Three-dimensional Bioprinting Research Articles

Three-Dimensional Bioprinting of Tarsal Plates with Adipose-Derived Mesenchymal Stem Cells: Evaluation of Meibomian Gland Reconstruction in a Rat Model

Background: The aim of this study was to develop 3D-bioprinted scaffolds embedded with human adipose-derived stem cells (hADSCs) to reconstruct the tarsal plate in a rat model. Methods: Scaffolds were printed using a 3D bioprinter with a bioink composed of atelocollagen and alginate. hADSCs (5 × 105 cells/mL) were embedded within the bioink. A total of 30 male Sprague Dawley (SD) rats (300 g) were divided into three groups: group 1 (normal control, n = 10), group 2 (3D-bioprinted scaffolds, n = 10), and group 3 (3D-bioprinted scaffolds with hADSCs, n = 10). Four weeks after surgery, a histopathological analysis was performed using hematoxylin and eosin (H&E) staining, Masson’s trichrome (MT) staining, and immunofluorescence staining. Gene expression of SREBP-1, PPAR-γ, FADS-2, and FAS was assessed via real-time polymerase chain reaction (PCR). Results: No abnormalities were observed in the operated eyelids of any of the 30 rats. The histopathological analysis revealed lipid-secreting cells resembling meibocytes in both group 2 and group 3, with more pronounced meibocyte-like cells in group 3. Immunofluorescence staining for phalloidin expression showed a significant increase in group 3. Additionally, the RNA expression of SREBP-1, PPAR-γ, FADS-2, and FAS, all related to lipid metabolism, was elevated in group 3. Conclusions: The 3D-printed scaffolds combined with hADSCs were effective for tarsal plate reconstruction, with the hADSCs notably contributing to the generation of cells associated with lipid metabolism.

Comparison study on hyaline cartilage versus fibrocartilage formation in a pig model by using 3D-bioprinted hydrogel and hybrid constructs

Cartilage tissue engineering (CTE) with the help of engineered constructs has shown promise for the regeneration of hyaline cartilage, where fibrocartilage may also be formed due to the biomechanical loading resulting from the host weight and movement. Previous studies have primarily reported on hyaline cartilage formationin vitroand/or in small animals, while leaving the fibrocartilage formation undiscovered. In this paper, we, at the first time, present a comparison study on hyaline cartilage versus fibrocartilage formation in a large animal model of pig by using two constructs (namely hydrogel and hybrid ones) engineered by means of three-dimensional (3D) bioprinting. Both hydrogel and hybrid constructs were printed from the bioink of alginate (2.5%) and ATDC5 cells (chondrogenic cells at a cell density of 5 × 106cells ml-1), with the difference in that in the hybrid construct, there was a polycaprolactone (PCL) strand printed between every two bioink strands, which were strategically designed to shield the force imposed on the cells within the bioink strands. Both hydrogel and hybrid constructs were implanted into the chondral defects created in the articular cartilage of weight-bearing portions of pig stifle joints; the cartilage formation was examined at one- and three-months post-implantation, respectively, by means of Safranin O, Trichrome, immunofluorescent staining, and synchrotron radiation-based (SR) inline phase contrast imaging microcomputed tomography (inline-PCI-CT). Glycosaminoglycan (GAG) and collagen type II (Col II) secretion were used to evaluate the hyaline cartilage formation, while collagen type I (Col I) was used to indicate fibrocartilage given that Col I is low in hyaline cartilage but high in fibrocartilage. Our results revealed that cartilage formation was enhanced over time in both hydrogel and hybrid constructs; particularly, the hydrogel construct exhibited more cartilage formation at both one- and three-months post-implantation, while hybrid constructs tended to have less fibrocartilage formed in a long time period. Also, the result from the inline-PCI-CT revealed that the inline-PCI-CT was able to provide not only the information seen in other histology images, but also high-resolution details of biomaterials and regenerating cartilage. This would represent a significant advance toward the non-invasive assessment of cartilage formation regeneration within large animal models and eventually in human patients.

