Digital light processing (DLP)‐based (bio)printing strategies for tissue modeling and regeneration

Digital light processing (DLP)‐based bioprinting technology has recently aroused considerable concerns as a strategy to deliver biomedical materials and/or specific cells to create sophisticated structures for various tissue modeling and regeneration. In this review, we display a concise introduction of DLP bioprinting, and a further discussion on the design and manufacture of DLP (bio)printer with varied bioinks and their biomedical applications toward drug screening, disease modeling, tissue repair, and regenerative medicine. Finally, the advantages, challenges, and perspectives of the DLP printing platforms are detailed. It is believed that DLP bioprinting will play a decisive role in the field of tissue model and regenerative medicine, mainly due to its time‐efficient, higher resolution, and amenability to automation for various tissue needs.


INTRODUCTION
Three-dimensional (3D) bioprinting that fabricates various 3D high-resolution objects in free forms has been widely selected to create a 3D tissue model for simulating the human microphysiological environment. [1,2] Among all the 3D bioprinting strategies, the extrusion-based 3D bioprinting technology has been most widely adopted for creating the 3D printing hydrogels tissue model. [3][4][5] In term of extrusion-based 3D bioprinting, this strategy mainly builds 3D tissue structures by directly extruding various function F I G U R E 1 (A) Brief comparison of 3D extrusion-based bioprinting and digital light processing (DLP)-based bioprinting, and the representative applications of the DLP bioprinting strategies for tissue model. (B) The statistics for the number of papers on DLP printing tissue model published from 2016 to 2022. (C) Radar chart of the number of articles related to DLP printing tissue model in different disciplines from 2016 to 2022 (data source: Web of Science, searched using "DLP printing" AND "tissue" as keywords, data collected on August 3, 2022) by using light from a digital micromirror device (DMD) projector ( Figure 1A), so DLP technology with a much faster printing and production speed maybe become an novel strategy to obtain complicated structure than that of extrusion-based bioprinting methods. [12][13][14] Currently, 3D bioprinting improved the application of tissue structure in drug screening, [15,16] disease modeling, [17,18] and tissue repair and regenerative medicine. [19,20] Most of importantly, heart, [21,22] blood vessels, [23,24] bone, [25,26] cartilage, [27,28] liver, [1,29,30] lung, [1,31] eye, [32,33] neuronal tissue, [34,35] and pancreatic tissue [36] have been successfully designed and developed through 3D extrusion-based bioprinting and DLPbased bioprinting strategies ( Figure 1A). However, the resolution of extrusion-based 3D bioprinting structure is relatively limited, and the resolution of conventional extrusion printing is usually less than 200 μm, and the obtained structure is also easy to cause stress relaxation and permanent deformation due to the different crosslinking rate during the printing process. [37,38] Therefore, the 3D bioprinting tissue model needs various stiffness (rigid porous biomaterial scaffolds, such as bone, [39][40][41] and soft hydrogel, such as cartilage, [42] muscle, [43,44] liver, [45] vessels, [46] and ear [47] for loading bearing and carrying bioagents to mimicry of natural tissues requires. [48,49] Recently, with novel extracellular matrix's (ECM) improvement, the 3D bioprinting strategy has been widely used to create the multi-material components of 3D tissue constructs in the field of tissue engineering. [48,50] However, the current 3D extrusion-based bioprinting strategies have several limitations in integrating soft and rigid multifunctional biomaterials. [51] TA B L E 1 Summary of the digital light processing (DLP) bioprinting-related works Bottom-up DLP printer decellularized extracellular matrix (dECM), methacrylated gelatin (GelMA) and hiHep cells 365-nm UV light -Liver regeneration [79] DLP-stereolithography (DLP-SLA) and molten material extrusion hybprinter HUVEC-laden PEGDA and C3H10T1/2 fibroblast-laden collagen Visible light 100 Vascularized tissue engineering applications [24] Fluid-supported liquid interface polymerization (FLIP) DLP printer GelMA loaded GFP-expressing mouse adults fibroblast 405-nm UV light 62.5 Biomedical applications [91] A custom-built, multi-material DLP 3D printer GelMA and PEGDA 380-nm UV light 15 Vascularized micro-tissues and drug screening platforms [63] Gradient DLP 3D printer GelMA and PEGDA 450-nm blue light 38 Tissue regeneration [50] A smartphone-enabled portable DLP 3D printer GelMA and PEGDA 450-nm blue light 49 Bone repair [69] Digital near-infrared photopolymerization (DNP) printer GelMA 980-nm NIR light 100 Tissue regeneration [87] To address these issues, the DLP 3D printing technology as an effective strategy allows the design and develop complex tissue scaffolds that enable the encapsulation of various types of stem cells into multi-materials with various stiffness. [52][53][54] The stiffness of the printed scaffold can be designed and developed by adjusting the light exposure time and lightness within desired regions for encapsulating multiple cell types. [30,55,56] Moreover, multiple ECM components can be considered as an ideal bioink to recapitulate the complex native microenvironment to simulate different targeted tissues for cell proliferation and differentiation. [57][58][59] Figure 1B exhibits the number of papers published on the DLP printing tissue model since 2016, reflecting the numbers of research article have been increasing sharply, and covered various fields ( Figure 1C).
Of note, DLP (bio)printers have been widely designed and applied in drug screening, [60] disease modeling, [33] and tissue regeneration. [61] For example, Gou et al. designed and developed a liver-inspired 3D tissue model which allows toxins to be trapped efficiently. [62] In order to simulate fluid-solid interactions that regulate drug entry into microscopic tissues, Bhusal et al. designed and developed a multi-material DLP-based bioprinter to build a hydrogel-based microfluidic chip. [63] Moreover, Yang et al. also designed and fabricated heart valve hydrogels of heterogeneous structures with complex shapes via the DLP printing method, which showed the high fatigue resistance mainly attributed to the stiffness of the skeleton and matrix materials. [22] In addition, another DLP bioprinting of hydrogel-based hepatic construct was used to create personalized liver models for pathophysiological studies and early drug screening. [64][65][66] Generally, a variety of advantages can be offered by DLP bioprinting modalities, including ease to operate, [67,68] relatively cost-effectiveness, [69,70] and excellent accuracy/efficiency. [71][72][73] In this review, we concentrate on the various 3D DLP (bio)printers with various bioinks by different photocrosslinking methods to create the tissue model for drug screening, disease modeling, tissue repair, and regenerative medicine (Table 1).

