Silk Fibroin Enhances Cytocompatibilty and Dimensional Stability of Alginate Hydrogels for Light-Based Three-Dimensional Bioprinting.

Three-dimensional (3D) bioprinting is a technology under active study for use in tissue engineering and regenerative medicine. Bioink comprises cells and polymers and is the essential material for 3D bioprinting. The characteristics of the bioink affect its printability, gelation behavior, and cell compatibility. In this study, alginate derivatives were synthesized to induce rapid gelation, and a bioink was prepared by mixing these alginate derivatives with silk fibroin to enhance cell compatibility. A low-concentration (3 wt %) alginate/silk fibroin (Alg/SF) bioink was pregelated by the ionic cross-linking of Alg to increase the viscosity for 3D printing. The rheological and mechanical properties were analyzed using a rheometer and a texture meter, respectively. Analysis of cell viability and proliferation using fibroblasts (NIH-3T3) in the bioinks showed that the Alg/SF bioink has improved cytocompatibility compared to that of conventional Alg bioinks, making it a promising material for tissue engineering.

Co-culture of smooth muscle cells and endothelial cells on three-dimensional bioprinted polycaprolactone scaffolds for cavernosal tissue engineering.

PURPOSE: In vitro evaluation of polycaprolactone (PCL) scaffolds fabricated by a three-dimensional (3D) printing technique for tissue engineering applications in the corpus cavernosum. MATERIALS AND METHODS: PCL scaffolds were fabricated by use of a 3 D bioprinting system. The 3D-printed scaffolds had interconnected structures for cell ingrowth. Human aortic smooth muscle cells (haSMCs) were seeded on the scaffold and cultured for 5 days, and then human umbilical vein endothelial cells (HUVECs) were also added on the scaffolds and co-cultured with haSMCs for up to 7 days. The ability of these scaffolds to support the growth of HUVECs and haSMCs was investigated in vitro. 3 D strand-deposited scaffolds were characterized by scanning electron microscopy (SEM) images and porosity measurement. RESULTS: SEM images showed the surface of the PCL scaffolds to be well covered by HUVECs and haSMCs. Immunofluorescent staining of α-flk1 and α-smooth muscle actin on the HUVECs and haSMCs seeded scaffolds confirmed that the cells remained viable and proliferated throughout the time course of the culture. CONCLUSION: 3 D bioprinting of a PCL scaffold is feasible for co-culturing of HUVECs and haSMCs. This was a preliminary study to investigate the possibility of fabrication of tissue-engineered corpus cavernosum.

Characterization and preparation of bio-tubular scaffolds for fabricating artificial vascular grafts by combining electrospinning and a 3D printing system.

The last decade has seen artificial blood vessels composed of natural polymer nanofibers grafted into human bodies to facilitate the recovery of damaged blood vessels. However, electrospun nanofibers (ENs) of biocompatible materials such as chitosan (CTS) suffer from poor mechanical properties. This study describes the design and fabrication of artificial blood vessels composed of a blend of CTS and PCL ENs and coated with PCL strands using rapid prototyping technology. The resulting tubular vessels exhibited excellent mechanical properties and showed that this process may be useful for vascular reconstruction.

Tissue-engineered tracheal reconstruction using three-dimensionally printed artificial tracheal graft: preliminary report.

Three-dimensional printing has come into the spotlight in the realm of tissue engineering. We intended to evaluate the plausibility of 3D-printed (3DP) scaffold coated with mesenchymal stem cells (MSCs) seeded in fibrin for the repair of partial tracheal defects. MSCs from rabbit bone marrow were expanded and cultured. A half-pipe-shaped 3DP polycaprolactone scaffold was coated with the MSCs seeded in fibrin. The half-pipe tracheal graft was implanted on a 10 × 10-mm artificial tracheal defect in four rabbits. Four and eight weeks after the operation, the reconstructed sites were evaluated bronchoscopically, radiologically, histologically, and functionally. None of the four rabbits showed any sign of respiratory distress. Endoscopic examination and computed tomography showed successful reconstruction of trachea without any collapse or blockage. The replaced tracheas were completely covered with regenerated respiratory mucosa. Histologic analysis showed that the implanted 3DP tracheal grafts were successfully integrated with the adjacent trachea without disruption or granulation tissue formation. Neo-cartilage formation inside the implanted graft was sufficient to maintain the patency of the reconstructed trachea. Scanning electron microscope examination confirmed the regeneration of the cilia, and beating frequency of regenerated cilia was not different from those of the normal adjacent mucosa. The shape and function of reconstructed trachea using 3DP scaffold coated with MSCs seeded in fibrin were restored successfully without any graft rejection.

