only Kerecis® was tested in one group, Kerecis® and ProHeal® were compared in the other. In both groups, the application time of the dressing material was one to two days after split-thickness skin grafting to the donor sites. The Kerecis®-treatment group experienced faster healing than the other treatment group. In particular, the average wound healing time using the Kerecis® treatment and the ProHeal® treatment was 10.7 ± 1.5 days and 13.1 ± 1.4 days, respectively. We believe that the faster healing of the Kerecis® treatment, compared to that of the ProHeal® treatment, maybe due to the synergistic effect of the unique biophysical structure and the bioactive components of acellular fish skin.

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.

Response to Letter to Editor "Comment on 'A photo-crosslinkable cartilage-derived extracellular matrix bioink for auricular cartilage tissue engineering' by Visscher et al."

STATEMENT OF SIGNIFICANCE:  .