DESIGN AND MANUFACTURING OF DLP PRINTERS
A conventional DLP printer should include a digital projection device, a light source system, a vat to hold the liquid bioink, a high precision z-axis movement device, and a program to control individual movements and other auxiliary equipment. [63,69] Compared with standard extrusion-based printers, the DLP printer allows the production of the fine microstructures developed via the quickly projecting digital patterns onto a photopolymerizable bioink. [74][75][76][77][78] Although the basic design of DLP printer is simple, various modifications of DLP printer have been made for different printing purposes (Table 1 and Figure 2).

Customized bottom-up 3D DLP bioprinter
Mao and co-workers designed and manufactured a bottomup DLP 3D bioprinter that used photocurable GelMA with liver decellularized ECM (dECM) and human-induced hepatocytes (hiHep cells) to fabricate a liver microtissue structure ( Figure 2A). [79] The developed DLP bioprinter included a UV light source with a 365-nm wavelength and 2.25 mW/cm 2 light intensity, a DMD based on a Texas Instruments DLP module with a resolution of 1920 × 1080, and a z-axis mobile platform. Moreover, the liver microtissue fabricated by DLP printing presented an excellent hepatic function which could be considered an effective platform for creating a functional liver tissue model as a substitute for patients with F I G U R E 2 Schematic of diverse digital light processing (DLP) printers. (A) Common DLP printer. Reproduced with permission. [79] (B) DNP printer. Reproduced with permission. [87] (C) DLP-SLA/molten material extrusion hybprinter. Reproduced with permission. [24] (D) FLIP DLP printer. Reproduced with permission. [91] (E) Multimaterial DLP-based printer. Reproduced with permission. [63] (F) Gradient DLP 3D printer. Reproduced with permission. [50] (G) A smartphone-enabled portable DLP 3D printer. Reproduced with permission [69] liver failure. However, bioinks with excellent biocompatibility for the co-culture of multiple cells need to be designed and developed due to the multicellular component of the human organ, which enables carrying messages between different cells in liver tissue engineering. [30,80,81] Additionally, in vivo studies of liver microtissues with optimized geometry and function are needed to further understand how to develop implantable liver replacements, and the co-culture of cells with multiple roles will also be conducted.

Digital NIR photopolymerization bioprinter
Since UV light is limited by its tissue-penetration ability, NIR light has an excellently penetrating ability for the deep tissue and has been applied for controlled drug release, photodynamic therapy, [82] photothermal therapy, [83] in vivo imaging, [84] 3D image visualization, [85] and optogenetics in vivo. [86] Chen et al. designed a digital NIR photopolymerization-based bioprinting platform for creating complex constructs by using up-conversion nano-particle @lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) nanoparticle-initiated NIR polymerization ( Figure 2B). [87] Through this system, living tissue models could be noninvasively bioprinted in vivo from the subcutaneously injected bioinks for tissue repair or organ reconstruction, indicating expected clinical application. This work confirms that the noninvasive in vivo 3D bioprinting strategy would start a novel avenue for tissue repair and regeneration.

DLP-stereolithography/molten material extrusion hybprinter
Despite excessive advances in creating tissue modeling, the systems and current 3D bioprinting techniques fall short in such rigid and soft multifunctional components' integration. [88] Yang et al. developed a novel 3D hybrid bioprinting device (Hybprinter) that enables the integration of soft and rigid bioinks to obtain tissue structure by employing DLP-stereolithography (SLA) printing and molten material extrusion (MME) techniques ( Figure 2C). [24] In their work, the Hybprinter generated a multiphasic hybrid structure with unique control through the deposition of stiff thermoplastic material by using MME to control the porosity, and then gelation of gentle photo-crosslinking hydrogel (PEGDA) component in the objective areas through DLP-SLA technology. Furthermore, the living cells within the hydrogel throughout layer-by-layer manufacture and postseeding formed multiple cells spatial distributions in the construct with high cell viability. [89]