Hyaluronic acid/polyphenol sunscreens with broad-spectrum UV protection properties from tannic acid and quercetin.

Currently, commercial sunscreens cause a number of biotoxicity and environmental issues, making it imperative to develop biocompatible alternatives. In this study, we aimed to develop an alternative sunscreen from two ecofriendly and biocompatible natural polyphenolic compounds, tannic acid (TA) and quercetin (Que). The sunscreen was prepared through a simple process using an oil-in-water emulsion as the medium and hyaluronic acid (HA) as the base polymer to improve biocompatibility. The HA/TA/Que. sunscreen prepared in this study exhibits 0 % transmittance in the UVB region and <15 % transmittance in the UVA region, resulting in excellent sun-protection properties (SPF 30). Remarkably, the as-prepared HA/TA/Que. sunscreen has a suitable viscosity and similar UV protection properties to those of commercial sunscreens. The HA/TA/Que. sunscreen also exhibits 90.4 % antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl, demonstrating an ability to effectively capture reactive oxygen species that directly affect the skin. In addition, the cell viability was >90 % at a concentration of 50 µg/mL after 7 days, indicating excellent cytocompatibility. Owing to its various advantageous features, the HA/TA/Que. sunscreen with excellent sun protection properties and multiple functionalities is expected to resolve many environmental and biological issues caused by commercial sunscreens.

Microfluidics-assisted fabrication of natural killer cell-laden microgel enhances the therapeutic efficacy for tumor immunotherapy.

Recently, interest in cancer immunotherapy has increased over traditional anti-cancer therapies such as chemotherapy or targeted therapy. Natural killer (NK) cells are part of the immune cell family and essential to tumor immunotherapy as they detect and kill cancer cells. However, the disadvantage of NK cells is that cell culture is difficult. In this study, porous microgels have been fabricated using microfluidic channels to effectively culture NK cells. Microgel fabrication using microfluidics can be mass-produced in a short time and can be made in a uniform size. Microgels consist of photo cross-linkable polymers such as methacrylic gelatin (GelMa) and can be regulated via controlled GelMa concentrations. NK92 cell-laden three-dimensional (3D) microgels increase mRNA expression levels, NK92 cell proliferation, cytokine release, and anti-tumor efficacy, compared with two-dimensional (2D) cultures. In addition, the study confirms that 3D-cultured NK92 cells enhance anti-tumor effects compared with enhancement by 2D-cultured NK92 cells in the K562 leukemia mouse model. Microgels containing healthy NK cells are designed to completely degrade after 5 days allowing NK cells to be released to achieve cell-to-cell interaction with cancer cells. Overall, this microgel system provides a new cell culture platform for the effective culturing of NK cells and a new strategy for developing immune cell therapy.

BMP-2-immobilized PCL 3D printing scaffold with a leaf-stacked structure as a physically and biologically activated bone graft.

Although three-dimensional (3D) printing techniques are used to mimic macro- and micro-structures as well as multi-structural human tissues in tissue engineering, efficient target tissue regeneration requires bioactive 3D printing scaffolds. In this study, we developed a bone morphogenetic protein-2 (BMP-2)-immobilized polycaprolactone (PCL) 3D printing scaffold with leaf-stacked structure (LSS) (3D-PLSS-BMP) as a bioactive patient-tailored bone graft. The unique LSS was introduced on the strand surface of the scaffold via heating/cooling in tetraglycol without significant deterioration in physical properties. The BMP-2 adsorbed on3D-PLSS-BMPwas continuously released from LSS over a period of 32 d. The LSS can be a microtopographical cue for improved focal cell adhesion, proliferation, and osteogenic differentiation.In vitrocell culture andin vivoanimal studies demonstrated the biological (bioactive BMP-2) and physical (microrough structure) mechanisms of3D-PLSS-BMPfor accelerated bone regeneration. Thus, bioactive molecule-immobilized 3D printing scaffold with LSS represents a promising physically and biologically activated bone graft as well as an advanced tool for widespread application in clinical and research fields.