3D printed collagen scaffold for heart valve repair

Three-dimensional (3D) bioprinting has emerged as a promising approach for the development of functional tissues and organs, including the heart valves. In this study, we investigated the interaction of 3D printed collagen scaffolds with H9c2(2–1) and NIH/3T3 cells to improve heart-valve repair strategies. Type I collagen was extracted from rat tails, characterized using SDS-PAGE and Raman spectroscopy, and used as a biomaterial ink for 3D printing. The rheological properties were evaluated. The FRESH technique was used to support the printed construct. In vitro assessments were performed to determine the cell viability and distribution within the scaffold. These results demonstrated the successful extraction and characterization of Type I collagen, which exhibited suitable rheological properties for 3D bioprinting. The printed collagen scaffolds supported the growth and distribution of H9c2(2–1) and NIH/3T3 cells, indicating their potential application in heart valve repair. This study highlights the importance of collagen as a biomaterial in 3D bioprinting and provides insights into the development of advanced strategies for heart valve repair.

3D Bioprinting in Cancer Modeling and Biomedicine: From Print Categories to Biological Applications.

The continuous interaction between tumor cells and the local microenvironment plays a decisive role in tumor development. Selecting effective models to simulate the tumor microenvironment to study the physiological processes of tumorigenesis and progression is extremely important and challenging. Currently, three-dimensional (3D) bioprinting technology makes it possible to replicate a physiologically relevant tumor microenvironment and induce genomic and proteomic expression to better mimic tumors in vivo. Meanwhile, it plays a crucial role in the prevention and treatment of human diseases, contributing to drug delivery and drug screening, tissue development and regenerative medicine. This paper provides an overview of the categories of 3D bioprinting technology, and the recent advances in the bioinks required for printing. In addition, we summarize the current tumor models based on 3D bioprinting and provide an assessment of possible future biological applications.

Heterotopically differentiated PDLSCs-laden 3D bioprinting scaffolds for concurrent oral hard and soft tissue regeneration

Following dental extraction, the alveolar bone and gingival tissues could undergo varying degrees of resorption, affecting subsequent implant integration and aesthetic outcomes. Adequate volume of both hard and soft tissues is essential for optimal results. Three-dimensional (3D) bioprinting technology offers the advantages of biomimicry, personalization, and precise spatial distribution, which are pivotal for enhancing the success and esthetics of dental restorations. In this study, we fabricated a construct with a natural transition and varying material concentrations by 3D bioprinting, comprising an upper layer of Collagen/Alginate/periodontal ligament stem cells (PDLSCs) and a lower layer of Collagen/nano-hydroxyapatite (nHA)/Alginate/PDLSCs. Characterization of the physicochemical properties revealed that the incorporation of nHA significantly enhanced the mechanical properties of both the bioink and the construct. Flow cytometry analysis confirmed the stemness of PDLSCs. Scanning electron microscopy (SEM) revealed that the construct possesses satisfactory pore density and a natural transition at the stratification point. The construct displayed good cell viability and proliferation, with the cellular movement observed at the stratification interface after bioprinting. Differentiation staining and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) results demonstrated that PDLSCs within the 3D construct are capable of both osteogenic and fibroblastic differentiations. Ectopic transplantation in mice confirmed the biocompatibility of the construct. A rat tooth extraction model validated the construct's effectiveness in the integrated regeneration of both hard and soft tissues in alveolar ridge preservation (ARP). In conclusion, this personalized, concentration-varied 3D construct exhibits excellent biocompatibility and tissue preservation effects, holding significant potential for clinical application.  

3D Nanofiber-Assisted Embedded Extrusion Bioprinting for Oriented Cardiac Tissue Fabrication.

Three-dimensional (3D) bioprinting technology stands out as a promising tissue manufacturing process to control the geometry precisely with cell-loaded bioinks. However, the isotropic culture environment within the bioink and the lack of topographical cues impede the formation of oriented cardiac tissue. To overcome this limitation, we present a novel method named 3D nanofiber-assisted embedded bioprinting (3D-NFEP) to fabricate cardiac tissue with an oriented morphology. Aligned 3D nanofiber scaffolds were fabricated by divergence electrospinning, which provided structural support for printing of the low-viscosity bioink and structural induction to cardiomyocytes. Cells adhered to the aligned fibers after hydrogel degradation, and a high degree of cell alignment was observed. This technology was also demonstrated as a feasible solution for multilayer cell printing. Therefore, 3D-NFEP was demonstrated as a promising method for bioprinting oriented cardiac tissue with low-viscosity bioink and is expected to be applied for structured and cardiac tissue engineering.

Advances and Challenges of Bioassembly Strategies in Neurovascular In Vitro Modeling: An Overview of Current Technologies with a Focus on Three-Dimensional Bioprinting.