Fluid-supported liquid interface polymerization DLP printer
Although the excellent cell biocompatibility of bioinks, the capability to build these tissue models with complex structures that use extrusion-based printing is restrained mainly due to their low mechanical stiffness. [90] Beh et al. developed a continuous fluid-supported liquid interface polymerization DLP printing platform which consisted of a 405 nm UV projector with the H-Nu or LAP UV initiators and photo-initiator systems ( Figure 2D), that used a fluid support technique to print soft structures with complex geometry, which can further reduce the printing time to as little as minutes for certain formulations. [91] After printing, the support fluid was easily removed by rinsing. This technique avoids inadvertent adhesion that affects normal resin-based 3D printer, and facilitates rapid, continuous printing smooth objects along the z-axis at 200 mm/h without layering artifacts. In this work, a complex structure (create centimeter-scale, cell-laden hydrogels) with free-standing channel networks (500 μm-thick walls) was designed and manufactured to support cellular metabolism by nutrient diffusion, which overcomes the main nutrient supply issue in bioprinting.

Multimaterial DLP-based printer
Recent progress in hydrogel engineering and DLP-based bioprinting technology has achieved fast developments in the field of the design and manufacture of organs-on-chips. [92,93] However, existing multimaterial printers are not enabling to continuously build the cell-laden structure with clinically relevant sizes and multicomponent biomaterials. [94][95][96] Thus, Bhusal et al. designed a multimaterial, DLP-based printer for speedy, hydrogel-based microfluidic chips via one-step prototyping strategy for the objective microtissue ( Figure 2E). [63] The printer with a small sample scale, low reagent consumption, low expense, and ease of operation is capable of bi-axial movements that are sustained by stepper motors and z-axis movement by an excellent precision linear stage. The rotation of the upper table allowed bioink exchange for multimaterial bioprinting ( Figure 2E). In their work, a composite hydrogel bioink based on PEGDA and GelMA was selected to create the hydrogel-based microfluidic chips for analyzing drug mass transport, which offered a valuable tool for the rapid integration of micro-tissue models into organs-on-chips and drug screening platforms. In addition, another advantage of multi-material printing is that it enables reproduce the gradient architecture biologic tissues which widely distributed in human tissued from the neural tube's polarization to the osteochondral interface's architecture, which have crucial influences in tissue growth, physiology, and disease statuses. [97,98] More importantly, the various gradient generation mainly relies on the multimaterial capacity during the DLP 3D printing. Mian and co-workers first time innovated a gradient DLP 3D printer through integrating a microfluidic chaotic mixer chip consisting of multiple inlets, a microchannel, and an ink vat to create multifunctional graded scaffolds ( Figure 2F). As described in Figure 2F, the microfluidic mixer chip produced the homogenous graded bioinks by adjusting the inlet flow speed on-the-fly in real time. Once the various bioinks with accurate volume were injected into the microfluidic mixer chip and filled the vat, the construction platform was approached at the printing level by projecting the predesigned patterns at the ending region of the mixer using the 450 nm blue light. After the photo-crosslinking of each layer, the build platform lifted, and the uncured hydrophilic ink residue at the bottom of vat was rapidly evacuated from the outlet mainly attributed to the hydrophobic modification of the outlet area, which primarily addressed the problems of cross-contamination issue during the ink exchanges, and as well as the challenge associated with significant bioink waste observed in the other multimaterial DLP bioprinting methods. [99,100] As a result, this DLP 3D gradient printing device enables to develop the 3D structures with biological gradient features in superior resolution and faster speed.

2.6
A smartphone-enabled portable DLP 3D printer The current limitations of 3D printing platform are bulky, and the operation process is inconvenient. In the case of limited resources, the technology cannot be immediately used in the point-of-care settings. Li et al. first designed and manufactured a portable DLP 3D printer, which was established based on a custom-written smartphone-operated app and a smartphone-powered projector, with full sizes of 10 cm × 20 cm × 20 cm in width, length, and height, respectively ( Figure 2G). [69] The smartphone-enabled DLP printer included a smartphone-powered projector typically for outputting the designed patterns and a custom-built smartphone app to handle the printing platform. In their work, the smartphone-enabled DLP printer could achieve a comparable resolution of commercial printers. Notably, the in situ smartphone-enabled DLP bioprinting platform allowed directly bioprint cell-laden hydrogel scaffold connected to the surrounding tissues closely within the boundaries of the injured porcine muscle. Therefore, due to its portable and assemble characteristics, the smartphone-based DLP printer displays a potentially significant advantage under limited resources for in vivo bioprinting in the future.

DLP BIOPRINTING FOR IN VITRO TISSUE MODELING AND TISSUE REGENERATION
Due to its excellent features, DLP bioprinting technology and printer have been widely investigated in biofabrication and in vitro tissue modeling, [101,102] which can obtain smooth 3D objects with perfect structure than extrusion printing by continuously refreshing the projected optical patterns and moving the stage with the printed object. [103,104] Therefore, DLP bioprinting has drawn significant interest, which is mainly attributed to its excellent resolution, accuracy, and ability to precisely manipulate biomaterials, cells, and therapeutic molecules in 3D environments. [43,105] Owing to all these excellent features, the DLP-based printer has been progressively considered to serve as a potentially biofabrication platform for creating 3D tissue or disease models for tissue repair and regeneration, and drug screening ( Table 2).