Decellularized matrix bioink with gelatin methacrylate for simultaneous improvements in printability and biofunctionality.

Gelatin methacrylate (GelMA) bioink has been widely used in bioprinting because it is a printable and biocompatible biomaterial. However, it is difficult to print GelMA bioink without any temperature control because it has a thermally-sensitive rheological property. Therefore, in this study, we developed a temperature-controlled printing system in real time without affecting the viability of the cells encapsulated in the bioink. In addition, a skin-derived decellularized extracellular matrix (SdECM) was printed with GelMA to better mimic the native tissue environment compared with solely using GelMA bioink with the enhancement of structural stability. The temperature setting accuracy was calculated to be 98.58 ± 1.8 % for the module and 99.48 ± 1.33 % for the plate from 5 °C to 37 °C. The group of the temperature of the module at 10 °C and the plate at 20 °C have 93.84 % cell viability with the printable range in the printability window. In particular, the cell viability and proliferation were increased in the encapsulated fibroblasts in the GelMA/SdECM bioink, relative to the GelMA bioink, with a morphology that significantly spread for seven days. The gene expression and growth factors related to skin tissue regeneration were relatively upregulated with SdECM components. In the bioprinting process, the rheological properties of the GelMA/SdECM bioink were successfully adjusted in real time to increase printability, and the native skin tissue mimicked components providing tissue-specific biofunctions to the encapsulated cells. The developed bioprinting strategies and bioinks could support future studies related to the skin tissue reconstruction, regeneration, and other medical applications using the bioprinting process.

3D-printed versatile biliary stents with nanoengineered surface for anti-hyperplasia and antibiofilm formation.

Biliary strictures are characterized by the narrowing of the bile duct lumen, usually caused by surgical biliary injury, cancer, inflammation, and scarring from gallstones. Endoscopic stent placement is a well-established method for the management of biliary strictures. However, maintaining optimal mechanical properties of stents and designing surfaces that can prevent stent-induced tissue hyperplasia and biofilm formation are challenges in the fabrication of biodegradable biliary stents (BBSs) for customized treatment. This study proposes a novel approach to fabricating functionalized polymer BBSs with nanoengineered surfaces using 3D printing. The 3D printed stents, fabricated from bioactive silica poly(ε-carprolactone) (PCL) via a sol-gel method, exhibited tunable mechanical properties suitable for supporting the bile duct while ensuring biocompatibility. Furthermore, a nanoengineered surface layer was successfully created on a sirolimus (SRL)-coated functionalized PCL (fPCL) stent using Zn ion sputtering-based plasma immersion ion implantation (S-PIII) treatment to enhance the performance of the stent. The nanoengineered surface of the SRL-coated fPCL stent effectively reduced bacterial responses and remarkably inhibited fibroblast proliferation and initial burst release of SRL in vitro systems. The physicochemical properties and biological behaviors, including in vitro biocompatibility and in vivo therapeutic efficacy in the rabbit bile duct, of the Zn-SRL@fPCL stent demonstrated its potential as a versatile platform for clinical applications in bile duct tissue engineering.

Longitudinal long term follow up investigation on the carcinogenic impact of polyhexamethylene guanidine phosphate in rat models.

