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Special Section: Bioprinting of 3D Functional Tissue Constructs

Regular Section

Original research article

by Hang Liu, Fan Wu, Renwei Chen, Yanan Chen, Kai Yao, Zengping Liu, Bhav Harshad Parikh, Linzhi Jing, Tiange Liu, Xinyi Su, Jie Sun, Dejian Huang

Age-related macular degeneration (AMD) is the leading cause of visual loss and affects millions of people worldwide. Dysfunction of the retinal pigment epithelium (RPE) is associated with the pathogenesis of AMD. The purpose of this work is to build and evaluate the performance of ultrathin scaffolds with an electrohydrodynamic jet (EHDJ) printing method for RPE cell culture. We printed two types of ultrathin (around 7 μm) polycaprolactone scaffolds with 20 μm and 50 μm pores, which possess mechanical properties resembling that of native human Bruch’s membrane and are biodegradable. Light microscopy and cell proliferation assay showed that adult human retinal pigment epithelial (ARPE-19) cells adhered and proliferated to form a monolayer on the scaffolds. The progress of culture matured on the scaffolds was demonstrated by immunofluorescence (actin, ZO-1, and Na+/K+-ATPase) and Western blot analysis of the respective proteins. The RPE cells cultured on EHDJ-printed scaffolds with 20 μm pores presented higher permeability, higher transepithelial potential difference, and higher expression level of Na+/K+-ATPase than those cultured on Transwell inserts. These findings suggest that the EHDJ printing can fabricate scaffolds that mimic Bruch’s membrane by promoting maturation of RPE cells to form a polarized and functional monolayered epithelium with potential as an in vitro model for studying retinal diseases and treatment methods.

Original research article

by Xiaomin Duan, Wei Wang, Wenping Ma, Zhenhui Mao, Fangliang Xing, Xin Zhao

It is technically challenging for pediatric anesthesiologists to use bronchial blocker (BB) to isolate the lungs of infants during thoracoscopic surgery. Further, BB currently sold in the market cannot match the anatomical characteristics of the infants, especially on the right main bronchus. It may easily cause poor exhaustion of the right upper lobe, which leads to interference with the thoracoscopic surgical field. The two dimensional reconstruction data of 124 normal infants’ airways were extracted from the medical image database of Beijing Children’s Hospital for statistical analysis. After using linear fitting and goodness-of-fit test, a good linear relationship was detected between infant age and various parameters related to aid in designing a new BB for infants (R2=0.502). According to the growth and development rate of infants, the DICOM files of airway CT scan of 7 infants aged 30, 60, 90, 120, 180, 270, and 360 days were selected to print non-transparent convex and transparent concave 3D models. The non-transparent convex model was precisely measured to obtain the important parameters for BB design infants only, to complete the design of BB, to generate the sample, and to verify the blocking effect of produced sample in transparent concave three-dimensional (3D) model.

Original research article

by Reza Noroozi, Farzad Tatar, Ali Zolfagharian, Roberto Brighenti, Mohammad Amin Shamekhi, Abbas Rastgoo, Amin Hadi, Mahdi Bodaghi

Tissue engineering, whose aim is to repair or replace damaged tissues by combining the principle of biomaterials and cell transplantation, is one of the most important and interdisciplinary fields of regenerative medicine. Despite remarkable progress, there are still some limitations in the tissue engineering field, among which designing and manufacturing suitable scaffolds. With the advent of additive manufacturing (AM), a breakthrough happened in the production of complex geometries. In this vein, AM has enhanced the field of bioprinting in generating biomimicking organs or artificial tissues possessing the required porous graded structure. In this study, triply periodic minimal surface structures, suitable to manufacture scaffolds mimicking bone’s heterogeneous nature, have been studied experimentally and numerically; the influence of the printing direction and printing material has been investigated. Various multi-morphology scaffolds, including gyroid, diamond, and I-graph and wrapped package graph (I-WP), with different transitional zone, have been three-dimensional (3D) printed and tested under compression. Further, a micro-computed tomography (μCT) analysis has been employed to obtain the real geometry of printed scaffolds. Finite element analyses have been also performed and compared with experimental results. Finally, the scaffolds’ behavior under complex loading has been investigated based on the combination of μCT and finite element modeling.