DLP bioprinting for human cardiovascular research
Cardiovascular disease is the leading cause of global death. [75,106,107] In order to overcome the shortage of donor heart tissue for heart transplantation, various engineering strategies have been widely developed to manufacture heart tissue by creating implantable therapeutic heart patches/components. [108][109][110] Wang et al. developed a novel DLP printing platform to produce the 4D NIR light-sensitive cardiac tissue model with aligned microgrooves structure and adjustable curvature ( Figure 3A-i). [111] Their results demonstrated that the NIR-responsive 4D cardiac tissue constructs exhibited a remotely controllable and dynamic transformation in a spatiotemporal manner. Then, the co-culture of human inducible pluripotent stem cell-cardiomyocyte (hiPSC-CMs), hMSCs, and hECs showed evenly cell distribution, especially the hiPSC-CMs possessed good myocardial maturation on the 4D curved heart with the optimized microgroove structure ( Figure 3A-ii). Moreover, immunofluorescence staining results indicated that the 4D cardiac tissue supplied a promising approach to generating a dynamic cardiac tissue construct with adjustable curvature and aligned myofibers primarily attributed to the myocardial maturation and increased sarcomere density (Figure 3A-iii). This research offers a promising approach for constructing a complicated heart tissue model with consistent cell distribution, which can further be improved by exploring the 4D-induced mechanical forces for better cardiac function in the future.
In another work, Yu et al. presented photocrosslinkable tissue-specific dECM bioinks for bioprinting patient-specific tissues with high control over complicated microarchitecture and mechanical properties by a DLP bioprinting strategy ( Figure 3B-i). [45] The dECM bioink provided favorable conditions for maintaining tissue-specific maturation of hiPSC-derived cells. In this work, fluorescence images of hiPSC-CMs showed more actin and α-actin expressions, as well as higher cell density, compared to cells bioprinted in type I collagen controls ( Figure 3B-ii). Therefore, the dECM-based cardiac tissue model, as a potential platform for biologically related living human tissue, opens the door to the subsequent study of the functional maturity and longterm stability of hiPSC-derived cells in complex bionic tissue systems for biological disease mechanisms, developing personalized medicine, as well as for diagnostic drug screening applications.

DLP bioprinting for human liver research
The liver plays a crucial function in the synthesis of important proteins and the metabolism of xenobiotics. [112] The dysfunctions of the liver are closely connected to drug-induced toxicity and disease development. [113,114] For these particular reasons, the in vitro liver models generated by the 3D printing approach have been widely investigated to serve as a replacement for animal models in pathophysiological studies, drug screening, and hepatotoxicity prediction. [115][116][117] The preservation and functional maturation of hepatic cells that are derived from human induced pluripotent stem cells (hiPSCs) is crucial to personalization and in vitro drug screening of disease analysis. Ma et al. proposed a hydrogelbased 3D liver model that combined hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) with human umbilical vein endothelial cells (HUVECs) and adipose-derived stem cells (ADSCs) embedded in a microscale hexagonal architecture through the rapid, DLP-based 3D bioprinting technology ( Figure 4A). [30] As shown in Figure 4A-ii, after bioprinting, cells appeared as individual spheroids of their respective structures ( Figure 4A-ii a-c), with red fluorescently labeled HUVECs and ADSCs aligned with the hydrogel pattern, further illustrating that the sinus-like structure within the hepatic lobules. In the bright field, the bioprinted structures gradually became blurred over time, but the images were clearly discernible under the fluorescent field, indicating that the entire structure was not lose the inherent patterns which TA B L E 2 Summary of the digital light processing (DLP) bioprinting-related applications
Spinal cord injury repair [35] GelMA + LAP + NGCs LAP Repair large-gap nerve injuries [34] predesigned for various cell types ( Figure 4A-ii a). Moreover, the greater spheroid formation was obtained in the tri-culture system of hiPSC-HPCs, HUVECs, and ADSCs, compared with the hiPSC-HPC-only sample. The larger spheroid size indicates that a bigger range of cellular connections and possibly excellent function ( Figure 4A-ii d). Therefore, their results suggest that the design and bioprinting of 3D in vitro liver models with hiPSC-HPCs in lobular structures and associated supporting cells can promote the maturation and functional preservation of hiPSC-HPCs. Considering bioink plays a pivotal role in tissue functions, Yu et al. designed another liver model using photocrosslinkable dECM bioinks via a DLP-based continuous 3D bioprinter ( Figure 4B). [45] In this work, the liver structures bioprinted with dECM and GelMA exhibited the multicellular spheroids and larger hiPSC-HPC aggregates compared to the control group (collafen I and GelMA) ( Figure 4B-ii). Meanwhile, the enhanced maturation of hiPSC-HPCs in liver tissue model was further demonstrated by the increased expressions of transthyretin (TTR) and albumin (ALB) than the dECM group ( Figure 4B-iii).