Polyhexamethylene guanidine phosphate (PHMG-p) is a major component in humidifier disinfectants, which cause life-threatening lung injuries. However, to our knowledge, no published studies have investigated associations between PHMG-p dose and lung damage severity with long-term follow-up. Therefore, we evaluated longitudinal dose-dependent changes in lung injuries using repeated chest computed tomography (CT). Rats were exposed to low (0.2 mg/kg, n = 10), intermediate (1.0 mg/kg, n = 10), and high (5.0 mg/kg, n = 10) doses of PHMG-p. All rats underwent repeated CT scans after 10 and 40 weeks following the first exposure. All CT images were quantitatively analyzed using commercial software. Inflammation/fibrosis and tumor counts underwent histopathological evaluation. In both radiological and histopathologic results, the lung damage severity increased as the PHMG-p dose increased. Moreover, the number, size, and malignancy of the lung tumors increased as the dose increased. Bronchiolar-alveolar hyperplasia developed in all groups. During follow-up, there was intergroup variation in bronchiolar-alveolar hyperplasia progression, although bronchiolar-alveolar adenomas or carcinomas usually increase in size over time. Thirty-three carcinomas were detected in the high-dose group in two rats. Overall, lung damage from PHMG-p and the number and malignancy of lung tumors were shown to be dose-dependent in a rat model using repeated chest CT scans during a long-term follow-up.

Highly gallol-substituted, rapidly self-crosslinkable, and robust chitosan hydrogel for 3D bioprinting.

Although three-dimensional (3D) bioprinting is a promising technology for reconstructing artificial tissues and organs using bioink, there is a lack of a bioink that satisfies all requirements, including printability, gelation, mechanical properties, and cytocompatibility, Herein, a novel self-crosslinkable bioink derived from chitosan (CS) and gallic acid (GA) is presented. 3D printed scaffolds with excellent shape fidelity are realized by systematically analyzing the self-crosslinking mechanism of hydrogel formation from CS-GA conjugates and by optimizing various parameters of the printing process. The CS-GA hydrogel forms rapidly in a physiological pH without any chemical crosslinking agent. In addition, the CS-GA hydrogel exhibited various physical and chemical intermolecular interactions, fast gelation rates, and excellent mechanical properties (>337 kPa). Moreover, the CS-GA hydrogel singificantly improves the cell viability (>92 %) and proliferation of the bioink. Therefore, the self-crosslinkable CS-GA bioink has great potential to overcome the limitations of conventional bioinks.

Peptide derived from stromal cell-derived factor 1δ enhances the in vitro expression of osteogenic proteins via bone marrow stromal cell differentiation and promotes bone formation in in vivo models.

Mesenchymal stem cells (MSCs) rely on chemokines and chemokine receptors to execute their biological and physiological functions. Stromal cell-derived factor-1 (SDF-1) is upregulated in injury sites, where it acts as a chemotactic agent, attracting CXCR4-expressing MSCs, which play a pivotal role in the healing and regeneration of tissue throughout the body. Furthermore, SDF-1 expression has been observed in regions experiencing inflammation-induced bone destruction and fracture sites. In this study, we identified a novel peptide called bone-forming peptide-5 (BFP-5), derived from SDF-1δ, which can promote the osteogenesis of MSCs as well as bone formation and healing. Multipotent bone marrow stromal cells treated with BFP-5 showed enhanced alizarin red S staining and higher alkaline phosphatase (ALP) activity. Moreover, ALP and osterix proteins were more abundantly expressed when cells were treated with BFP-5 than SDF-1α. Histology and microcomputed tomography data at 12 weeks demonstrated that both rabbit and goat models transplanted with polycaprolactone (PCL) scaffolds coated with BFP-5 showed significantly greater bone formation than animals transplanted with PCL scaffolds alone. These findings suggest that BFP-5 could be useful in the development of related therapies for conditions associated with bones.

Assessment of Esophageal Reconstruction via Bioreactor Cultivation of a Synthetic Scaffold in a Canine Model.

OBJECTIVES: Using tissue-engineered materials for esophageal reconstruction is a technically challenging task in animals that requires bioreactor training to enhance cellular reactivity. There have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in initial epithelialization in the special environment of peristalsis. The purpose of this study was to evaluate the potential of an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a double-layered polymeric scaffold and a custom-designed mesenchymal stem cell-based bioreactor system in a canine model. METHODS: We fabricated a novel double-layered scaffold as a tissue-engineered esophagus using an electrospinning technique. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. After 3 days of cultivation using the bioreactor system, tissue-engineered artificial esophagus was transplanted into a partial esophageal defect (5×3 cm-long resection) in a canine model. RESULTS: Scanning electron microscopy (SEM) showed that the electrospun fibers in a tubular scaffold were randomly and circumferentially located toward the inner and outer surfaces. Complete recovery of the esophageal mucosa was confirmed by endoscopic analysis and SEM. Esophagogastroduodenoscopy and computed tomography also showed that there were no signs of leakage or stricture and that there was a normal lumen with complete epithelialization. Significant regeneration of the mucosal layer was observed by keratin-5 immunostaining. Alpha-smooth muscle actin immunostaining showed significantly greater esophageal muscle regeneration at 12 months than at 6 months. CONCLUSION: Custom-designed bioreactor cultured electrospun polyurethane scaffolds can be a promising approach for esophageal tissue engineering.