Original research article

by Chanh Trung Nguyen, Van Thuy Duong, Chang Ho Hwang, Kyo-in Koo

Rapid construction of pre-vascular structure is highly desired for engineered thick tissue. However, angiogenesis in free-standing scaffold has been rarely reported because of limitation in growth factor (GF) supply into the scaffold. This study, for the 1st time, investigated angiogenic sprouting in free-standing two-vasculature-embedded scaffold with three different culture conditions and additional GFs. A two-core laminar flow device continuously extruded one vascular channel with human umbilical vein endothelial cells (HUVECs) and a 3 mg/ml type-1 collagen, one hollow channel, and a shell layer with 2% w/v gelatin-alginate (70:30) composite. Under the GF flowing condition, angiogenic sprouting from the HUVEC vessel had started since day 1 and gradually grew toward the hollow channel on day 10. Due to the medium flowing, the HUVECs showed elongated spindle-like morphology homogeneously. Their viability has been over 80% up to day 10. This approach could apply to vascular investigation, and drug discovery further, not only to the engineered thick tissue.

Original research article

by Youwen Yang, Chenrong Ling, Mingli Yang, Liuyimei Yang, Dongsheng Wang, Shuping Peng, Cijun Shuai

Magnesium (Mg) degrades too fast in human body, which limits its orthopedic application. Single-phase Mg-based supersaturated solid solution is expected to possess high corrosion resistance. In this work, rare earth scandium (Sc) was used as alloying element to prepare Mg(Sc) solid solution powder by mechanical alloying (MA) and then shaped into implant using selective laser melting (SLM). MA utilizes powerful mechanical force to introduce numerous lattice defects, which promotes the dissolution of Sc in Mg matrix and forms supersaturated solid solution particles. Subsequently, SLM with fast heating and cooling rate maintains the original supersaturated solid solution structure. Immersion tests revealed that high Sc content significantly enhanced the corrosion resistance of Mg matrix because of the formation of protective corrosion product film, which was also proved by the electrochemical impedance spectroscopy measurements. Thereby, Mg(Sc) alloy showed a relatively low degradation rate of 0.61 mm/year. In addition, cell tests showed that the Mg(Sc) exhibited favorable biocompatibility and was suitable for medical application.

Original research article

by Salwa Alshehri, Ram Karan, Sarah Ghalayini, Kowther Kahin, Zainab Khan, Dominik Renn, Sam Mathew, Magnus Rueping, Charlotte A. E. Hauser

Three-dimensional (3D) bioprinting has emerged as a promising method for the engineering of tissues and organs. Still, it faces challenges in its widespread use due to issues with the development of bioink materials and the nutrient diffusion barrier inherent to these scaffold materials. Herein, we introduce a method to promote oxygen diffusion throughout the printed constructs using genetically encoded gas vesicles derived from haloarchaea. These hollow nanostructures are composed of a protein shell that allows gases to permeate freely while excluding the water flow. After printing cells with gas vesicles of various concentrations, the cells were observed to have increased activity and proliferation. These results suggest that air-filled gas vesicles can help overcome the diffusion barrier throughout the 3D bioprinted constructs by increasing oxygen availability to cells within the center of the construct. The biodegradable nature of the gas vesicle proteins combined with our promising results encourage their potential use as oxygen-promoting materials in biological samples.

Original research article

by Leliang Zheng, Yancheng Zhong, Tiantian He, Shuping Peng, Liuyimei Yang

Tumor recurrence and bacterial infection are common problems during bone repair and reconstruction after bone tumor surgery. In this study, silver-anchored MoS2 nanosheets (Ag@PMoS2) were synthesized by in situ reduction, then a composite polymer scaffold (Ag@PMoS2/PGA) with sustained antitumor and antibacterial activity was successfully constructed by selective laser sintering technique. In the Ag@PMoS2 nanostructures, silver nanoparticles (Ag NPs) were sandwiched between adjacent MoS2 nanosheets (MoS2 NSs), which restrained the restacking of the MoS2 NSs. In addition, the MoS2 NSs acted as steric hindrance layers, which prevented the aggregation of Ag NPs. More importantly, MoS2 NSs can provide a barrier layer for Ag NPs, hindering Ag NPs from reacting with the external solution to prevent its quick release. The results showed that Ag@PMoS2/PGA scaffolds have stronger photothermal effect and antitumor function. Meanwhile, the Ag@PMoS2/PGA scaffolds also demonstrated slow control of silver ion (Ag+) release and more efficient long-term antibacterial ability. Besides, composite scaffolds have been proved to kill the MG-63 cells by inducing apoptosis and inhibit bacterial proliferation by upregulating the level of bacterial reactive oxygen species. This kind of novel bifunctional implants with antitumor and antibacterial properties provides better choice for the artificial bone transplantation after primary bone tumor resection.