DLP bioprinting for lung research
Lung is an important respiratory organ of human body. Lung cancer and other lung diseases are among the leading causes of death induced by cancer worldwide. [118,119] Therefore, early screening and personalized treatment of lung diseases are great importance for preventing disease progression. [120,121] Compared with extrusion-based bioprinting, the DLP-based bioprinting technique shows advantages in cytocompatibility, high printing accuracy, and fast fabrication speed, thus providing a quick and effective method for generating cell-encapsulated hydrogel structures to mimic lung tissue. [122][123][124] Hu et al. utilized a PEO-GelMA two-phase bioink to prepare 3D lung cancer tissue models in vitro by using the DLP 3D bioprinting ( Figure 5A). [31] In this study, the LL/2 lung cancer cells cultured in 2D condition (NS group) showed patellar growth after 48 h, while the same cells cultured in 3D-bioprinted samples presented spherical growth after 48 h, and cell pseudopodia were not very obvious. After 48 h treatment with Y27632, the cell densities did not change significantly in 2D and 3D environments. Additionally, after 48 h treatment with PTX, the cell density of LL/2 cells in 2D microenvironment obviously decreased, while the cell density in 3D microenvironment had little change than the control group. Finally, when treated with the PTX and Y27632 combination for 48 h, the cell den-sities and cell morphology of LL/2 cells were significantly reduced in 3D and 2D environments. Compared with the 3D culture microenvironment, the cell morphological changes were more evident in the 2D culture condition. More importantly, 3D culture modulated actin cytoskeleton aggregation through the ROCK pathway, mimicking tumor tissue, and thus altered the sensitivity of lung cancer cells to paclitaxel ( Figure 5B). Thus, this porous 3D lung prepared by 3D bioprinting technology is a valuable strategy for lung cancer cell culture, showing a potential application in cancer studies and drug development in the future.

DLP (bio)printing for cartilage and bone research
Treatment of cartilage and bone defects caused by traumatic injury or disease is still a challenging field in clinical application due to its poorly regenerative abilities. [125][126][127] The activity of chondrocytes will determine the success of cartilage deposition and repair. [128,129] DLP bioprinting technology is an effective method for fabricating cell scaffolds with suitable microenvironmental conditions in tissue engineering and regenerative medicine. [130][131][132] Jiang et al. designed and manufactured a platelet-rich plasma (PRP)-GelMA hydrogel model by the DLP printing technique for osteochondral defect repair in an injured rabbit model ( Figure 6A). [28] As shown in Figure 6A, the PRP-GelMA group presented partially soft tissue structure in the wound area, while untreated and pure GelMA groups showed small holes at 6 weeks of this section. At 12 weeks, the wound repair was still inhibited in untreated group; the wound surface was covered with irregular soft tissues. The pure GelMA group still had partial defects, and cartilage tissues were found in the treatment groups. At 18 weeks after surgery, smooth cartilaginous restorations in the PRP-GelMA group had fused with the original tissue, significantly cleft and defects were found in the GelMA and untreated group. In addition to the repair of cartilage, the images of micro-computed tomography (micro-CT) reconstruction presented that the clefts in the GelMA group were bigger than those in the PRP-GelMA group, despite the fact that the defects were filled with mature bone tissue into both groups. Therefore, the PRP-GelMA hydrogel demonstrated a good biocompatibility which can create a reparative environment for osteochondral regeneration and repair. However, further investigations are needed to determine this excellent biomaterial and its potential clinical application in osteochondral regeneration. Reproduced with permission. [111] (B) Scanningless and continuous 3D bioprinting of human heart tissues with decellularized extracellular matrix. (i) Schematic of the rapid 3D bioprinting process to fabricate dECM tissue structure with tissue-specific hiPSCs. (ii) Representative images of immunohistochemical staining of the bioprinted heart (α-actinin, actin) tissue structure and their respective collagen I controls after 7 days. Reproduced with permission [45] Due to the higher resolution and fast printing speed, DLPprinted platforms ease to produce the precise structures similar to native tissues and good cytocompatibility. [133,134] In Hong et al.'s work, a silk fibroin with glycidyl-methacrylate (Silk-GMA) structure printed via DLP approach was applied to cartilage regeneration ( Figure 6B). [10] In this work, the endoscopic observation confirmed that the inner size of transplanted Silk-GMA hydrogel loaded with chondrocytes gradually increased, and the surrounding injured tissues also grew to the surgical site of the transplanted artificial trachea  [30] (B) (i) Schematic illustration of heart tissue modeling through a light-based approach. (ii) Representative confocal images of immunohistochemical staining of the DLP printed liver (E-cadherin [E-cad]), albumin (ALB), and their respective collagen I control. (iii) Gene expression of early and late tissue-specific markers for the DLP printed liver tissue structure relative to collagen I control. Reproduced with permission [45] after 6 weeks ( Figure 6B-i). Additional, histological results of H&E and MT staining revealed new cartilage regenerated in the graft ( Figure 6B-ii). Therefore, functional and efficient engineered cartilage of Silk-GMA with chondrocytes can be produced by DLP printing platforms for the in vivo transplantation. These results further illustrated that Silk-GMA tissue model prepared by DLP printing could not only provide potential application for cartilage generation in vitro and in vivo but also demonstrate good biocompatibility and superior mechanical properties for the defective tissues repair and regeneration. Further studies on the regeneration of various types of stem cells and Silk-GMA-based hydrogel are needed. Moreover, Rajput et al. also designed and developed an DLP 3D bioprinting bone tissue scaffolds with tunable mechanical properties and architecture from photocurable silk fibroin. [135] This work also demonstrates the potential of silk fibroin-derived bioinks for DLP-based 3D bioprinting of scaffolds in the field of bone tissue engineering.  [31] Bone tissue, as a supporting structure for the human body, plays a vital role in protecting internal tissues or organs. [136] However, the diversity of hierarchical structure, the high mechanical performance, and the various types of bone-resident cells are the primary issues for creating the biomimetic bone scaffolds for bone regeneration. [137] Zhang et al. successfully designed and prepared a bone-like scaffold with integrated layered Haversian bone structure by DLP 3D printing technology ( Figure 6C-i). [26] They established a rabbit bone marrow-derived MSC-rabbit aortic EC (RBMSC-RAEC) co-culture platform to treating rabbit femoral defects.
In vivo experiments of rabbit femur defects also confirmed that the RBMSC-RAEC co-culture platform can promote the various new bone and blood vessels generated in Haversian canals than in the single culture group and the cell-free group ( Figure 6C-ii). In addition, the Haversian bone-like scaffolds have been shown to benefit early bone formation. Therefore, this study proposed a strategy for designing biomimetic bone regeneration functionalized materials. However, more bone-resident cells, including osteoblasts, osteoclasts, and macrophages, need to be further introduced into the coculture platform, and the mechanism of multicell synergy is also required to be further investigated in the future.