A bioactive microparticle-loaded osteogenically enhanced bioprinted scaffold that permits sustained release of BMP-2.

Extrusion-based bioprinting technology is widely used for tissue regeneration and reconstruction. However, the method that uses only hydrogel as the bioink base material exhibits limited biofunctional properties and needs improvement to achieve the desired tissue regeneration. In this study, we present a three-dimensionally printed bioactive microparticle-loaded scaffold for use in bone regeneration applications. The unique structure of the microparticles provided sustained release of growth factor for > 4 weeks without the use of toxic or harmful substances. Before and after printing, the optimal particle ratio in the bioink for cell viability demonstrated a survival rate of ≥ 85% over 7 days. Notably, osteogenic differentiation and mineralization-mediated by human periosteum-derived cells in scaffolds with bioactive microparticles-increased over a 2-week interval. Here, we present an alternative bioprinting strategy that uses the sustained release of bioactive microparticles to improve biofunctional properties in a manner that is acceptable for clinical bone regeneration applications.

NK cells encapsulated in micro/macropore-forming hydrogels via 3D bioprinting for tumor immunotherapy.

BACKGROUND: Patients face a serious threat if a solid tumor leaves behind partial residuals or cannot be completely removed after surgical resection. Immunotherapy has attracted attention as a method to prevent this condition. However, the conventional immunotherapy method targeting solid tumors, that is, intravenous injection, has limitations in homing in on the tumor and in vivo expansion and has not shown effective clinical results. METHOD: To overcome these limitations, NK cells (Natural killer cells) were encapsulated in micro/macropore-forming hydrogels using 3D bioprinting to target solid tumors. Sodium alginate and gelatin were used to prepare micro-macroporous hydrogels. The gelatin contained in the alginate hydrogel was removed because of the thermal sensitivity of the gelatin, which can generate interconnected micropores where the gelatin was released. Therefore, macropores can be formed through bioprinting and micropores can be formed using thermally sensitive gelatin to make macroporous hydrogels. RESULTS: It was confirmed that intentionally formed micropores could help NK cells to aggregate easily, which enhances cell viability, lysis activity, and cytokine release. Macropores can be formed using 3D bioprinting, which enables NK cells to receive the essential elements. We also characterized the functionality of NK 92 and zEGFR-CAR-NK cells in the pore-forming hydrogel. The antitumor effects on leukemia and solid tumors were investigated using an in vitro model. CONCLUSION: We demonstrated that the hydrogel encapsulating NK cells created an appropriate micro-macro environment for clinical applications of NK cell therapy for both leukemia and solid tumors via 3D bioprinting. 3D bioprinting makes macro-scale clinical applications possible, and the automatic process shows potential for development as an off-the-shelf immunotherapy product. This immunotherapy system could provide a clinical option for preventing tumor relapse and metastasis after tumor resection. Micro/macropore-forming hydrogel with NK cells fabricated by 3D bioprinting and implanted into the tumor site.

A wearable colorimetric sweat pH sensor-based smart textile for health state diagnosis.