Bioassembly encompasses various techniques such as bioprinting, microfluidics, organoids, and self-assembly, enabling advances in tissue engineering and regenerative medicine. Advancements in bioassembly technologies have enabled the precise arrangement and integration of various cell types to more closely mimic the complexity functionality of the neurovascular unit (NVU) and that of other biodiverse multicellular tissue structures. In this context, bioprinting offers the ability to deposit cells in a spatially controlled manner, facilitating the construction of interconnected networks. Scaffold-based assembly strategies provide structural support and guidance cues for cell growth, enabling the formation of complex bio-constructs. Self-assembly approaches utilize the inherent properties of cells to drive the spontaneous organization and interaction of neuronal and vascular components. However, recreating the intricate microarchitecture and functional characteristics of a tissue/organ poses additional challenges. Advancements in bioassembly techniques and materials hold great promise for addressing these challenges. The further refinement of bioprinting technologies, such as improved resolution and the incorporation of multiple cell types, can enhance the accuracy and complexity of the biological constructs; however, developing bioinks that support the growth of cells, viability, and functionality while maintaining compatibility with the bioassembly process remains an unmet need in the field, and further advancements in the design of bioactive and biodegradable scaffolds will aid in controlling cell adhesion, differentiation, and vascularization within the engineered tissue. Additionally, integrating advanced imaging and analytical techniques can provide real-time monitoring and characterization of bioassembly, aiding in quality control and optimization. While challenges remain, ongoing research and technological advancements propel the field forward, paving the way for transformative developments in neurovascular research and tissue engineering. This work provides an overview of the advancements, challenges, and future perspectives in bioassembly for fabricating neurovascular constructs with an add-on focus on bioprinting technologies.

Gel-Based Suspension Medium Used in 3D Bioprinting for Constructing Tissue/Organ Analogs.

Constructing tissue/organ analogs with natural structures and cell types in vitro offers a valuable strategy for the in situ repair of damaged tissues/organs. Three-dimensional (3D) bioprinting is a flexible method for fabricating these analogs. However, extrusion-based 3D bioprinting faces the challenge of balancing the use of soft bioinks with the need for high-fidelity geometric shapes. To address these challenges, recent advancements have introduced various suspension mediums based on gelatin, agarose, and gellan gum microgels. The emergence of these gel-based suspension mediums has significantly advanced the fabrication of tissue/organ constructs using 3D bioprinting. They effectively stabilize and support soft bioinks, enabling the formation of complex spatial geometries. Moreover, they provide a stable, cell-friendly environment that maximizes cell viability during the printing process. This minireview will summarize the properties, preparation methods, and potential applications of gel-based suspension mediums in constructing tissue/organ analogs, while also addressing current challenges and providing an outlook on the future of 3D bioprinting.

3D bioprinting of fish skin-based gelatin methacryloyl (GelMA) bio-ink for use as a potential skin substitute.

Gelatin methacryloyl (GelMA), typically derived from mammalian sources, has recently emerged as an ideal bio-ink for three-dimensional (3D) bioprinting. Herein, we developed a fish skin-based GelMA bio-ink for the fabrication of a 3D GelMA skin substitute with a 3D bioprinter. Several concentrations of methacrylic acid anhydride were used to fabricate GelMA, in which their physical-mechanical properties were assessed. This fish skin-based GelMA bio-ink was loaded with human adipose tissue-derived mesenchymal stromal cells (ASCs) and human platelet lysate (HPL) and then printed to obtain 3D ASCs + HPL-loaded GelMA scaffolds. Cell viability test and a preliminary investigation of its effectiveness in promoting wound closure were evaluated in a critical-sized full thickness skin defect in a rat model. The cell viability results showed that the number of ASCs increased significantly within the 3D GelMA hydrogel scaffold, indicating its biocompatibility property. In vivo results demonstrated that ASCs + HPL-loaded GelMA scaffolds could delay wound contraction, markedly enhanced collagen deposition, and promoted the formation of new blood vessels, especially at the wound edge, compared to the untreated group. Therefore, this newly fish skin-based GelMA bio-ink developed in this study has the potential to be utilized for the printing of 3D GelMA skin substitutes.

Revolutionizing Intervertebral Disc Regeneration: Advances and Future Directions in Three-Dimensional Bioprinting of Hydrogel Scaffolds.