DLP bioprinting for eye repair
Conjunctiva is an important component of the ocular surface. It is a layer of mucous membrane covering the inner side of the upper and lower eyelids and in front of the eyeball. It has functions of lubrication, mechanical support, and immune responses. [138][139][140] Inflammation of the conjunctiva from disease or injury and damage can lead to various symptoms, such as dry eye and visual impairment. [141,142] In order to investigate the mechanism of conjunctival disease, 3D-engineered models obtained by DLP bioprinting strategy have become an effective strategy to create ocular surface disease modeling for high-throughput drug screening. [33,[143][144][145][146][147] To obtain a 3D-engineered models, Zhong et al. designed and produced a rabbit-derived conjunctival stem cells (CjSCs) loaded GelMA hydrogel model via DLP-based bioprinting for injectable delivery ( Figure 7A). [143] In their work, the stiffness of the hydrogel micro-constructs could be adjusted by changing the light exposure time, which could maintain the viability and stem cell behavior of encapsulated CjSCs for dynamic suspension culture of CjSCs. Moreover, GelMA hydrogel with micro-constructs allowed easy injection onto the wound surface by using a F I G U R E 6 Digital light processing (DLP) bioprinting for cartilage, bone, and tooth repair. (A) Macro and Micro-CT observation (n = 5) of osteochondral defect repair at 6, 12, and 18 weeks with pure GelMA and PRP-GelMA scaffolds. Reproduced with permission. [28] (B) DLP 3D printing silk fibroin hydrogel for cartilage tissue engineering. (i) Schematic summaries of Silk-GMA hydrogel transplantation loaded chondrocytes and endoscopy was used to observe rabbit trachea transplantation at 6 weeks. (ii) In vivo histological analysis of cartilage tissue regeneration by Silk-GMA hydrogel loaded chondrocytes after transplantation 6 weeks. Reproduced with permission. [10] (C) Multicell transfer of Haversian bone-like scaffolds in bone regeneration by 3D printing. (i) 3D printed Haversian bone-like scaffolds for multi-cell delivery. (ii) The Haversian bone-like bioceramic scaffolds can promote the regeneration of new bone and new blood vessels in rabbit femur defects model. Reproduced with permission [26] 30-gauge syringe needle. It showed no influence on structural deformation and cell viability, suggesting the possibility for subconjunctival delivery and immobilization to the target subconjunctival region in an in vitro rabbit eyeball model. Therefore, the novel cell culture method with DLP bioprinting technology can be selected to develop clinically relevant hydrogel micro-constructs for the treatment of ocular surface diseases.
In addition, the same team also developed multimaterials GelMA bioinks composing hCjSCs, immune cells, and vascular cells to realize multicell co-culture to create a 3D multicellular in vitro pterygium model through DLP bioprinting. [33] In this work, the bioprinted model enabled to reproduce the disease microenvironment of pterygium and showed consistent upregulation of genes associated with interleukin cascade, tumor necrosis factor signaling, and other inflammatory responses. It is essential that this work is the first demonstrated 3D pterygium model, which may be considered as an effective platform to studies personalized medicine and drug screening in the future.