Sweat pH is an important indicator for diagnosing disease states, such as cystic fibrosis. However, conventional pH sensors are composed of large brittle mechanical parts and need additional instruments to read signals. These pH sensors have limitations for practical wearable applications. In this study, we propose wearable colorimetric sweat pH sensors based on curcumin and thermoplastic-polyurethane (C-TPU) electrospun-fibers to diagnose disease states by sweat pH monitoring. This sensor aids in pH monitoring by changing color in response to chemical structure variation from enol to di-keto form via H-atom separation. Its chemical structure variation changes the visible color due to light absorbance and reflectance changes. Furthermore, it can rapidly and sensitively detect sweat pH due to its superior permeability and wettability. By O2 plasma activation and thermal pressing, this colorimetric pH sensor can be easily attached to various fabric substrates such as swaddling and patient clothing via surface modification and mechanical interlocking of C-TPU. Furthermore, the diagnosable clothing is durable and reusable enough to neutral washing conditions due to the reversible pH colorimetric sensing performance by restoring the enol form of curcumin. This study contributes to the development of smart diagnostic clothing for cystic fibrosis patients who require continuous sweat pH monitoring.

Correction: A wearable colorimetric sweat pH sensor-based smart textile for health state diagnosis.

Correction for 'A wearable colorimetric sweat pH sensor-based smart textile for health state diagnosis' by Ji-Hwan Ha et al., Mater. Horiz., 2023, 10, 4163-4171, https://doi.org/10.1039/d3mh00340j.

Generation of human induced pluripotent stem cells from osteoarthritis patient-derived synovial cells.

OBJECTIVE: This study was undertaken to generate and characterize human induced pluripotent stem cells (PSCs) from patients with osteoarthritis (OA) and to examine whether these cells can be developed into disease-relevant cell types for use in disease modeling and drug discovery. METHODS: Human synovial cells isolated from two 71-year-old women with advanced OA were characterized and reprogrammed into induced PSCs by ectopic expression of 4 transcription factors (Oct-4, SOX2, Klf4, and c-Myc). The pluripotency status of each induced PSC line was validated by comparison with human embryonic stem cells (ESCs). RESULTS: We found that OA patient-derived human synovial cells had human mesenchymal stem cell (MSC)-like characteristics, as indicated by the expression of specific markers, including CD14-, CD19-, CD34-, CD45-, CD44+, CD51+, CD90+, CD105+, and CD147+. Microarray analysis of human MSCs and human synovial cells further determined their unique and overlapping gene expression patterns. The pluripotency of established human induced PSCs was confirmed by their human ESC-like morphology, expression of pluripotency markers, gene expression profiles, epigenetic status, normal karyotype, and in vitro and in vivo differentiation potential. The potential of human induced PSCs to differentiate into distinct mesenchymal cell lineages, such as osteoblasts, adipocytes, and chondrocytes, was further confirmed by positive expression of markers for respective cell types and positive staining with alizarin red S (osteoblasts), oil red O (adipocytes), or Alcian blue (chondrocytes). Functional chondrocyte differentiation of induced PSCs in pellet culture and 3-dimensional polycaprolactone scaffold culture was assessed by chondrocyte self-assembly and histology. CONCLUSION: Our findings indicate that patient-derived synovial cells are an attractive source of MSCs as well as induced PSCs and have the potential to advance cartilage tissue engineering and cell-based models of cartilage defects.

Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model.

The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects.

An osteogenic bioink composed of alginate, cellulose nanofibrils, and polydopamine nanoparticles for 3D bioprinting and bone tissue engineering.

Bioprinting is an emerging technology for manufacturing cell-laden three-dimensional (3D) scaffolds, which are used to fabricate complex 3D constructs and provide specific microenvironments for supporting cell growth and differentiation. The development of bioinks with appropriate printability and specific bioactivities is crucial for bioprinting and tissue engineering applications, including bone tissue regeneration. Therefore, to produce functional bioinks for osteoblast printing and bone tissue formation, we formulated various nanocomposite hydrogel-based bioinks using natural and biocompatible biomaterials (i.e., alginate, tempo-oxidized cellulose nanofibrils (TOCNF), and polydopamine nanoparticles (PDANPs)). Rheological studies and printability tests revealed that bioinks containing 1.5% alginate and 1.5% TOCNF in the presence or absence of PDANP (0.5%) are suitable for 3D printing. Furthermore, in vitro studies of 3D-printed osteoblast-laden scaffolds indicated that the 0.5% PDANP-incorporated bioink induced significant osteogenesis. Overall, the bioink consisting of alginate, TOCNF, and PDANPs exhibited excellent printability and bioactivity (i.e., osteogenesis).