Hydrogels are multifunctional platforms. Through reasonable structure and function design, they use material engineering to adjust their physical and chemical properties, such as pore size, microstructure, degradability, stimulus-response characteristics, etc. and have a variety of biomedical applications. Hydrogel three-dimensional (3D) printing has emerged as a promising technique for the precise deposition of cell-laden biomaterials, enabling the fabrication of intricate 3D structures such as artificial vertebrae and intervertebral discs (IVDs). Despite being in the early stages, 3D printing techniques have shown great potential in the field of regenerative medicine for the fabrication of various transplantable tissues within the human body. Currently, the utilization of engineered hydrogels as carriers or scaffolds for treating intervertebral disc degeneration (IVDD) presents numerous challenges. However, it remains an indispensable multifunctional manufacturing technology that is imperative in addressing the escalating issue of IVDD. Moreover, it holds the potential to serve as a micron-scale platform for a diverse range of applications. This review primarily concentrates on emerging treatment strategies for IVDD, providing an in-depth analysis of their merits and drawbacks, as well as the challenges that need to be addressed. Furthermore, it extensively explores the biological properties of hydrogels and various nanoscale biomaterial inks, compares different prevalent manufacturing processes utilized in 3D printing, and thoroughly examines the potential clinical applications and prospects of integrating 3D printing technology with hydrogels.

On the Relation between the Viscoelastic Properties of Granular Hydrogels and Their Performance as Support Materials in Embedded Bioprinting.

Granular hydrogels, formed by jamming microgels suspension, are promising materials for three-dimensional bioprinting applications. Despite their extensive use as support materials for embedded bioprinting, the influence of the particle's physical properties on the macroscale viscoelasticity on one hand and on the printing performance on the other hand remains unclear. Herein, we investigate the linear and nonlinear rheology of κ-carrageenan granular hydrogel through small- and large-amplitude oscillatory shear measurements. We tuned the granular hydrogel's properties by changing the stiffness (soft, medium, stiff) and the packing density of the individual microgels. Characterizations in the linear viscoelasticity regime revealed that the storage modulus of granular hydrogels is not a simple function of microgel stiffness and depends on the microgel packing density. At larger strains, increasing the microgel stiffness reduced the energy dissipation of the granular beds and increased the solid-fluid transition point. To understand how the different rheological properties of granular support materials influence embedded bioprinting, we examined the printing fidelity and cellular filament shrinkage within the granular beds. We found that microgels with low packing density diminished the printing quality, while stiff microgels promoted filament roughness. In addition, we found that highly packed stiff microgels significantly reduced the postprinting contraction of cellular filaments. Overall, this work provides a comprehensive knowledge of the rheology of granular hydrogels that can be used to rationally design support beds for bioprinting applications with specific characteristics.

In vitro vascularization of 3D cell aggregates in microwells with integrated vascular beds

Most human tissues possess vascular networks supplying oxygen and nutrients. Engineering of functional tissue and organ models or equivalents often require the integration of artificial vascular networks. Several approaches, such as organs on chips and three-dimensional (3D) bioprinting, have been pursued to obtain vasculature and vascularized tissues in vitro. This technical feasibility study proposes a new approach for the in vitro vascularization of 3D microtissues. For this, we thermoform arrays of round-bottom microwells into thin non-porous and porous polymer films/membranes and culture vascular beds on them from which endothelial sprouting occurs in a Matrigel-based 3D extra cellular matrix. We present two possible culture configurations for the microwell-integrated vascular beds. In the first configuration, human umbilical vein endothelial cells (HUVECs) grow on and sprout from the inner wall of the non-porous microwells. In the second one, HUVECs grow on the outer surface of the porous microwells and sprout through the pores toward the inside. These approaches are extended to lymphatic endothelial cells. As a proof of concept, we demonstrate the in vitro vascularization of spheroids from human mesenchymal stem cells and MG-63 human osteosarcoma cells. Our results show the potential of this approach to provide the spheroids with an abundant outer vascular network and the indication of an inner vasculature.

3D Bioprinting Using Universal Fugitive Network Bioinks.