Original research article

by Lina Dong, Mujiao Liang, Zhongwei Guo, Anyang Wang, Gangpei Cai, Tianying Yuan, Shengli Mi, Wei Sun

In nature, many biological tissues are composed of oriented structures, which endow tissues with special properties and functions. Although traditional hydrogels can achieve a high level of biomimetic composition, the orderly arrangement of internal structures remains a challenge. Therefore, it is of great significance to synthesize hydrogels with oriented structures easily and quickly. In this study, we first proposed and demonstrated a fabrication process for producing a well-ordered and dual-responsive cellulose nanofibers + hyaluronic acid methacrylate (CN+HAMA) hydrogels through an extrusion-based three-dimensional (3D) printing process. CN in the CN+HAMA hydrogels are directionally aligned after extrusion due to shear stress. In addition, the synthesized hydrogels exhibited responsive behaviors to both temperature and ultraviolet light. Since the temperature-responsiveness is reversible, the hydrogels can transit between the gelation and solution states while retaining their original qualities. Furthermore, the developed well-oriented CN+HAMA hydrogels induced directional cell growth, paving the way for potential applications in ordered biological soft-tissue repair.

Original research article

by Huilin Tang, Fei Bi, Guoqing Chen, Shuning Zhang, Yibing Huang, Jiahao Chen, Li Xie, Xiangchen Qiao, Weihua Guo

Three-dimensional (3D) bioprinting is an emerging method for tissue regeneration. However, promoting the epithelial-mesenchymal interaction (EMI), while maintaining the characteristics of epithelial cells has always been a challenge in tissue engineering. Since EMI acts as a critical factor in bone regeneration, this study aims to promote EMI by recombining epithelial and mesenchymal cells through 3D bioprinting. Hertwig’s epithelial root sheath (HERS) is a transient structure appeared in the process of tooth root formation. Its epithelial characteristics are easy to attenuate under appropriate culture environment. We recombined HERS cells and dental papilla cells (DPCs) through 3D bioprinting to simulate the micro-environment of cell-cell interaction in vivo. HERS cells and DPCs were mixed with gelatin methacrylate (GelMA) separately to prepare bio-inks for bioprinting. The cells/GelMA constructs were transplanted into the alveolar socket of Sprague-Dawley rats and then observed for 8 weeks. Hematoxylin and eosin staining, Masson staining, and immunohistochemical analysis showed that dimensional cultural pattern provided ideal environment for HERS cells and DPCs to generate mineralization texture and promote alveolar bone regeneration through their interactions. 3D bioprinting technology provides a new way for the co-culture of HERS cells and DPCs and this study is inspiring for future research on EMI model.

Original research article

by Jingge Ma, Jinfu Wu, Hongjian Zhang, Lin Du, Hui Zhuang, Zhaowenbin Zhang, Bing Ma, Jiang Chang, Chengtie Wu

Deep burn injury always causes severe damage of vascular network and collagen matrix followed by delayed wound healing process. In this study, natural diatomite (DE) microparticles with porous nanostructure were separated based on the particles size through a dry sieving method and combined with gelatin methacryloyl (GelMA) hydrogel to form a bioactive composite ink. The DE-containing inorganic/organic composite scaffolds, which were successfully prepared through three-dimensional (3D) printing technology, were used as functional burn wound dressings. The scaffolds incorporated with DE are of great benefit to several cellular activities, including cell spreading, proliferation, and angiogenesis-related gene expression in vitro, which can mainly be attributed to the positive effect of bioactive silicon (Si) ions released from the embedded DE. Moreover, due to establishment of bioactive ionic environment, the deep burn wounds treated with 3D-printed DE incorporated scaffolds exhibited rapid wound healing rate, enhanced collagen deposition, and dense blood vessel formation in vivo. Therefore, the present study demonstrates that the cost-effective DE can be used as biocompatible Si source to significantly promote the bioactivities of wound dressings for effective tissue regeneration.

Original research article

by Yi-Chao Hunag, Chun-Ming Chang, Shao-Fu Huang, Chia-Heng Hong, Chun-Li Lin

In this study, we developed a modularized proximal interphalangeal (PIP) joint implant that closely resembles the anatomical bone articular surface and cavity contour based on computed tomography (CT) image reconstruction. Clouds of points of 48 groups reconstructed phalanx articular surfaces of CT images, including the index, middle, ring, and little fingers, were obtained and fitted to obtain the articular surface using iterative closest points algorithm. Elliptical-cone stems, including the length, the major and minor axis at the stem metaphyseal/diaphyseal side for the proximal and middle phalanxes, were designed. The resurfacing PIP joint implant components included the bi-condylar surface for the proximal phalanx with elliptical-cone stem, ultra-high molecular weight polyethylene bi-concave articular surface for middle phalanx with hook mechanism, and the middle phalanx with elliptical-cone stem. Nine sets of modularized designs were made to meet the needs of clinical requirements and the weakness structure from the nine sets, that is, the worst structure case combination was defined and manufactured using titanium alloy three-dimensional (3D) printing. Biomechanical tests including anti-loosening pull-out strength for the proximal phalanx, elliptical-cone stem, and articular surface connection strength for the middle phalanx, and static/dynamic (25000 cycles) dislocation tests under three daily activity loads for the PIP joint implant were performed to evaluate the stability and anti-dislocation capability. Our experimental results showed that the pull-out force for the proximal phalanx implant was 727.8N. The connection force for the hook mechanism to cone stem of the middle phalanx was 49.9N and the hook mechanism was broken instead of stem pull out from the middle phalanx. The static dislocation forces/dynamic fatigue limits (pass 25000 cyclic load) of daily activities for piano-playing, pen-writing, and can-opening were 525.3N/262.5N, 316.0N/158N, and 115.0N/92N, respectively, and were higher than general corresponding acceptable forces of 19N, 17N, and 45N from the literatures. In conclusion, our developed modularized PIP joint implant with anatomical articular surface and elliptical-cone stem manufactured by titanium alloy 3D printing could provide enough joint stability and the ability to prevent dislocation.

Original research article

by Jianyu He, Jinglin Wang, Yuan Pang, Hang Yu, Xueqian Qin, Ke Su, Tao Xu, Haozhen Ren

Three-dimensional (3D) bioprinting technology is an effective method for exploring the biological functions of hepatocytes by building biomimetic 3D microenvironments. Various hepatic tissue models have been developed for disease modeling, drug screening, and tissue regeneration using 3D bioprinting technology. Human-induced pluripotent stem cells (hiPSCs) are a promising cell source for the generation of functional hepatocytes for bioprinting. In this study, we introduced hiPSC-derived hepatocytes (hiPSC-Heps) as mature hepatocytes for the bioprinting of a 3D hepatic tissue model. The 3D-printed (3DP) model facilitated the formation of hiPSC-Hep spheroids with higher viability and proliferation than the commonly used non-printed sandwich-cultured model. hiPSC-Heps in the 3DP model exhibited higher mRNA expression of liver-specific functions than those in the two-dimensional-cultured model. Moreover, enhanced secretion of liver function-related proteins, including α-1-antitrypsin, albumin, and blood urea nitrogen, was observed in the 3DP model. For the evaluation of acetaminophen-induced hepatotoxicity, the 3DP model exhibited a favorable drug response with upregulation of the drug metabolism-related gene cytochrome P450-1A2 (CYP1A2). Overall, the bioprinted hepatic tissue model showed great biofunctional and drug-responsive performance, which could be potentially applied in in vitro toxicological studies.

Original research article

by Marisela Rodriguez-Salvador, Itzel Fox-Miranda, Baruc Emet Perez-Benitez, Jose Ricardo Lopez-Robles

Tissue spheroids represent an innovative solution for tissue engineering and regenerative medicine. They constitute an in vitro three-dimensional cell culture model capable of mimicking the complex composition of a native tissue on a micro-scale; this model can function as a building block and be assembled into larger tissue constructs. Due to the potential tissue spheroids have for the evolution of the health industry, there is a need to assess the research dynamics of this field. Thus far, there have been no studies on their use as building blocks. To fill this gap, a study was performed to characterize the evolution of research where tissue spheroids were used as building blocks to generate tissue constructs. A scientometric analysis of the literature regarding tissue spheroid technologies was developed by quantification of bibliometric performance indicators. For this purpose, articles published during the period January 1, 2015 – December 31, 2021, from the Scopus database were organized and analyzed. The main subject areas, countries, cities, journals, institutions, and top-cited articles as well as the types of techniques, cells, culture time, and principal applications were identified. This research supports the definition and growth of research and development strategies for new technologies such as tissue spheroids.