F I G U R E 7
Digital light processing (DLP) bioprinting for eye tissue modeling. (A) Hydrogel micro-constructs loaded with CjSCs were prepared by rapid DLP bioprinting for injection delivery to ocular surface under conjunctiva. Reproduced with permission. [33] (B) 3D printed bionic epithelium/stroma double hydrogel for implantation corneal regeneration. (i) Schematic diagram of ink components and network formation in PEGDA-GelMA hydrogel. (ii) DLP bioprinted double dome-shaped corneal scaffold and its application in the rabbit keratectomy model. Reproduced with permission [144] In another work, He et al. designed and developed a biomimetic epithelium/stroma bilayer hydrogel tissue model based on PEGDA-GelMA bioink loaded with cells (rabbit corneal epithelial cells [rCECs] and rabbit adipose-derived mesenchymal stem cells [rASCs]) for corneal regeneration ( Figure 7B). [144] In this work, the bilayer cells-laden corneal scaffold with high fidelity and robustly surgical operation capability can be bioprinted ( Figure 7B). Their results also further confirmed that the 3D-printed bilayer cells-laden corneal scaffold effectively achieved corneal defects closure, re-epithelialization, and stromal regeneration distribute to the precise localization of cells in epithelia and stroma layer.
In the future, the suturing resistance of the hydrogel should be further improved. Clinically, biomimetic endodermis construction should also be considered to achieve total corneal regeneration.

3.6
DLP bioprinting brain tumor for drug screening DLP-based 3D bioprinting, as a fast microfabrication method compatible with various light-sensitive bioink, has been widely used and developed for cancer tissue models. [92,148] Glioblastoma multiforme (GBM) is the most common primary brain tumors which seriously threatens the life and health of patients due to its high cellular and molecular heterogeneity, hypervascularization, and inherent drug resistance. [149][150][151] Organoids, as an excellent platform for self-assembled 3D tissue models, have been widely designed and developed for various cancer modeling, including pancreatic cancer, bladder cancer, and GBM. [152][153][154] However, innate variation and limited control over structure in the process of self-assembly limit its further application. [155] In Tang et al.'s work, the GBM model based on native ECM derivatives (GelMA and HAMA) were designed and fabricated through the rapid DLP 3D bioprinting system ( Figure 8A). [156] The GBM model consisted of patient-derived glioblastoma stem cells, macrophages, astrocytes, and neural stem cells ( Figure 8A-i). As shown in Figure 8A-ii, the dextran molecules marked with fluorescence quickly diffused into the DLP-bioprinted GBM model, illustrating the drug compounds had excellent dispersibility in the 3D hydrogel model. Additionally, their results also demonstrated that erlotinib and gefitinib have significantly target effects of reducing EGFR activity in various culture system. Moreover, they indicated that the designed and developed 3D bioprinting hydrogel model could form GBM drug responses using gene expression data from a drug-sensitive model of 3D tetra-culture and enabled to predict drug sensitivity and resistance based on transcriptional characteristics of a 3D tetra-culture model. In conclusion, the 3D-bioprinted GBM structure can be considered as an effective and physiologically biomimicry brain tumor model, which provides a platform for revealing the multicellular interactions in brain tumor biology in the future.
In addition, the same team also designed and developed a biomimetic tri-regional GBM model based on the brain tumors-specific ECM-derived bioinks composed of glycidyl methacrylate hyaluronic acid and GelMA using DLP 3D bioprinting strategy ( Figure 8B). [157] In this work, they obtained three different zones with various stiffnesses due to the different bioprint parameters and bioink formulations, which was effective to build a highly matched microenvironment with brain tumor (Figure 8B-ii). Moreover, their result demonstrated that the 3D-bioprinted model has a positive effect on enhancing the resistance of tumor cells to temozolomide compared to spherically cultured cells. Therefore, the 3D GBM model with various stiffnesses enables coordinate the biophysical properties and biochemical characteristics to bionic real organizational microenvironment and analyzes the biophysical impacts on other tumor-stromal interactions in the future.

DLP printing for nervous tissue repair
Peripheral nerve injury is a serious medical issue caused by trauma, iatrogenic injury, or congenital defect that, resulting in total or partial loss of sensory and motor function. [158][159][160] Current bioprinting approaches to obtain specific functional tissues lack suitable biomanufacturing techniques to construct complex 3D microstructures, which are critical for guiding cell growth and promoting tissue maturation. [161][162][163] Compared to other 3D bioprinting technologies, DLP printing, as nozzle-free printing technology, can further improve the fabrication of complex 3D structures by enabling structural diversification of digital masks. [164,165] Koffler et al. designed and created a complex spinal cord structure for tissue repair using a DLP-based 3D printing strategy using the ink consisting of PEGDA and GelMA ( Figure 9A). [35] The structure was printed to be the same dimensions as rodent spinal cord in 1.6 s, which exhibited a comparable elastic modulus with a normal spinal cord ( Figure 9A-i). In their work, hydrogel scaffolds were further loaded with neural progenitor cells after printing, to support axonal regeneration and the formation of new "neural relays" at the site of complete spinal cord damage in rodents. They also demonstrated that the damaged host axons can be regenerated onto the 3D biomimetic scaffold to extend axons from the scaffold to the host spinal cord below the injury site, restoring synaptic transmission and significantly improving functional outcomes. (Figure 9A-ii). Therefore, 3D-printed spinal cord scaffolds can promote the tissue regeneration and guide the cell growth along the structural cues.
In another research, Ye et al. designed and developed a multichannel nerve guidance conduit (NGCs) for peripheral nerve tissue repair and regeneration through DLP 3D bioprinting with GelMA ink ( Figure 9B-i). [34] The results of the co-culture of PC12 cells proved that printed NGCs have excellent biocompatibility to improve the survival, proliferation, and migration of neural cells in the direction of longitudinal channel ( Figure 9B-ii). Their research also confirmed that the material has been used to differentiate stem cells into neurons on prefabricated conduits ( Figure 9B-iii). Therefore, the designed and developed GelMA NGCs using DLP printing technology present great application potential for nerve regeneration.