Three-dimensional (3D) bioprinting has emerged with potential for creating functional 3D tissues with customized geometries. However, the limited availability of bioinks capable of printing 3D structures with both high-shape fidelity and desired biological environments for encapsulated cells remains a key challenge. Here, we present a 3D bioprinting approach that uses universal fugitive network bioinks prepared by loading cells and hydrogel precursors (bioink base materials) into a 3D printable fugitive carrier. This approach constructs 3D structures of cell-encapsulated hydrogels by printing 3D structures using fugitive network bioinks, followed by cross-linking printed structures and removing the carrier from them. The use of the fugitive carrier decouples the 3D printability of bioinks from the material properties of bioink base materials, making this approach readily applicable to a range of hydrogel systems. The decoupling also enables the design of bioinks for the biological functionality of the final printed constructs without compromising the 3D printability. We demonstrate the generalizable 3D printability by printing self-supporting 3D structures of various hydrogels, including conventionally non-3D printable hydrogels and those with a low polymer content. We conduct preprinting screening of bioink base materials through 3D cell culture to select bioinks with high cell compatibility. The selected bioinks produce 3D constructs of cell-encapsulated hydrogels with both high-shape fidelity and high cell viability and proliferation. The universal fugitive network bioink platform could significantly expand 3D printable bioinks with customizable biological functionalities and the adoption of 3D bioprinting in diverse research and applied settings.

Three-Dimensional Bioprinting as a Tool for Tissue Engineering: A Review

Three-Dimensional Bioprinting as a Tool for Tissue Engineering: A Review

Innovative Strategies in 3D Bioprinting for Spinal Cord Injury Repair.

Spinal cord injury (SCI) is a catastrophic condition that disrupts neurons within the spinal cord, leading to severe motor and sensory deficits. While current treatments can alleviate pain, they do not promote neural regeneration or functional recovery. Three-dimensional (3D) bioprinting offers promising solutions for SCI repair by enabling the creation of complex neural tissue constructs. This review provides a comprehensive overview of 3D bioprinting techniques, bioinks, and stem cell applications in SCI repair. Additionally, it highlights recent advancements in 3D bioprinted scaffolds, including the integration of conductive materials, the incorporation of bioactive molecules like neurotrophic factors, drugs, and exosomes, and the design of innovative structures such as multi-channel and axial scaffolds. These innovative strategies in 3D bioprinting can offer a comprehensive approach to optimizing the spinal cord microenvironment, advancing SCI repair. This review highlights a comprehensive understanding of the current state of 3D bioprinting in SCI repair, offering insights into future directions in the field of regenerative medicine.

3D Bioprinting of Engineered Living Materials with Extracellular Electron Transfer Capability for Water Purification.

Attention is widely drawn to the extracellular electron transfer (EET) process of electroactive bacteria (EAB) for water purification, but its efficacy is often hindered in complex environmental matrices. In this study, the engineered living materials with EET capability (e-ELMs) were for the first time created with customized geometric configurations for pollutant removal using three-dimensional (3D) bioprinting platform. By combining EAB and tailored viscoelastic matrix, a biocompatible and tunable electroactive bioink for 3D bioprinting was initially developed with tuned rheological properties, enabling meticulous manipulation of microbial spatial arrangement and density. e-ELMs with different spatial microstructures were then designed and constructed by adjusting the filament diameter and orientation during the 3D printing process. Simulations of diffusion and fluid dynamics collectively showcase internal mass transfer rates and EET efficiency of e-ELMs with different spatial microstructures, contributing to the outstanding decontamination performances. Our research propels 3D bioprinting technology into the environmental realm, enabling the creation of intricately designed e-ELMs and providing promising routes to address the emerging water pollution concerns.

A biomimetic skin microtissue biosensor for the detection of fish parvalbumin

In this paper, a biomimetic skin microtissue biosensor was developed based on three-dimensional (3D) bioprinting to precisely and accurately determine fish parvalbumin (FV). Based on the principle that allergens stimulate cells to produce ONOO− (peroxynitrite anion), a screen-printed electrode for the detection nanomolar level ONOO− was innovatively prepared to indirectly detect FV based on the level of ONOO− release. Gelatin methacryloyl (GelMA), RBL-2H3 cells, and MS1 cells were used as bio-ink for 3D bioprinting. The high-throughput and standardized preparation of skin microtissue was achieved using stereolithography 3D bioprinting technology. The printed skin microtissues were put into the self-designed 3D platform that integrated cell culture and electrochemical detection. The experimental results showed that the sensor could effectively detect FV when the optimized ratio of RBL-2H3 to MS1 cells and allergen stimulation time were 2:8 and 2 h, respectively. The linear detection range was 0.125–3.0 μg/mL, and the calculated lowest detection limit was 0.122 μg/mL. In addition, the sensor had excellent selectivity, specificity, stability, and reliability. Thus, this study successfully constructed a biomimetic skin microtissue electrochemical sensor for PV detection.

Developing Patient-Derived 3D-Bioprinting models of pancreatic cancer

IntroductionPancreatic cancer (PC) remains a challenging malignancy, and adjuvant chemotherapy is critical in improving patient survival post-surgery. However, the intrinsic heterogeneity of PC necessitates personalized treatment strategies, highlighting the need for reliable preclinical models. ObjectivesThis study aimed to develop novel patient-derived preclinical PC models using three-dimensional bioprinting (3DP) technology. MethodsPatient-derived PC models were established using 3DP technology. Genomic and histological analyses were performed to characterize these models and compare them with corresponding patient tissues. Chemotherapeutic drug sensitivity tests were conducted on the PC 3DP models, and correlations with clinical outcomes were analyzed. ResultsThe study successfully established PC 3DP models with a modeling success rate of 86.96%. These models preserved genomic and histological features consistent with patient tissues. Drug sensitivity testing revealed significant heterogeneity among PC 3DP models, mirroring clinical variability, and potential correlations with clinical outcomes. ConclusionThe PC 3DP models demonstrated their utility as reliable preclinical tools, retaining key genomic and histological characteristics. Importantly, drug sensitivity profiles in these models showed potential correlations with clinical outcomes, indicating their promise in customizing treatment strategies and predicting patient prognoses. Further validation with larger patient cohorts is warranted to confirm their potential clinical utility.

Optimizing biomaterial inks: A study on the printability of Carboxymethyl cellulose-Laponite nanocomposite hydrogels and dental pulp stem cells bioprinting

Tissue engineering approaches require biocompatible materials with precise pre-designed geometry, shape fidelity, and promote cellular functions. Addressing these requirements, our study focused on developing an optimized bioink formulation using carboxymethyl cellulose (CMC) and Laponite hydrogels tailored for extrusion-based three-dimensional bioprinting. To this, we investigated the rheological properties and filament behavior before and during printing. As Laponite concentration increased in CMC solutions, it improved shear-thinning behavior, viscosity, and storage modulus, resulting in well-defined filament characteristics with lower diffusion rates, excellent shape fidelity, and robust printability. Thus, we achieved a suitable biomaterial ink formulation with concentrations of 1 wt% of CMC and 4 wt% of Laponite (1C4L). Subsequently, a statistical analysis guided us to select the optimal parameters for large-scale construct printing: a nozzle speed of 5 mm/s, a print distance of 0.41 mm, and an extrusion multiplier of 1.35. After that, we enhanced the structural integrity of printed hydrogels through ionic crosslinking with calcium chloride (CaCl2) and citric acid (CA), revealing higher-strength hydrogels at higher concentrations of CaCl2. Finally, we have confirmed the groundbreaking potential of our bioink by integrating dental pulp mesenchymal stem cells (DPSC) into the 1C4L ink. Our bioprinted constructs showed optimized swelling, non-toxic effects, and retained excellent shape fidelity, crucial for creating anatomically accurate tissues. Our findings provide crucial insights linking the rheological analysis, the bioprinting process, and the biological properties of hydrogels, paving the way for their use for tissue engineering and other biomedical applications.

Composite bioink incorporating cell-laden liver decellularized extracellular matrix for bioprinting of scaffolds for bone tissue engineering

The field of bone tissue engineering (BTE) has witnessed a revolutionary breakthrough with the advent of three-dimensional (3D) bioprinting technology, which is considered an ideal choice for constructing scaffolds for bone regeneration. The key to realizing scaffold biofunctions is the selection and design of an appropriate bioink, and existing bioinks have significant limitations. In this study, a composite bioink based on natural polymers (gelatin and alginate) and liver decellularized extracellular matrix (LdECM) was developed and used to fabricate scaffolds for BTE using 3D bioprinting. Through in vitro studies, the concentration of LdECM incorporated into the bioink was optimized to achieve printability and stability and to improve the proliferation and osteogenic differentiation of loaded rat bone mesenchymal stem cells (rBMSCs). Furthermore, in vivo experiments were conducted using a Sprague Dawley rat model of critical-sized calvarial defects. The proposed rBMSC-laden LdECM–gelatin–alginate scaffold, bioprinted layer-by-layer, was implanted in the rat calvarial defect and the development of new bone growth was studied for four weeks. The findings showed that the proposed bioactive scaffolds facilitated angiogenesis and osteogenesis at the defect site. The findings of this study suggest that the developed rBMSC-laden LdECM–gelatin–alginate bioink has great potential for clinical translation and application in solving bone regeneration problems.