Original research article

by Lanlan Dong, Zhengzhe Han, Xiang Li

Hydrogels have become an attractive option for tissue repair. A novel multifunctional hydrogel was developed using a two-step method involving photopolymerization and tannic acid (TA) solution incubation. The mechanical properties of this hydrogel were enhanced by the multi-hydrogen bond interaction between the TA and N-acryloyl glycinamide/gelatin methacrylate (NAGA/GelMA). The compressive modulus was doubled. The compressive strengths of the hydrogel were 5.5 MPa. The swelling rate was reduced by a factor of three. The adhesion strength of the composite hydrogel reached 80 KPa. The TA-mediated NAGA/GelMA/Laponite composite hydrogel exhibited excellent anti-fatigue and anti-oxidation properties, as well as printability. In vitro experiments indicated that the TA-mediated hydrogel facilitated the proliferation of bone marrow mesenchymal stem cells and osteogenic and chondrogenic differentiation. The developed multifunctional composite hydrogel has great potential for osteochondral defect repair under osteoarthritis conditions.

Original research article

by Anja Kriegel, Christian Schlosser, Tanja Habeck, Christoph Dahmen, Hermann Götz, Franziska Clauder, Franz Paul Armbruster, Andreas Baranowski, Philipp Drees, Pol Maria Rommens, Ulrike Ritz

The use of bioactive molecules is a promising approach to enhance the bone healing properties of biomaterials. The aim of this study was to define the role of bone sialoprotein (BSP) immobilized in collagen type I in various settings. In vitro studies with human primary osteoblasts in mono- or in co-culture with endothelial cells demonstrated a slightly increased gene expression of osteogenic markers as well as an increased proliferation rate in osteoblasts after application of BSP immobilized in collagen type I. Two critical size bone defect models were used to analyze bone regeneration. BSP incorporated in collagen type I increased bone regeneration only marginally at one concentration in a calvarial defect model. To induce the mechanical stability, three-dimensional printing was used to produce a stable porous cylinder of polylactide. The cylinder was filled with collagen type I and immobilized BSP and implanted into a femoral defect of critical size in rats. This hybrid material was able to significantly induce bone regeneration. Our study clearly shows the osteogenic effect of BSP when combined with collagen type I as carrier and thereby offers various approaches and options for its use as bioactive molecule in bone substitute materials.

Original research article

by Jiahua Zheng, Yibin Liu, Chenxiao Hou, Zhongkang Li, Shaopeng Yang, Xiao Liang, Liang Zhou, Jiangbo Guo, Jingkun Zhang, Xianghua Huang

Fertility preservation is becoming a clinical duty in practice. Three-dimensional (3D) bioprinting technology is potentially realize ovarian morphological repair and reproductive endocrine function rebuild. There is no published work on 3D bioprinting ovary using a decellularized extracellular matrix (dECM)-based bioink, though dECM is the preferred matrix choice for an artificial ovary. The study aimed to explore swine ovarian dECM-based bioink to fabricate 3D primary ovarian cells (POCs)-laden structures for mouse ovarian failure correction. In this study, the ovarian dECM was converted to dECM-based bioink by dECM solution mixed with a seaweed gelatin blend solution of bioink that was characterized using scanning electron microscopy, circular dichroism, rheology, hematoxylin and eosin staining, and immunohistochemistry. The 3D scaffolds were, then, printed with or without POCs by the extrusion 3D bioprinter. The laden POCs viability was detected with the live/dead assay kit. A female castrated mouse model was established, and the mice were treated with five different methods. The results revealed that the 3D scaffold encapsulating POCs group had more positive signals in neoangiogenesis, cell proliferation and survival than the 3D scaffold group, and ensured sex hormone secretion. Meanwhile, the expression of germ cells in the 3D scaffold encapsulating POCs group was more intensely than the non-printed hydrogel encapsulating POCs group. The work shows that the 3D bioprinting ovary employing ovarian dECM-based bioink is a promising approach for ovarian failure correction.

Review article

by Qiushi Liang, Yuanzhu Ma, Xudong Yao, Wei Wei

Chondral lesions caused by stressors, such as injury or inflammation, lead to osteoarthritis (OA). OA is a degenerative joint disease that has become a challenge worldwide. As the articular cartilage is incapable of self-regeneration due to the absence of vessels and nerves, novel cartilage repair techniques are urgently needed. Three-dimensional (3D) bioprinting, which allows the precise control of internal architecture and geometry of printed scaffolds, has stepped up to be a promising strategy in cartilage restoration. With regards to 3D bioprinting, bioinks with proper chemical and mechanical properties play one of the most critical roles in designing successful cartilage tissue constructs. In particular, hydrogels as 3D hydrophilic cross-linked polymer networks are highly recommended as bioinks because of their fine biocompatibility, easy fabrication, and tunable mechanical strength. Herein, we highlight the widely used polymers for hydrogel preparation and further provide a non-exhaustive overview of various functional modified additives (such as cells, drugs, bioactive factors and ceramic) to exploit the unique properties suitable for bioprinted cartilage. Finally, a prospective on future development for 3D-bioprinting in cartilage repair is elucidated in this review.

Review article

by Shenglong Li, Siyu Liu, Xiaohong Wang

In the past several decades, three-dimensional (3D) printing has provided some viable tissues and organs for repairing or replacing damaged tissues and organs. However, the construction of sufficient vascular networks in a bioartificial organ has proven to be challenging. To make a fully functional bioartificial organ with a branched vascular network that can substitute its natural counterparts, various studies have been performed to surmount the limitations. Significant progress has been achieved in 3D printing of vascularized liver, heart, bone, and pancreas. It is expected that this technology can be used more widely in other bioartificial organ manufacturing. In this review, we summarize the specific applications of 3D printing vascularized organs through several rapid prototyping technologies. The limitations and future directions are also discussed.

Perspective article

by Victor M. Villapún, Luke N. Carter, Steven Avery, Alba González-Álvarez, James W. Andrews, Sophie Cox

Additive manufacturing (AM) technologies have disrupted many supply chains by making new designs and functionalities possible. The opportunity to realize complex customized structures has led to significant interest within healthcare; however, full utilization critically requires the alignment of the whole supply chain. To offer insights into this process, a survey was conducted to understand the views of different medical AM stakeholders. The results highlighted an agreement between academics, designers, manufacturers, and medical experts, that personalization and design control are the main benefits of AM. Interestingly, surface finish was consistently identified as an obstacle. Nevertheless, there was a degree of acceptance that post-processing was necessary to achieve appropriate quality control. Recommendations were made for extending the use of in situ process monitoring systems to support improved reproducibility. Variations in the future vision of AM were highlighted between stakeholder groups and areas of interest for development noted for each stakeholder. Collectively, this survey indicates that medical stakeholders agree on the capabilities of AM but have different priorities for its implementation and progression. This highlights a degree of disconnection among the supply chain at a ground level; thus, collaboration on AM specific standards and enhancement of communication between stakeholders from project inception is recommended.

Clinical case study

by Xianglin Hu, Shachar Kenan, Mo Cheng, Weiluo Cai, Wending Huang, Wangjun Yan

Three dimensional (3D)-printing technology facilitates complex spine surgery with unique advantages in artificial vertebral body design and manufacturing. In this study, we aimed to demonstrate how a 3D-printed spinal implant is utilized in the management of multi-level spinal tumors and integrates with comprehensive oncologic treatment. Eight spinal or paraspinal tumor patients requiring spinal reconstruction after total en bloc spondylectomy were selected as candidates for 3D-printed titanium artificial vertebral body implants. All patients underwent surgery on three or more vertebral segments or complex spinal junction segments. The clinical, oncological, and surgical characteristics of patients were collected. Of the eight candidates, seven suffered from pain and/or limb disorder. Six underwent successful 3D-printed spinal implantation, while two failed due to implant mismatching and were converted to conventional reconstruction. Of the six patients undergoing 3D-printed spinal implant surgery: (i) Five had recurrent tumors; (ii) three underwent neoadjuvant therapy; (iii) the median surgery time was 414 min; (iv) the median blood loss was 2150 ml; (v) the median blood transfusion was 2000 ml; (vi) the median length of hospital stay was 9 days; (vii) four patients received adjuvant therapy after surgery; and (viii) all patients experienced no pain, moved freely, and had no local recurrence at a median of 11.5 months post-operative follow-up. Spinal reconstruction with a 3D-printed titanium artificial vertebral body allows for total en bloc resection of complex multi-level spinal tumors. Combined with neoadjuvant and adjuvant therapy, these patients had excellent postoperative outcomes, long-term normal spinal function, and associated low local recurrence probability.