CONCLUSIONS, CHALLENGES, AND PERSPECTIVE
DLP bioprinting platforms can generate highly complex tissue structures with high surface quality since it has benefits such as rapid crosslinking in a complete layer, high resolution, and ease of operation. Therefore, it has been investigated in the areas of drug screening, disease modeling, tissue repair, and regenerative medicine. However, the current DLP printer for tissue repair and regeneration are not without their challenges, which means that all aspects of the higher printing resolution, printing efficiency, biocompatibility, and full sized need be further improved.
In currently research, the microscale printing resolution can be designed and developed by adjusting the material components printed and the cell contents into the printing inks. However, the special capillary network with the higher printing resolution is still in great challenge to reproduce in some highly complex tissue modeling or structure. Moreover, the higher printing efficiency also be considered as a main challenge for printing bionic organ/tissue modeling. Another technological challenges is the viability of the various cells into the prepared printing mixture solution which always produce a certain of negative consequences for metabolically active cell types, such as liver cells. In addition, the DLP printer with different light resource need to further design and develop to improve the biocompatibility of DLP 3D printing platforms and decrease the effect on cell The 3D printed GBM model serves as a platform for drug reaction modeling. Reproduced with permission. [156] (B) GBM tissue models printed by 3D DLP have regionally different biophysical properties. (i) 3D DLP printed GBM tissue model schematic, stiffness of each area is different. (ii) Representative 3D DLP printed GBM tissue model under different stiffness conditions of day 0 bright field image and SEM, and the time axis of GBM growth and angiogenesis in GBM tissue models by 3D DLP printed was explained. Reproduced with permission [157] viability. Finally, the full size of the bionic organ/tissue modeling are still challenges to present full physiological function of the bioprinted tissue constructs modeling for clinical and commercial applications.
Notably, the morphologies of the circulatory and pulmonary systems containing such prescribed biomimetic and multi-vascular architectures are physically and evolutionarily entangled, and their organizational structure is extremely complex. Then, the prime users of DLP (bio)printer need to reproduce the internal microstructure accurately, and thus, it is very vital to design and manufacture printing systems that are easy to operate and does not require personnel with  [34] bioprinting expertise to obtain fine print file during bioprinting. In addition, the stability, consistency, and repeatability of the DLP printer equipment in the bioprinting process are crucial, which can be enhanced by intelligent manufacturing technology that offsets the resolution decreases when printing a large proportion of the tissue model.
There are also great limitations on the printing materials (bioink) used for DLP 3D bioprinting. Due to the requirements on biomaterials to possess specific qualities, the common types of materials used for 3D bioprinting are reduced to only a few. Bioink, as a critical component of the DLP bioprinting system, should avoid cell toxicity from the photo-crosslinking materials loaded cells in the bioprinting process. Furthermore, the new bioinks should also meet the unique requirements for traditional bioinks, such as printability, mechanical strength, biodegradability, and cellular compatibility, in addition to ensuring the speed of photopolymerization and the fidelity of printed structure according to the type of tissue to be repaired or regenerated. In recent research, some researchers has make a great deal of effort to develop multimaterial bioinks for DLP 3D bioprinting platforms. [50,63] However, developing 3D-printable and cell-compatible materials with adjustable biochemical properties to recapitulate the native tissue microenvironment also need to further address in future.
The biggest and most important challenge is the practical DLP 3D printed tissue models application in the field of drug screening and disease modeling, patient-specific cell sources (such as human induced pluripotent stem cells (iPSC)derived cells and primary diseased cells from patients). Current studies already demonstrated the use of iPSC-derived cells to build various tissue modeling for tissue repair and regeneration. [166][167][168][169][170] However, the iPSC-derived cells were not a complete replacement for normal maturation cells to reproduce the functional level of health cells still be considered as a huge challenge for the organ/tissue modeling and regeneration. Moreover, the most direct and effective way is to obtain the primary cells directly from the patient, which was not widely performed in current work mainly due to the finiteness of the patient's cells. Therefore, the tissue modeling was built by using primary diseased cells from patients to provide more reliable data results on the development of personalized disease modeling and drug screening.
In summary, DLP printing technology provides the possibility to develop in vitro tissue models with physiological relevant cell composition, material properties, complex micro-structures and proper vascularization, but this is only the front end of the development. With further developments of advanced manufacturing and technology, we believe that DLP bioprinting will permit patient-specific tissue that models in a fast, convenient, and accurate in developing sophisticated in vitro disease models and precision medicine.

A C K N O W L E D G M E N T S
The authors acknowledge funding support from the Natural Science Foundation of China (grant number: 22005077), Heilongjiang Provincial Universities Basal Research Foundation-Youth Innovation Talent Project (grant number: 145109210), and Natural Science Foundation of Heilongjiang Province of China (grant number: LH2021B032).

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest.