Development and Application of a Mechanical Arm-Based In Situ 3D Bioprinting Method for the Repair of Skin Wounds

Development and Application of a Mechanical Arm-Based In Situ 3D Bioprinting Method for the Repair of Skin Wounds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Application of a Mechanical Arm-Based In Situ 3D Bioprinting Method for the Repair of Skin Wounds Yichen Luo, Xue xusong, Yuanzhong Gao, Jien Ma, Xuhui Zhou, Qi Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3575253/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Current treatments for skin wounds typically involve multiple surgical procedures that require complex processes and expensive costs, making it difficult to achieve timely treatment in field environments. We developed an innovative in situ printing method, utilizing robotic arm control, to address the significant challenges of large-scale skin wound repair resulting from natural disasters such as earthquakes, fires, and explosions during relief efforts. Our portable 3D printing equipment, which integrates debridement, precise 3D scanning and modeling of wounds, and compatibility with cell-loaded bioink, facilitates rapid repair of large-area skin wounds in specialized field environments. Compared with traditional methods, this in situ printing method has significant advantages, including the ability to customize treatment according to the unique needs of the wound, achieve rapid healing, and the potential to reduce the total cost. We conducted experiments on rats with full-thickness dorsal skin defects and compared the performance of in situ bioprinting method with commercial skin defect repair dressings. Our results demonstrate that the in situ bioprinted skin achieved faster wound healing and more uniform re-epithelialization than the commercial dressing treatment. This study demonstrates the potential of in situ bioprinting method as a promising and effective strategy for rapid skin wound healing, especially for patients in remote environments where traditional wound treatment methods may not be readily available or practical. Wound repair In situ 3D bioprinting Mechanical arm control Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Article Highlights The development of a portable 3D skin printer that can operate in harsh field environments and meet the customized needs of individuals quickly. The printer utilizes the flexibility of a mechanical arm, providing a larger printing area and smaller size that can be easily collapsed into a suitcase-sized package when not in use. The portable printer offers significant advantages over traditional skin injury treatment methods, contributing to expedited skin wound recovery. 1. Introduction In modern disaster relief scenarios, various natural disasters, including fires, earthquakes, and explosions, can cause severe skin damage to victims, with an increasing frequency of occurrence. According to the World Health Organization, approximately 11 million skin injury patients worldwide require treatment, of which 300 thousand die due to a lack of effective treatment [ 1 , 2 ]. Timely and proper treatment of severe skin damage is critical to minimize long-term impacts on patients' lives. When patients suffer from third-degree burns or deep second-degree burns, their skin lacks protection from the epidermis, making them vulnerable to severe infections that can endanger their lives. Large wound typically face difficulties healing without skin grafting, and even when recovery occurs, it is frequently accompanied by increased scarring and contracture, ultimately affecting skin function and appearance [ 3 ]. Prompt treatment of wounds, particularly those resulting from explosive injuries (e.g., burns and lacerations), is essential to protect underlying tissues from bacterial infection. Rapid interventions, such as applying skin substitutes to wounds, can reduce healing time and patient pains. However, wounds sustained in harsh environments may not receive timely professional medical care, increasing the risk of infection, hemorrhagic shock, amputation, and even death. Due to accidents, trauma, or diseases that can cause significant damage to the skin, it is crucial to prevent infection and regenerate the wound through rapid repair [ 4 ]. Traditional skin wound treatments, such as split skin grafts (SSGs), are effective for skin repair but pose additional challenges, including pain and donor site limitation, particularly for patients with large burn areas and limited healthy skin for reconstruction [ 5 , 6 ]. Tissue engineering techniques offer more complex biological skin equivalents as alternatives to autografts [ 7 ]. Engineered skin substitutes, composed of synthetic or biological scaffolds, aim to improve wound healing; however, they are expensive to produce and may lead to scarring post-healing [ 8 , 9 ]. Grafts containing both keratinocytes and fibroblasts have been shown to enhance skin regeneration in burn wounds and facilitate recovery in chronic diabetic foot ulcers compared to standard therapies [ 10 ]. Unfortunately, creating custom-sized skin substitutes for wounds with large and irregular shapes remains challenging [ 11 ]. Additionally, the equipment and complex procedures required for producing skin substitutes necessitate a sterile, controlled environment and flat platform [ 12 , 13 ], rendering them unsuitable for use in harsh field conditions. Compared to manual cell seeding or cell spraying [ 14 ], bioprinting offers the advantage of layer-by-layer free-form manufacturing, enabling targeted cell delivery to specific sites [ 15 ]. This technology has been employed in various applications, including in situ skin printing systems [ 16 – 19 ]. Binder from the Wake Forest University has developed an in situ skin printing system that moves a 3D printing system over a patient’s damaged skin and prints bioink directly to the wounds [ 20 ]. This system combines a laser-based wound scanning system with a cartridge-based delivery system, allowing for customized treatment plans based on individual patient needs. Cathal D O’Connell from the University of Wollongong has designed a handheld skin 3D printer with two ink tanks [ 21 , 22 ]. The operator manually performs in situ patient skin printing in a process akin to pen writing, with a pedal controlling the pneumatic extrusion pressure regulating the thickness of the printed lines. However, the above in situ printing methods have certain drawbacks, such as large device volume or low hand-held precision. This article presents the design and proof of concept for a portable skin wound repair 3D printing system tailored to field environments. By employing instant wound scanning and skin printing treatments, the healing time for skin repair is significantly reduced. The system directly prints skin cells into the wound, forming a layered skin structure by placing cells in specific wound areas, which achieves the purpose of rapidly covering the wound and providing growth factors to meet the urgent needs of field environments. 2. Methods 2.1 Portable bioprinter work area analysis Based on the three-DOF robot arm, coordinate system was established according to the D-H notation to analyze the kinematics positive solution (Fig. 1 a). The coordinate of end effector vector was derivated by the basic kinematics equation: $${}^{i}p={}_{i+1}{}^{i}Tp\left(n=3\right)$$ and transferred to the equation: $${}_{3}{}^{0}T=\left[\begin{array}{cc}\begin{array}{cc}{c}_{1}{c}_{23}& -{c}_{1}{s}_{23}\\ {s}_{1}{c}_{23}& -{s}_{1}{s}_{23}\end{array}& \begin{array}{cc}{s}_{1}& {c}_{1}({a}_{1}+{a}_{2}{c}_{2}+{a}_{3}{c}_{23})\\ {-c}_{1}& {s}_{1}({a}_{1}+{a}_{2}{c}_{2}+{a}_{3}{c}_{23})\end{array}\\ \begin{array}{cc}{s}_{23}& {c}_{23}\\ 0 & 0\end{array}& \begin{array}{cc}0& {a}_{2}{s}_{2}+{a}_{3}{s}_{23}+{d}_{1}\\ 0& 1\end{array}\end{array}\right]$$ In which, \({s}_{i}=sin{\theta }_{i}\) , \({c}_{i}=cos{\theta }_{i}\) , \({s}_{ij}=sin{(\theta }_{i}+{\theta }_{j})\) , \({c}_{ij}=cos{(\theta }_{i}+{\theta }_{j})\) , \({a}_{1}\) , \({a}_{2}\) , \({a}_{3}\) , \({\theta }_{1}\) , \({\theta }_{2}\) , \({\theta }_{3}\) represent length and rotation angle of the base, major arm, minor arm, respectively. \({d}_{1}\) represents the vertical distance of rotating axis between base and major arm. The joint rotation angles of base, major arm and minor arm were randomly discretized within limited area: $${\theta }_{i}={{\theta }_{i\text{m}\text{i}\text{n}}+({\theta }_{i\text{m}ax}-\theta }_{i\text{m}\text{i}\text{n}})\times rand(N, 1)$$ Then the equation of pose coordinates \({\left[x,y,z,\xi \right]}^{T}\) were obtained by the kinematics positive solution. And space clouds model was generated through the MATLAB software to calculate the 2D and 3D work area of the portable bioprinter. Scanning accuracy test A black and white checkerboard featuring 2 x 2 cm squares was printed using an Epson LaserJet Pro M452 printer (Japan) as a 2D template. The scanning system captured four images from different angles to reconstruct the checkerboard model. The average distance of the squares' intersection points from the restored model was calculated and compared to the template for final accuracy assessment. An irregular 3D structure was designed using SolidWorks software and subsequently printed by a CeraRay DLP printer (Xunshi Tech, China) with a printing accuracy of 10µm, serving as a 3D template. The scanning system took 24 photos from different angles to reconstruct the template model, which was then compared with the original model using Cloud Compare software to analyze the scanning system's accuracy. Cell culture Murine fibroblasts (L929) and human foreskin fibroblasts (HFF-1) used for cell printing were purchased from Human Fenghui Biotechnology Co., Ltd, China. Both L929 and HFF-1 were cultured in Dulbecco's Modified Eagle Medium/F12(Gibco, USA) supplemented with 10% v/v FBS and 1% P/S. Primary fibroblasts for animal experiments were harvest from neonatal rats. The skin was removed from the rat's back, trimmed into 15–25 mm 2 pieces after excising the subcutaneous tissue, and rinsed with PBS solution. The skin tissue was treated with 0.25% trypsin (Gibco, USA) at 4℃ for 16h to separate the epidermis from the dermis. The remaining dermis pieces were placed into 6-well plates with a small volume of Dulbecco's Modified Eagle Medium (Gibco, USA) containing 10% v/v FBS and 1% P/S. After incubating for 24h, the medium volume was adjusted to normal growth conditions. Following one week of culturing, proliferating cells were transferred to 25 cm 2 flasks for further incubation. Cells were cultured in an incubator set at 37℃ with a 5% CO2 environment, and the culture medium was replaced every two days. Cells were passaged at a ratio of 1:3, and all cells used in this study were maintained within passage 8. 2.2 Material The primary materials utilized in the experiment included sodium alginate (SA), gelatin, Gelatin methacryloyl(GelMA), and fibroblasts. All materials and reagents were of chemical grade. GelMA was purchased from Suzhou intelligent manufacturing research institute with a grafting rate at 60% and stored at -20℃. lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP, TCI, Japan) was dissolved in the DMEM culture medium at a final concentration of 0.5% w/v to obtain the pre-solvent for bioink and stored at -20℃ before the experiment. For the printability test, GelMA and sodium alginate (Sigma, USA) were added to the pre-solvent in 37℃ for 3h to reach their final concentrations, respectively. For the animal examination, the pre-solvent was sterilized using a 0.22µm sterilizing filter (Millipore, USA). GelMA and SA were sterilized under UV light exposure for 12h prior to being added to the pre-solvent to achieve a specific concentration. Bioink printability test Bioinks of varying concentrations were added into syringes at room temperature (25℃) with a continuous extrusion speed of 0.15mm/s and 27G needles to test the printability of mixed bioink. GelMA and SA were added to pre-solvent to achieve the final concentration of 2/5% SA/GelMA hydrogel. The hydrogel was loaded into a syringe and printed using our portable bioprinter in a grid pattern to assess the relationship between filament width and nozzle velocity at an extrusion speed of 0.18 mm/s, employing needles ranging from 25G to 30G. After printing, the entire printed structure was exposed to 405 nm visible light for 1 minute to facilitate complete photocrosslinking. HFF-1 and HUVECs were added to the sterilized 2/5% SA/GelMA bioink at final concentration of 1×10 6 cells/ml and 5×10 5 cells/ml, respectively. The bioink was then printed using the portable bioprinter in a grid pattern to analyze the machine's cell printability, with printing parameters including a 25G needle, 0.18 mm/s extrusion speed, and 8 mm/s nozzle velocity. After the printing process, the printed scaffold was exposed to 405 nm visible light for 1 minute for complete photocrosslinking and subsequently stained with a live-dead cell staining kit (Biovision, USA) after a 30-minute incubation. Animals 12 adult Sprague-Dawley (SD) rats, weighing 250 ± 30g, were purchased from Shanghai SLAC Laboratory Animal Co., LTD, and randomly assigned to 3 experimental groups, with 4 rats in each group. Prior to grafting, the dorsal skin of each rat was clipped, and a full-thickness skin defect wound (2 × 2 cm) was created. The animal study was approved by the Ethics Committee of Zhejiang University. In situ bioprinting for rats The dorsal skin defects in the rats were photographed from 36 angles to obtain comprehensive information, followed by the reconstruction of 3D wound models. Bioprinter pathways were automatically generated to fill the defects. For the first in situ bioprinting group, a 2/5% SA/GelMA bioink containing primary rat fibroblasts (1 × 10^6 cells/ml) was loaded into the syringe of the portable bioprinter and directly printed into the rats' defects. During the process, printing parameters were maintained at an extrusion speed of 0.15 mm/s and a nozzle velocity of 5 mm/s, using a 27G needle. After in situ printing, the printed tissue was crosslinked using 405 nm light for 1 minute. For the commercial dressing group, the dressing was extruded to uniformly fill the rat defects and allowed to solidify for 10 minutes. All groups received a breathable dressing (3M) to cover the dorsal defects, preventing infection from environmental exposure. Throughout the feeding and recovery process, all groups were treated equally. Wound analysis The wounds were photographed every 7 days and the wounds closure rate was calculated using ImageJ software. The wound closure rate was determined by the formula: 1- (wound size/original wound size) Histological examination Skin samples were collected on day 28 post-treatment. The harvested tissues were fixed in 4% paraformaldehyde phosphate buffer solution (XBIO, USA) for 24h and subsequently dehydrated through a series of alcohol solutions (75%, 85%, 95%, and 100%). The dehydrated samples were embedded in molten paraffin and frozen at -20℃ for 2h. The frozen samples were sectioned at a thickness of 4–5 \(\mu m\) . The paraffin sections were floated on 40℃ water for flattening, placed onto glass slides, and baked at 60℃ for 2h. After baking, the paraffin sections were stored at 4℃. To remove the paraffin, slides were treated with xylene and subsequently washed with a series of gradient alcohol solutions (100%, 95%, 90%, 80%, and 70%) followed by distilled water. The slides were then stained with Harris hematoxylin (Servicebio, China) for 5 minutes, washed with distilled water, and stained with eosin solution (Servicebio, China) for 2 minutes. The stained samples were dehydrated using gradient alcohol solutions (95% and 100%) and cleared with xylene. Structural images of the samples were captured using a NIKON DS-U3 microscope. Immunohistochemistry examination Paraffin sections were deparaffinized using xylene and washed with gradient alcohol (100%, 95%, 90%, 80%, and 70%) followed by distilled water. The sections were submerged in citric acid repair solution (pH 6.0) (G1202, Servicebio, China) for antigen retrieval and then dried at high temperature. Subsequently, the sections were washed three times with PBS (pH 7.4) (G0002, Servicebio, China) for 5 minutes per wash. After blocking with 3% BSA (G5001, Servicebio, China) for 30 minutes, the sections were incubated with primary antibodies overnight at 4℃. The sections were then washed three times with PBS (5 minutes per wash) and incubated with secondary antibodies for 1 hour. The primary antibodies used were mouse anti-rat endothelial cell marker (CD31) (GB12063, Servicebio, China), and the secondary antibodies were goat anti-mouse (GB25301, Servicebio, China) conjugated to Alexa Fluor 488. After washing three times with PBS (5 minutes per wash), DAPI (G1012, Servicebio, China) was added to the samples and incubated for 10 minutes, followed by an additional three PBS washes (10 minutes per wash). Images of the samples were captured using a NIKON ECLIPSE C1 microscope. 3. Results 3.1 Portable Bioprinter Design and Structure Currently, most 3D bioprinters on the market are bulky and unsuitable for specialized extreme environments such as field operations, training, and survival scenarios. The primary challenges in developing portable skin 3D printing equipment include portable design, cell survival rate, and database establishment [ 23 ]. First, portable 3D skin printing equipment necessitates a smaller volume and weight. In contrast to existing 3D bioprinting equipment [ 24 – 26 ], portable 3D bioprinters consist of printing nozzles, motion modules, and storage modules for printing materials (cells, biomaterials, growth factors, etc.). Integrated design is a major challenge, requiring precise planning for the printer's structure and the positioning of each component module. To ensure the necessary functions for rapid skin wound repair in field environments, the device should be as miniaturized and portable as possible. Traditional gantry structures achieve motion control by separately controlling the movements of the x-axis, y-axis, and z-axis. This approach has simple movement forms, convenient control, and low manufacturing costs, but the large frame does not meet the miniaturization requirements for the 3D printing system. We selected a more flexible mechanical arm structure for the portable bioprinter with a three-degree-of-freedom actuator, utilizing three stepper motors for motion control. Compared to the traditional gantry structure, the mechanical arm occupies less space when not in use and provides a larger printing area through arm extension during operation. The designed robotic arm features a connecting rod structure, with motors on both sides controlling the movement of the boom and forearm, respectively. Mounting holes at the base's four corners facilitate subsequent assembly, while those at the end of the robotic arm enable secure attachment of the printer head and scanning device, ensuring smooth movement. Figure 1 b and 1 c illustrates the three-axis robotic arm structure. After using the Monte Carlo method to simulate the working area of the robotic arm (Fig. 1 d and 1 e), we found that the manipulator-type structure offers a significantly larger printable range than the traditional gantry structure (Table 1 ). Table 1 Printing volume rate for bioprinters Allevi 3 bioprinter[ 27 ] SUNP biomaker[ 28 ] Portable bioprinter Dimensions(mm) 467 × 388 × 360 400 × 400 × 500 485 × 450 × 300 Work area(mm) 90 × 60 × 130 130 × 90 × 50 5.274e7(mm 3 ) Printing volume rate 1.08% 0.73% 80.55% The bioink is extruded into a filament through the printing nozzle, and the nozzle's directional movement achieves the desired structure and shape. Therefore, to ensure extrusion accuracy and structural requirements, the precision and stability of the printer head are critical factors. A syringe is fixed in the printer head and pushed precisely by a high-precision linear motor with a 1.25µm control accuracy (Fig. 1 f). Bioink filament extrusion is regulated by controlling the input pulses through the linear motor per unit time to adjust the material's extrusion speed at the nozzle. In combination with the nozzle's movement speed, the bioink filaments are controlled with high accuracy. This design enables the printer to achieve a printing accuracy of tens of microns with stable movement, meeting design requirements. To ensure timely and urgent rescue in remote environments and achieve automatic in situ printing with minimal intervention, the printer must quickly collect comprehensive wound information. Complete and accurate data enables the 3D reconstruction model to better represent the wound's state, facilitating an orderly treatment process. It is essential to integrate a scanning module capable of capturing wound information from multiple angles within the printer's limited space. A small 360-degree servo is fixed beneath the mechanical arm's end effector using a mounting frame and connected to a camera through two rotatable connectors. When the wound needs to be photographed from different angles for scanning, the servo motor can be controlled to rotate a specific angle to obtain the required roll angle, aligning the camera with the wound as needed. The servo's movements are controlled by the printer's main controller, which is compatible with the mechanical arm's movements. As shown in Fig. 2 , the entire system is powered by a 12V lithium battery, with a voltage regulator chip providing stable voltage due to the varying requirements of different modules. The movement module comprises a three-axis motor for the robotic arm and a linear guide rail for the Z-axis to control the nozzle and camera. The temperature module consists of a constant-temperature box for bioink storage and a system cooling fan. The control module employs a Raspberry Pi to connect input and output devices via USB and High Definition Multimedia Interface (HDMI). When the PC is on the same local area network as the Raspberry Pi, the printer can be controlled through a Virtual Network Console (VNC), enabling remote monitoring and timely interventions. 3.2 3D Model Reconstruction Process To ensure the bioprinter can automatically select the printing system parameters based on the wound and reduce the workload of medical staff, the printer requires an integrated scanning module capable of performing high-precision 3D reconstruction of skin wounds to fill the wound defects. The Fig. 3 illustrates the entire 3D reconstruction process. The actuator at the end of the printer drives the camera to collect wound information from various angles, obtaining the necessary wound images for 3D reconstruction. An algorithm is then used to restore the 3D model of the skin wound. The system allows users to adjust the reconstructed model, mark the wound position, and define printing parameters such as filament distance and layer height. Finally, the user interface provides users with optional printer preset schemes, and upon confirmation, a customized printing program is executed on the wound. The bioink carrier used in this study offers appropriate support for maintaining cell viability, rapid crosslinking, and accurate deposition to promote the formation of multilayer cell-loaded skin structures. After obtaining the wound images, this paper employs the Scale Invariant Feature Transform (SIFT) feature matching algorithm to extract feature points from the image [ 29 , 30 ]. SIFT is an image local feature descriptor based on scale space that maintains image scaling, rotation, and even affine transformation, offering stability, uniqueness, abundance, high speed, and scalability. The algorithm uses an image pyramid to detect features in scale space, determining the position and scale of key points (Fig. 4 a). It then uses the gradient of the main direction within the key point field as the directional feature of the point, achieving operator independence from scale and direction. To determine the 3D position of a point in a scene, traditional photogrammetry methods require knowledge of the camera's 3D position and orientation, whereas the motion recovery structure method for sparse reconstruction does not. By matching feature points, multiple view images of an object can be connected to calculate the camera pose and 3D coordinates (Fig. 4 b). Multi-view geometry serves as the foundation for sparse reconstruction. The same image undergoes different projections in various views using matrices. Two views are initially selected, with new views added sequentially to calculate camera intrinsic parameters, pose, and other parameters. The spatial coordinates of the matching feature points are then determined through triangulation. During multi-view reconstruction, reprojection may occur (one feature point on one image can match feature points on multiple other images). In such cases, minimizing the weighted square and reprojection error related to the cost function requires bundle adjustment, which greatly benefits the accuracy of subsequent processing. Although sparse reconstruction using the incremental method can be time-consuming, the resulting reconstruction is more robust and the model's accuracy is higher, which is crucial for precise wound printing. For flat accuracy, we fabricated a standard chessboard with a 2cm side and utilized the scanning system of the printer for 3D reconstruction. The black and white intersection point set of the checkboard was obtained using the Harris corner detection algorithm [ 31 ], and was transformed to the X-Y plane through spatial transformation (Fig. 4 c). The average error between each point is indicative of the flat reconstruction accuracy of the scanning system. As for the 3D accuracy, we printed an irregular 3D sample using an SLA 3D printer (with an accuracy of 5µm) and also utilized the scanning system for 3D reconstruction. The accuracy of the reconstructed model was obtained using CloudCompare, an open-source 3D point cloud processing software licensed under the GPL, with the initial sample treated as a standard (Fig. 4 d). The reconstruction error below the model is relatively large, which can be attributed to the scanning system’s inability to fully capture the area due to the influence of the horizontal workbench. However, the accuracy above the model is maintained at a high level (Table 2 ). Table 2 Scanning accuracy test Mean distance Std deviation Flat scanning 25µm 25µm 3D scanning 37.2µm 105µm In order to obtain a processable wound model, it is necessary to generate a dense point cloud. During sparse reconstruction, the 3D coordinates of some feature points have already been determined. According to the principle of multi-view geometry, a point in space will have a projected point in the views of multiple images. When a matching point pair is identified, the 3D depth of the corresponding point can be calculated based on the camera pose information. The 3D reconstruction model is stored in the OBJ file format, which is a data format file that includes simple vertex coordinates, texture coordinates, and surface information. The 3D point coordinates are linked to the texture color by reading the information in the OBJ file, enabling the reconstructed 3D model to be visually displayed. The completed model includes both the wound area and non-wound part, making it difficult to accurately distinguish the wound edge from the normal area due to complex factors such as hair, light, stains, and tissue fluid leakage. Rough edge information is displayed in the user interface, and an edge search algorithm is employed to obtain the accurate wound boundary. After simulating the surface morphology, the approximate slope of the original skin morphology is fitted through the skin wound edge points, and the missing skin parts on the wound are reconstructed. For the simulated wound surface, the height of each layer from the lowest point of the wound is obtained by inputting the slice thickness, and the printing trajectory for each slice is autogenerated using a multi-layer path-filling algorithm (Fig. 4 e). The resulting printed model can fulfill the requirements of skin 3D printing, effectively covering the wound area to promote the healing rate of the injured part. 3.3 Bioprinter Validation in Hydrogel Printing Currently, the range of biomaterials used for human skin reconstruction is relatively limited, particularly in the field of 3D bioprinting, which demands exceptional printability [ 11 ]. Alginate, a non-toxic and degradable natural polymer material, is widely utilized in biofabrication research due to its easy crosslinking, excellent biocompatibility, biodegradability, and abundant sources [ 32 ]. The micro-porous environment created by alginate scaffolds can accommodate numerous cells and maintain cell phenotypes, promoting cell adhesion and nutrient penetration. Moreover, the products resulting from enzymatic degradation in the human body exhibit no toxic effects. Gelatin, a polypeptide molecular polymer substance obtained through partial collagen hydrolysis, preserves collagen's specific amino acid composition and displays strong biocompatibility, thanks to the presence of arginine-glycine-aspartic acid (RGD) sequences that enhance cell-material affinity. This attribute makes gelatin a popular choice in tissue engineering research [ 33 ]. GelMA is a biomaterial synthesized by grafting methacryloyl groups onto gelatin, preserving gelatin's biocompatibility and temperature sensitivity while adding photoreactive crosslinking functionality, making it an ideal bioink for 3D bioprinting [ 34 ]. Considering the characteristics and requirements of skin tissue, this project tested sodium alginate (SA) and GelMA for printing and ultimately selected a blended material as the printing bioink for skin wound repair. The printability of hydrogels was observed at the nozzle outlet at room temperature (25℃), and the extrusion conditions for different concentrations of mixed bioink were categorized into three types: 1)Unextrudable: The bioink cannot be extruded through the needle or forms wrinkled filaments at the needle outlet, resulting in discontinuous printed fibers. 2)Printable: The bioink forms regular and smooth filaments through the needle, enabling stable fiber printing on the platform. 3)Overextrudable: The bioink is extruded as spindle-shaped filaments, sometimes breaking into droplets, leading to stacked printed fibers. The printability experiment results are illustrated in the Fig. 5 . At low concentrations of GelMA and SA (0 ~ 4% GelMA and 0 ~ 1% SA), the solution's viscosity is too low to form regular bioink filaments, indicating poor printability for skin printing. However, when the GelMA/SA solution concentration (> 20% GelMA and > 4% SA) is too high, the bioink exhibits excessive viscosity, preventing smooth extrusion at the nozzle outlet and formation of a continuous tissue scaffold. Consequently, the GelMA and SA concentrations in the red and yellow areas of the Fig. 5 a are unsuitable for printing bioink. In this experiment, we determined that a specific range of mixed GelMA/SA hydrogel solution concentration (green area in the Fig. 5 a) offers suitable viscosity to form smooth and uniform filaments upon extrusion, as SA provides high viscosity for extrusion and GelMA enables easy photo-crosslinking to maintain a stable structure in the body. For this project, a 2/5% SA/GelMA hydrogel was selected as the printing bioink. Various nozzle moving speeds and constant extrusion speeds were tested to form hydrogel filaments with different needles. By adjusting the printing parameters, hydrogel lines with a diameter of 200–1000 µm were obtained, as shown in the Fig. 5 b and 5 c, which were deemed suitable for tissue reconstruction. The portable bioprinter was employed for cell printing and experimental research, which has been verified by different cell types. 2/5% SA / GelMA mixed with murine L929 and HFF-1 were used as printing bioink and printed to test cell viability. The experimental results, displayed in Fig. 5 d, 5 e and 5 f, demonstrated that the survival rates of L929 and HFF-1 cells were (94.91 ± 3.02%) and (89.3% ± 5.85%), respectively. These findings were quantitatively analyzed using ImageJ software. 3.4 Bioprinter Validation in Rat Wound Models Animal models serve as essential experimental tools in modern medicine, facilitating our understanding of the objective laws governing life sciences. In this study, we employed SD rats in skin damage repair experiments using dorsal skin reconstruction models. Artificially controlling the shape, size, and depth of wounds on rats allows for a highly versatile approach in animal experiments focused on skin defect repair [ 35 ]. SD rats were divided into three groups: in situ bioprinted skin containing primary fibroblasts, commercial skin dressing (Jihao, China), and an untreated group. A 2x2 cm dorsal wound was created on each rat's back, with three rats per group to ensure reliable results. The rat experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University and were performed according to the set regulations for animal experiments. We used our in situ skin bioprinter to print GelMA hydrogel containing primary fibroblasts from rats' dorsal tissues onto the experimental group's wounds, applied commercial wound treatment dressing to the control group, and left the untreated group as a blank comparison. We monitored the wounds and recorded data weekly. No inflammation occurred during the entire process, and most rats recovered within 28 days. On day 28, we evaluated skin slices stained with hematoxylin and eosin (H&E) and immunohistochemistry staining of CD31 to compare the healing effects among the three groups. As illustrated in Fig. 6 a, 6 b and 6 c, the three groups exhibited distinct healing trends. By day 7, the in situ bioprinting group's wound closure rate (33.02 ± 10.11%) was comparable to the commercial dressing group (26.07 ± 2.82%), but significantly different from the untreated group (13.16 ± 2.01%). By day 14, the in situ bioprinting group's wound closure rate (81.99 ± 3.89%) substantially surpassed that of the commercial dressing group (60.17 ± 3.19%) and the untreated group (25.27 ± 4.12%). This trend persisted until day 21, with closure rates of 95.93 ± 2.18%, 87.76 ± 2.33%, and 76.65 ± 1.56%, respectively. By day 28, the wounds in the in situ bioprinting group and the commercial dressing group had completely healed, while the untreated group's wounds maintained a closure rate of 90.65 ± 1.16%. According to other research on skin healing, skin scaffolds provide extracellular matrix (ECM) structures, functional cellular components, and other biological factors to enable and accelerate skin regrowth [ 36 – 38 ]. Fibroblasts secrete growth factors for skin reconstruction [ 20 ], which may have contributed to the rapid healing observed in the in situ bioprinting group. Figure 6 d presents the H&E staining results, which were evaluated to compare the reconstructed skin following different treatments. Native healthy rat skin tissue served as a control group to assess the healing effects of each experimental group. Morphological analysis revealed that the recovered skin in the in situ bioprinting group exhibited "ridge" patterns in the epidermis, similar to native skin. In the commercial dressing group, fewer "ridge" patterns were observed in the regenerated skin, while the untreated group displayed unreconstructed portions, indicating superior healing effects in the in situ bioprinting group. Figure 6 e presents the epidermal thickness of different groups, demonstrating that the regenerated skin in all three groups was considerably thicker than the native rat skin. The in situ bioprinting group had the thickest epidermis, while the commercial dressing group exhibited more uniform epidermis compared to the untreated group. During the skin wound recovery process, autologous cells migrate to the wound area and accelerate healing through cell proliferation, which may result in the increased thickness of the epidermis. In situ bioprinted skin may provide a better bioscaffold that fully covers the wound area, facilitating cell migration. Additionally, growth factors secreted by the cells contained within the in situ bioprinted skin may contribute to faster healing by attracting autologous cells. The immunohistochemical staining results of CD31 are displayed in Fig. 6 f and 6 g, illustrating the neovascularization within the regenerated skin. The number of microvessels in the in situ bioprinted skin group was significantly higher than in the commercial dressing group, while no obvious microvascular regeneration was observed in the untreated group. These findings were numerically analyzed using ImageJ software. Upon closer examination of the enlarged image, a distinct hollow vessel structure was evident in the in situ bioprinted group, with vessel diameters reaching 30–40 µm (Fig. 6 h). The experimental results demonstrate that our in situ bioprinted method can promote wound epithelialization. 4. Discussion In this study, we analyzed the risks associated with various skin injuries in harsh outdoor environments and proposed different treatment methods based on the severity of the injury. To address the challenges of treating skin wounds in the field, we developed a portable 3D skin printer that can capable to harsh environments and meet the needs of individuals quickly. Traditional bioprinters use gantry structures that are typically large in size and require strict operating conditions, making them either immobile or only mobile within the laboratory [ 12 , 20 , 39 ]. In contrast, our printer utilizes the flexibility of a mechanical arm, providing a larger printing area and smaller size that can be easily collapsed into a suitcase-sized package when not in use. This allows the printer to be moved and operated outside of laboratory settings. We also designed a portable bio-ink compatible with this printer and conducted a comparative test. The experimental results demonstrate that the portable printer offers significant advantages over traditional skin injury treatment methods. Our bioprinter system can rapidly and precisely deposit materials and cells onto specific areas of the wound with high accuracy using an extrusion method. In an animal study using a rat skin defect model, we demonstrated that our system effectively covers the wound with a skin structure composed of rat primary fibroblasts, contributing to expedited skin wound recovery. Wound area assessments over a four-week period showed that wounds treated with in situ bioprinted skin closed more quickly than those treated with commercial dressings. Compared to the use of robotic arms, another trend in portable printers is handheld devices that allow for direct writing of bio-materials containing specific living cells. These handheld bioprinters are smaller than robotic devices, allowing for adjustment of the printing process according to changes in the wound conditions [ 21 , 35 ]. However, the extracellular matrix used for tissue repair must be carefully considered, including shear-thinning property, hydrogel pore-size and rapid gelation [ 19 , 39 ]. Meanwhile, handheld bioprinters have lower precision and are difficult to use for manufacturing complex structures. They can only be used to repair simple structures, and require highly skilled operators, making them unsuitable in harsh field environments. In the future, we plan to use the printing system to investigate the printing properties of other biological materials and cell types to further accelerate wound healing. This technology has broad application prospects, including the potential to address the vast global demand for skin wound repair, reduce the secondary injuries and psychological pain experienced by skin wound patients due to skin transplantation, and find applications in drug testing and screening, as well as medical aesthetics. Furthermore, this technology could be applied to battlefield wound repair in even more challenging conditions, providing military personnel with essential equipment for emergency combat injury treatment and enhancing their ability to address sudden casualties during field operations. Declarations Author Contributions YCL and XZ wrote the manuscript; YZG designed the experiments; XHZ and QL assisted with experimental setup; JEM and HYY jointly supervised this work; BZ was in charge of the whole trial. All authors have read and agreed to the published version of the manuscript. Funding This study has been supported by the National Key Research and Development Program of China (Grant No. 2018YFA0703000). Availability of data and materials The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Ethical approval This article does not contain any studies with human participants, however, animal model was used. The study protocol for the experimental use of the animals was approved by the ethics committee of Zhejiang University according to the standard operating procedures (SOPs). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creativecommons.org/ licenses/ by/4. 0/. References Peck M D (2012) Epidemiology of burns throughout the World. Part II: Intentional burns in adults 38(5):630-637. https://doi.org/10.1016/j.burns.2011.12.028 Jiang Y F, Huang S, Fu X B, Liu H W, Ran X W, Lu S L, Hu D H, Li Q A, Zhang H W, Li Y, Wang R X, Xie T, Cheng B A, Wang L F, Liu Y, Ye X B, Han C M, Chen H D (2011) Epidemiology of chronic cutaneous wounds in China 19(2):181-188. https://doi.org/10.1111/j.1524-475X.2010.00666.x Singer, Adam J, Clark, Richard A F (1999) Cutaneous Wound Healing Min S J, Lee J S, Nah H, Moon H J, Lee S J, Kang H J, Hwang Y S, Kwon I K, Heo D N (2023) Degradable and Tunable Keratin-fibrinogen Hydrogel as Controlled Release System for Skin Tissue Regeneration 20(3):1049-1059. Liu T, Qiu C, Ben C, Li H H, Zhu S H (2019) One-step approach for full-thickness skin defect reconstruction in rats using minced split-thickness skin grafts with Pelnac overlay 7(1) https://doi.org/10.1186/s41038-019-0157-0 Mohamed Haflah N H, Ng M H, Mohd Yunus M H, Naicker A S, Htwe O, Abdul Razak K A, Idrus R (2018) Massive Traumatic Skin Defect Successfully Treated with Autologous, Bilayered, Tissue-Engineered MyDerm Skin Substitute: A Case Report 8(2):e38. https://doi.org/10.2106/jbjs.Cc.17.00250 Martin-Piedra M A, Santisteban-Espejo A, Moral-Munoz J A, Campos F, Chato-Astrain J, Garcia-Garcia O D, Sanchez-Porras D, Campos A (2020) An Evolutive and Scientometric Research on Tissue Engineering Reviews 26(9-10):569-577. https://doi.org/10.1089/ten.tea.2019.0247 Lesher A, Curry R H, Evans J, Smith V A, Fitzgerald M T, Cina R A, Streck C J, Hebra A (2011) Effectiveness of Biobrane for treatment of partial-thickness burns in children 46(9):1759-1763. Lal S, Barrow R E, Wolf S E, Chinkes D L, Hart D W, Heggers J P, Herndon D N (2000) Biobrane (R) improves wound healing in burned children without increased risk of infection 14(3):314-318. https://doi.org/10.1097/00024382-200014030-00013 Hayes D W, Webb G E, Mandracchia V J, John K J (2001) Full-thickness burn of the foot: successful treatment with Apligraf. A case report 18(1):179-188. Kirchmajer D M, Gorkin R, Panhuis M I H (2015) An overview of the suitability of hydrogel-forming polymers for extrusion-based 3D-printing 3(20):4105-4117. https://doi.org/10.1039/c5tb00393h Ozbolat I T (2015) Bioprinting scale-up tissue and organ constructs for transplantation 33(7):395-400. https://doi.org/10.1016/j.tibtech.2015.04.005 Li X, Lian Q, Li D, Xin H, Jia S (2017) Development of a Robotic Arm Based Hydrogel Additive Manufacturing System for In-Situ Printing 7(1):73. Lv J, Yu X, Shen Q, Zhu Y (2013) The Application Progress of Stem Cells in Construction and Regeneration of Engineering Tissue 32(1):99. Calvert P (2007) Printing cells 318(5848):208-209. https://doi.org/10.1126/science.1144212 Keriquel V, Guillemot F, Arnault I, Guillotin B, Miraux S, Amedee J, Fricain J C, Catros S (2010) In vivo bioprinting for computer- and robotic-assisted medical intervention: preliminary study in mice 2(1) https://doi.org/Artn 014101 10.1088/1758-5082/2/1/014101 Skardal A, Mack D, Kapetanovic E, Atala A, Jackson J D, Yoo J, Soker S (2012) Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Healing of Large Skin Wounds 1(11):792-802. https://doi.org/10.5966/sctm.2012-0088 Keriquel V, Oliveira H, Remy M, Ziane S, Delmond S, Rousseau B, Rey S, Catros S, Amedee J, Guillemot F, Fricain J C (2017) In situ printing of mesenchymal stromal cells, by laser-assisted bioprinting, for in vivo bone regeneration applications 7 https://doi.org/ARTN 1778 10.1038/s41598-017-01914-x Hakimi N, Cheng R, Leng L, Sotoudehfar M, Ba P Q, Bakhtyar N, Amini-Nik S, Jeschke M G, Gunther A (2018) Handheld skin printer: in situ formation of planar biomaterials and tissues 18(10):1440-1451. https://doi.org/10.1039/c7lc01236e Albanna M, Binder K W, Murphy S V, Kim J, Qasem S A, Zhao W, Tan J, El-Amin I B, Dice D D, Marco J, Green J, Xu T, Skardal A, Holmes J H, Jackson J D, Atala A, Yoo J J (2019) In Situ Bioprinting of Autologous Skin Cells Accelerates Wound Healing of Extensive Excisional Full-Thickness Wounds 9(1):1856. https://doi.org/10.1038/s41598-018-38366-w Oconnell C D, Bella C D, Thompson F W, Augustine C, Beirne S, Cornock R, Richards C J, Chung J H Y, Gambhir S, Yue Z (2016) Development of the Biopen: a handheld device for surgical printing of adipose stem cells at a chondral wound site 8(1):015019-015019. Duchi S, Onofrillo C, Oconnell C D, Blanchard R, Augustine C, Quigley A F, Kapsa R M I, Pivonka P, Wallace G G, Bella C D (2017) Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair 7(1):5837. Singh S, Choudhury D, Yu F, Mironov V, Naing M W (2020) In situ bioprinting - Bioprinting from benchside to bedside? 101:14-25. https://doi.org/10.1016/j.actbio.2019.08.045 Choudhury D, Anand S, Naing M W (2018) The arrival of commercial bioprinters - Towards 3D bioprinting revolution! 4(2) https://doi.org/10.18063/IJB.v4i2.139 Duan B, Hockaday L A, Kang K H, Butcher J T (2013) 3D Bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels 101(5):1255-1264. https://doi.org/10.1002/jbm.a.34420 Lee C H, Cook J L, Mendelson A, Moioli E K, Yao H, Mao J J (2010) Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study 376(9739):440-448. https://doi.org/10.1016/s0140-6736(10)60668-x Allevi (2023) Allevi 3. https://www.allevi3d.com/allevi-3/. Accessed on 7th Nov, 2023 BIOTECH S (2023) SUNP BIOMAKER 2i. http://www.sunpbiotech.cn/Printer-system/8.html. Accessed on 7th Nov, 2023 Lowe D G (1999) Object recognition from local scale-invariant features Lowe D G (2004) Distinctive Image Features from Scale-Invariant Keypoints 60(2):91-110. Harris C G, Stephens M J (1988) A combined corner and edge detector Agrawal P, Soni S, Mittal G, Bhatnagar A (2014) Role of Polymeric Biomaterials as Wound Healing Agents 13(3):180-190. https://doi.org/10.1177/1534734614544523 Daly A C, Prendergast M E, Hughes A J, Burdick J A (2021) Bioprinting for the Biologist 184(1):18-32. https://doi.org/10.1016/j.cell.2020.12.002 Xiao W Q, He J K, Nichol J W, Wang L Y, Hutson C B, Wang B, Du Y A, Fan H S, Khademhosseini A (2011) Synthesis and characterization of photocrosslinkable gelatin and silk fibroin interpenetrating polymer network hydrogels 7(6):2384-2393. https://doi.org/10.1016/j.actbio.2011.01.016 Di Bella C, Duchi S, O'Connell C D, Blanchard R, Augustine C, Yue Z L, Thompson F, Richards C, Beirne S, Onofrillo C, Bauquier S H, Ryan S D, Pivonka P, Wallace G G, Choong P F (2018) In situ handheld three-dimensional bioprinting for cartilage regeneration 12(3):611-621. https://doi.org/10.1002/term.2476 Jin G R, Prabhakaran M P, Ramakrishna S (2014) Photosensitive and Biomimetic Core-Shell Nanofibrous Scaffolds as Wound Dressing 90(3):673-681. https://doi.org/10.1111/php.12238 Bhardwaj N, Sow W T, Devi D, Ng K W, Mandal B B, Cho N J (2015) Silk fibroin-keratin based 3D scaffolds as a dermal substitute for skin tissue engineering (vol 7, pg 53, 2015) 7(1) https://doi.org/10.1039/c4ib90045f Zhong S P, Zhang Y Z, Lim C T (2010) Tissue scaffolds for skin wound healing and dermal reconstruction 2(5):510-525. https://doi.org/10.1002/wnan.100 Wang M, He J, Liu Y, Meng L, Li D, Jin Z, Jin Z The trend towards in vivo bioprinting Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 Jan, 2024 Reviewers agreed at journal 22 Dec, 2023 Reviews received at journal 12 Dec, 2023 Reviewers agreed at journal 03 Dec, 2023 Reviewers invited by journal 02 Dec, 2023 Editor assigned by journal 29 Nov, 2023 Submission checks completed at journal 25 Nov, 2023 First submitted to journal 07 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3575253","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":253100483,"identity":"cb18773d-890d-4bd7-bc4b-8f977e20723c","order_by":0,"name":"Yichen Luo","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yichen","middleName":"","lastName":"Luo","suffix":""},{"id":253100485,"identity":"78b5b05b-092a-48d6-8a8d-caaece29a076","order_by":1,"name":"Xue xusong","email":"","orcid":"","institution":"Zhejiang 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16:13:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3228090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe structure and workspace of printer.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e The D-H coordinate system of the robotic arm. \u003cstrong\u003eb\u003c/strong\u003e The structure model of the mechanical arm, which can be folded into the shape of \u003cstrong\u003ec\u003c/strong\u003e in non working state. \u003cstrong\u003ed\u003c/strong\u003e 2D workspace projection and \u003cstrong\u003ee\u003c/strong\u003e envelope curve of maximum printable space obtained through Monte Carlo simulation. \u003cstrong\u003ef \u003c/strong\u003eThe nozzle structure of the printer.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3575253/v1/699d8caabb5ef1d0898a4ed7.png"},{"id":47308007,"identity":"8f299f6d-9457-4544-b8b9-c4c0414c281a","added_by":"auto","created_at":"2023-11-29 16:13:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":631194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControl system of portable bioprinter. \u003c/strong\u003eThe system incorporates camera control to scan the wound and capture detailed photos, enabling the reconstruction of a 3D model. Through manipulation of a mechanical arm and a specialized nozzle, the system facilitates effective wound repair. Additionally, seamless communication with input/output devices is achieved via Bluetooth technology. To maintain optimal conditions for the preservation of bio-ink, a temperature-controlled chamber is utilized. Notably, the system is designed with portability in mind, as it relies on the power supply of lithium batteries.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3575253/v1/1e1ffe0780e9e285e16c9df2.png"},{"id":47308009,"identity":"51401656-9729-4852-b3ce-ff17637e3535","added_by":"auto","created_at":"2023-11-29 16:13:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7605198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D model reconstruction process.\u003c/strong\u003e The system employs a camera to collect images from various angles and utilizes advanced algorithms to reconstruct a three-dimensional model of the skin wound. This enables a comprehensive and accurate representation of the wound's structure and dimensions.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3575253/v1/4d104b92d85b26b58181cdeb.png"},{"id":47308923,"identity":"bbc166eb-2fb2-464b-9844-33c7af570693","added_by":"auto","created_at":"2023-11-29 16:21:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":549744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe processing of wound model. a\u003c/strong\u003e Image pyramids can be used to detect the position and scale of key points. \u003cstrong\u003eb\u003c/strong\u003e Calculate camera pose by matching feature points. Planar and 3D physical models are created and reconstructed using the scanning system to obtain \u003cstrong\u003ec\u003c/strong\u003e flat scanning accuracy and \u003cstrong\u003ed\u003c/strong\u003e 3D scanning accuracy. \u003cstrong\u003ee\u003c/strong\u003e Layered slicing process. (i) Wound model before and after surface coverage. (ii) Auto-designed slices and (iii) interlayer path filling.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3575253/v1/864a9c082d08b2b2e66b2db6.png"},{"id":47308008,"identity":"dc124729-81c0-4992-8952-932e41b5390c","added_by":"auto","created_at":"2023-11-29 16:13:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3610333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimentation of hydrogel printing. a\u003c/strong\u003e The hydrogel in green area shows great printability. \u003cstrong\u003eb\u003c/strong\u003e The diameter of hydrogel is significantly related to the size of needle and the speed of movement. \u003cstrong\u003ec\u003c/strong\u003e Line printed with SA/GelMA using appropriate parameters. Printing with the hydrogel of \u003cstrong\u003ed\u003c/strong\u003e murine fibroblasts (L929) and \u003cstrong\u003ee\u003c/strong\u003e human fibroblasts (HFF-1) shows \u003cstrong\u003ef\u003c/strong\u003e a high survival rate.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3575253/v1/1eabd31e9f273ece62577f12.png"},{"id":47308010,"identity":"44954bbe-ddaa-49f6-9a42-d4434859755e","added_by":"auto","created_at":"2023-11-29 16:13:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":15068669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepair experimentation of rat wound. a\u003c/strong\u003e Digital photos were taken for each of the time points (days 0, 7, 14, 21) and compiled for each of the wounds, \u003cstrong\u003eb\u003c/strong\u003e wound sizes for different treatments (*P\u0026lt;0.05, **P\u0026lt;0.01) and \u003cstrong\u003ec\u003c/strong\u003e wound closure over time demonstrates accelerated wound closure time. Epithelialization in the bioprinted skin was confirmed through \u003cstrong\u003ed\u003c/strong\u003e hematoxylin and eosin (H\u0026amp;E) staining results of different treatments and \u003cstrong\u003ee\u003c/strong\u003e Reconstructed epidermis thickness (**P\u0026lt;0.01, ***P\u0026lt;0.001). \u003cstrong\u003ef\u003c/strong\u003e The immunohistochemical staining of endothelial cell marker (CD 31) and \u003cstrong\u003eg\u003c/strong\u003e fluorescence intensity in different groups illustrates the neovascularization and displays the clear \u003cstrong\u003eh\u003c/strong\u003eluminal structure of regenerated vessels.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3575253/v1/8f681a7c6c66d448a57e0c58.png"},{"id":47310757,"identity":"87afe0f1-5ac4-4436-b3a6-1727da600a42","added_by":"auto","created_at":"2023-11-29 16:37:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4382154,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3575253/v1/8cdf549c-078a-4aef-89b6-a151a0516683.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Application of a Mechanical Arm-Based In Situ 3D Bioprinting Method for the Repair of Skin Wounds","fulltext":[{"header":"Article Highlights","content":"\u003cul\u003e\n \u003cli\u003eThe development of a portable 3D skin printer that can operate in harsh field environments and meet the customized needs of individuals quickly.\u003c/li\u003e\n \u003cli\u003eThe printer utilizes the flexibility of a mechanical arm, providing a larger printing area and smaller size that can be easily collapsed into a suitcase-sized package when not in use.\u003c/li\u003e\n \u003cli\u003eThe portable printer offers significant advantages over traditional skin injury treatment methods, contributing to expedited skin wound recovery.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eIn modern disaster relief scenarios, various natural disasters, including fires, earthquakes, and explosions, can cause severe skin damage to victims, with an increasing frequency of occurrence. According to the World Health Organization, approximately 11\u0026nbsp;million skin injury patients worldwide require treatment, of which 300 thousand die due to a lack of effective treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Timely and proper treatment of severe skin damage is critical to minimize long-term impacts on patients' lives. When patients suffer from third-degree burns or deep second-degree burns, their skin lacks protection from the epidermis, making them vulnerable to severe infections that can endanger their lives. Large wound typically face difficulties healing without skin grafting, and even when recovery occurs, it is frequently accompanied by increased scarring and contracture, ultimately affecting skin function and appearance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrompt treatment of wounds, particularly those resulting from explosive injuries (e.g., burns and lacerations), is essential to protect underlying tissues from bacterial infection. Rapid interventions, such as applying skin substitutes to wounds, can reduce healing time and patient pains. However, wounds sustained in harsh environments may not receive timely professional medical care, increasing the risk of infection, hemorrhagic shock, amputation, and even death. Due to accidents, trauma, or diseases that can cause significant damage to the skin, it is crucial to prevent infection and regenerate the wound through rapid repair [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Traditional skin wound treatments, such as split skin grafts (SSGs), are effective for skin repair but pose additional challenges, including pain and donor site limitation, particularly for patients with large burn areas and limited healthy skin for reconstruction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Tissue engineering techniques offer more complex biological skin equivalents as alternatives to autografts [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Engineered skin substitutes, composed of synthetic or biological scaffolds, aim to improve wound healing; however, they are expensive to produce and may lead to scarring post-healing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Grafts containing both keratinocytes and fibroblasts have been shown to enhance skin regeneration in burn wounds and facilitate recovery in chronic diabetic foot ulcers compared to standard therapies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Unfortunately, creating custom-sized skin substitutes for wounds with large and irregular shapes remains challenging [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, the equipment and complex procedures required for producing skin substitutes necessitate a sterile, controlled environment and flat platform [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], rendering them unsuitable for use in harsh field conditions.\u003c/p\u003e \u003cp\u003eCompared to manual cell seeding or cell spraying [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], bioprinting offers the advantage of layer-by-layer free-form manufacturing, enabling targeted cell delivery to specific sites [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This technology has been employed in various applications, including in situ skin printing systems [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Binder from the Wake Forest University has developed an in situ skin printing system that moves a 3D printing system over a patient\u0026rsquo;s damaged skin and prints bioink directly to the wounds [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This system combines a laser-based wound scanning system with a cartridge-based delivery system, allowing for customized treatment plans based on individual patient needs. Cathal D O\u0026rsquo;Connell from the University of Wollongong has designed a handheld skin 3D printer with two ink tanks [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The operator manually performs in situ patient skin printing in a process akin to pen writing, with a pedal controlling the pneumatic extrusion pressure regulating the thickness of the printed lines. However, the above in situ printing methods have certain drawbacks, such as large device volume or low hand-held precision. This article presents the design and proof of concept for a portable skin wound repair 3D printing system tailored to field environments. By employing instant wound scanning and skin printing treatments, the healing time for skin repair is significantly reduced. The system directly prints skin cells into the wound, forming a layered skin structure by placing cells in specific wound areas, which achieves the purpose of rapidly covering the wound and providing growth factors to meet the urgent needs of field environments.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Portable bioprinter work area analysis\u003c/h2\u003e \u003cp\u003eBased on the three-DOF robot arm, coordinate system was established according to the D-H notation to analyze the kinematics positive solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The coordinate of end effector vector was derivated by the basic kinematics equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$${}^{i}p={}_{i+1}{}^{i}Tp\\left(n=3\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eand transferred to the equation:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$${}_{3}{}^{0}T=\\left[\\begin{array}{cc}\\begin{array}{cc}{c}_{1}{c}_{23}\u0026amp; -{c}_{1}{s}_{23}\\\\ {s}_{1}{c}_{23}\u0026amp; -{s}_{1}{s}_{23}\\end{array}\u0026amp; \\begin{array}{cc}{s}_{1}\u0026amp; {c}_{1}({a}_{1}+{a}_{2}{c}_{2}+{a}_{3}{c}_{23})\\\\ {-c}_{1}\u0026amp; {s}_{1}({a}_{1}+{a}_{2}{c}_{2}+{a}_{3}{c}_{23})\\end{array}\\\\ \\begin{array}{cc}{s}_{23}\u0026amp; {c}_{23}\\\\ 0 \u0026amp; 0\\end{array}\u0026amp; \\begin{array}{cc}0\u0026amp; {a}_{2}{s}_{2}+{a}_{3}{s}_{23}+{d}_{1}\\\\ 0\u0026amp; 1\\end{array}\\end{array}\\right]$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn which, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({s}_{i}=sin{\\theta }_{i}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({c}_{i}=cos{\\theta }_{i}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({s}_{ij}=sin{(\\theta }_{i}+{\\theta }_{j})\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({c}_{ij}=cos{(\\theta }_{i}+{\\theta }_{j})\\)\u003c/span\u003e\u003c/span\u003e,\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({a}_{1}\\)\u003c/span\u003e \u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({a}_{2}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({a}_{3}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\theta }_{1}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\theta }_{2}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\theta }_{3}\\)\u003c/span\u003e\u003c/span\u003e represent length and rotation angle of the base, major arm, minor arm, respectively. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({d}_{1}\\)\u003c/span\u003e\u003c/span\u003e represents the vertical distance of rotating axis between base and major arm.\u003c/p\u003e \u003cp\u003eThe joint rotation angles of base, major arm and minor arm were randomly discretized within limited area:\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$${\\theta }_{i}={{\\theta }_{i\\text{m}\\text{i}\\text{n}}+({\\theta }_{i\\text{m}ax}-\\theta }_{i\\text{m}\\text{i}\\text{n}})\\times rand(N, 1)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThen the equation of pose coordinates \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\left[x,y,z,\\xi \\right]}^{T}\\)\u003c/span\u003e\u003c/span\u003e were obtained by the kinematics positive solution. And space clouds model was generated through the MATLAB software to calculate the 2D and 3D work area of the portable bioprinter.\u003c/p\u003e \u003cp\u003e \u003cb\u003eScanning accuracy test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA black and white checkerboard featuring 2 x 2 cm squares was printed using an Epson LaserJet Pro M452 printer (Japan) as a 2D template. The scanning system captured four images from different angles to reconstruct the checkerboard model. The average distance of the squares' intersection points from the restored model was calculated and compared to the template for final accuracy assessment.\u003c/p\u003e \u003cp\u003eAn irregular 3D structure was designed using SolidWorks software and subsequently printed by a CeraRay DLP printer (Xunshi Tech, China) with a printing accuracy of 10\u0026micro;m, serving as a 3D template. The scanning system took 24 photos from different angles to reconstruct the template model, which was then compared with the original model using Cloud Compare software to analyze the scanning system's accuracy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMurine fibroblasts (L929) and human foreskin fibroblasts (HFF-1) used for cell printing were purchased from Human Fenghui Biotechnology Co., Ltd, China. Both L929 and HFF-1 were cultured in Dulbecco's Modified Eagle Medium/F12(Gibco, USA) supplemented with 10% v/v FBS and 1% P/S.\u003c/p\u003e \u003cp\u003ePrimary fibroblasts for animal experiments were harvest from neonatal rats. The skin was removed from the rat's back, trimmed into 15\u0026ndash;25 mm\u003csup\u003e2\u003c/sup\u003e pieces after excising the subcutaneous tissue, and rinsed with PBS solution. The skin tissue was treated with 0.25% trypsin (Gibco, USA) at 4℃ for 16h to separate the epidermis from the dermis. The remaining dermis pieces were placed into 6-well plates with a small volume of Dulbecco's Modified Eagle Medium (Gibco, USA) containing 10% v/v FBS and 1% P/S. After incubating for 24h, the medium volume was adjusted to normal growth conditions. Following one week of culturing, proliferating cells were transferred to 25 cm\u003csup\u003e2\u003c/sup\u003e flasks for further incubation.\u003c/p\u003e \u003cp\u003eCells were cultured in an incubator set at 37℃ with a 5% CO2 environment, and the culture medium was replaced every two days. Cells were passaged at a ratio of 1:3, and all cells used in this study were maintained within passage 8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Material\u003c/h2\u003e \u003cp\u003eThe primary materials utilized in the experiment included sodium alginate (SA), gelatin, Gelatin methacryloyl(GelMA), and fibroblasts. All materials and reagents were of chemical grade. GelMA was purchased from Suzhou intelligent manufacturing research institute with a grafting rate at 60% and stored at -20℃. lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP, TCI, Japan) was dissolved in the DMEM culture medium at a final concentration of 0.5% w/v to obtain the pre-solvent for bioink and stored at -20℃ before the experiment.\u003c/p\u003e \u003cp\u003eFor the printability test, GelMA and sodium alginate (Sigma, USA) were added to the pre-solvent in 37℃ for 3h to reach their final concentrations, respectively.\u003c/p\u003e \u003cp\u003eFor the animal examination, the pre-solvent was sterilized using a 0.22\u0026micro;m sterilizing filter (Millipore, USA). GelMA and SA were sterilized under UV light exposure for 12h prior to being added to the pre-solvent to achieve a specific concentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioink printability test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBioinks of varying concentrations were added into syringes at room temperature (25℃) with a continuous extrusion speed of 0.15mm/s and 27G needles to test the printability of mixed bioink. GelMA and SA were added to pre-solvent to achieve the final concentration of 2/5% SA/GelMA hydrogel. The hydrogel was loaded into a syringe and printed using our portable bioprinter in a grid pattern to assess the relationship between filament width and nozzle velocity at an extrusion speed of 0.18 mm/s, employing needles ranging from 25G to 30G. After printing, the entire printed structure was exposed to 405 nm visible light for 1 minute to facilitate complete photocrosslinking.\u003c/p\u003e \u003cp\u003eHFF-1 and HUVECs were added to the sterilized 2/5% SA/GelMA bioink at final concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/ml and 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/ml, respectively. The bioink was then printed using the portable bioprinter in a grid pattern to analyze the machine's cell printability, with printing parameters including a 25G needle, 0.18 mm/s extrusion speed, and 8 mm/s nozzle velocity. After the printing process, the printed scaffold was exposed to 405 nm visible light for 1 minute for complete photocrosslinking and subsequently stained with a live-dead cell staining kit (Biovision, USA) after a 30-minute incubation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimals\u003c/b\u003e \u003c/p\u003e \u003cp\u003e12 adult Sprague-Dawley (SD) rats, weighing 250\u0026thinsp;\u0026plusmn;\u0026thinsp;30g, were purchased from Shanghai SLAC Laboratory Animal Co., LTD, and randomly assigned to 3 experimental groups, with 4 rats in each group. Prior to grafting, the dorsal skin of each rat was clipped, and a full-thickness skin defect wound (2 \u0026times; 2 cm) was created. The animal study was approved by the Ethics Committee of Zhejiang University.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn situ bioprinting for rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe dorsal skin defects in the rats were photographed from 36 angles to obtain comprehensive information, followed by the reconstruction of 3D wound models. Bioprinter pathways were automatically generated to fill the defects. For the first in situ bioprinting group, a 2/5% SA/GelMA bioink containing primary rat fibroblasts (1 \u0026times; 10^6 cells/ml) was loaded into the syringe of the portable bioprinter and directly printed into the rats' defects. During the process, printing parameters were maintained at an extrusion speed of 0.15 mm/s and a nozzle velocity of 5 mm/s, using a 27G needle. After in situ printing, the printed tissue was crosslinked using 405 nm light for 1 minute.\u003c/p\u003e \u003cp\u003eFor the commercial dressing group, the dressing was extruded to uniformly fill the rat defects and allowed to solidify for 10 minutes. All groups received a breathable dressing (3M) to cover the dorsal defects, preventing infection from environmental exposure. Throughout the feeding and recovery process, all groups were treated equally.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWound analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe wounds were photographed every 7 days and the wounds closure rate was calculated using ImageJ software. The wound closure rate was determined by the formula: 1- (wound size/original wound size)\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological examination\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSkin samples were collected on day 28 post-treatment. The harvested tissues were fixed in 4% paraformaldehyde phosphate buffer solution (XBIO, USA) for 24h and subsequently dehydrated through a series of alcohol solutions (75%, 85%, 95%, and 100%). The dehydrated samples were embedded in molten paraffin and frozen at -20℃ for 2h. The frozen samples were sectioned at a thickness of 4\u0026ndash;5\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu m\\)\u003c/span\u003e\u003c/span\u003e. The paraffin sections were floated on 40℃ water for flattening, placed onto glass slides, and baked at 60℃ for 2h. After baking, the paraffin sections were stored at 4℃.\u003c/p\u003e \u003cp\u003e To remove the paraffin, slides were treated with xylene and subsequently washed with a series of gradient alcohol solutions (100%, 95%, 90%, 80%, and 70%) followed by distilled water. The slides were then stained with Harris hematoxylin (Servicebio, China) for 5 minutes, washed with distilled water, and stained with eosin solution (Servicebio, China) for 2 minutes. The stained samples were dehydrated using gradient alcohol solutions (95% and 100%) and cleared with xylene. Structural images of the samples were captured using a NIKON DS-U3 microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemistry examination\u003c/b\u003e \u003c/p\u003e \u003cp\u003e Paraffin sections were deparaffinized using xylene and washed with gradient alcohol (100%, 95%, 90%, 80%, and 70%) followed by distilled water. The sections were submerged in citric acid repair solution (pH 6.0) (G1202, Servicebio, China) for antigen retrieval and then dried at high temperature. Subsequently, the sections were washed three times with PBS (pH 7.4) (G0002, Servicebio, China) for 5 minutes per wash. After blocking with 3% BSA (G5001, Servicebio, China) for 30 minutes, the sections were incubated with primary antibodies overnight at 4℃. The sections were then washed three times with PBS (5 minutes per wash) and incubated with secondary antibodies for 1 hour. The primary antibodies used were mouse anti-rat endothelial cell marker (CD31) (GB12063, Servicebio, China), and the secondary antibodies were goat anti-mouse (GB25301, Servicebio, China) conjugated to Alexa Fluor 488. After washing three times with PBS (5 minutes per wash), DAPI (G1012, Servicebio, China) was added to the samples and incubated for 10 minutes, followed by an additional three PBS washes (10 minutes per wash). Images of the samples were captured using a NIKON ECLIPSE C1 microscope.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Portable Bioprinter Design and Structure\u003c/h2\u003e \u003cp\u003eCurrently, most 3D bioprinters on the market are bulky and unsuitable for specialized extreme environments such as field operations, training, and survival scenarios. The primary challenges in developing portable skin 3D printing equipment include portable design, cell survival rate, and database establishment [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. First, portable 3D skin printing equipment necessitates a smaller volume and weight. In contrast to existing 3D bioprinting equipment [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR26\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e26\u003c/span\u003e], portable 3D bioprinters consist of printing nozzles, motion modules, and storage modules for printing materials (cells, biomaterials, growth factors, etc.). Integrated design is a major challenge, requiring precise planning for the printer's structure and the positioning of each component module. To ensure the necessary functions for rapid skin wound repair in field environments, the device should be as miniaturized and portable as possible.\u003c/p\u003e \u003cp\u003eTraditional gantry structures achieve motion control by separately controlling the movements of the x-axis, y-axis, and z-axis. This approach has simple movement forms, convenient control, and low manufacturing costs, but the large frame does not meet the miniaturization requirements for the 3D printing system. We selected a more flexible mechanical arm structure for the portable bioprinter with a three-degree-of-freedom actuator, utilizing three stepper motors for motion control. Compared to the traditional gantry structure, the mechanical arm occupies less space when not in use and provides a larger printing area through arm extension during operation.\u003c/p\u003e \u003cp\u003eThe designed robotic arm features a connecting rod structure, with motors on both sides controlling the movement of the boom and forearm, respectively. Mounting holes at the base's four corners facilitate subsequent assembly, while those at the end of the robotic arm enable secure attachment of the printer head and scanning device, ensuring smooth movement. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec illustrates the three-axis robotic arm structure. After using the Monte Carlo method to simulate the working area of the robotic arm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), we found that the manipulator-type structure offers a significantly larger printable range than the traditional gantry structure (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrinting volume rate for bioprinters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAllevi 3 bioprinter[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSUNP biomaker[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePortable bioprinter\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDimensions(mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e467 \u0026times; 388 \u0026times; 360\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e400 \u0026times; 400 \u0026times; 500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e485 \u0026times; 450 \u0026times; 300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWork area(mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90 \u0026times; 60 \u0026times; 130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 \u0026times; 90 \u0026times; 50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.274e7(mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePrinting volume rate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.08%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.73%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80.55%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe bioink is extruded into a filament through the printing nozzle, and the nozzle's directional movement achieves the desired structure and shape. Therefore, to ensure extrusion accuracy and structural requirements, the precision and stability of the printer head are critical factors. A syringe is fixed in the printer head and pushed precisely by a high-precision linear motor with a 1.25\u0026micro;m control accuracy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Bioink filament extrusion is regulated by controlling the input pulses through the linear motor per unit time to adjust the material's extrusion speed at the nozzle. In combination with the nozzle's movement speed, the bioink filaments are controlled with high accuracy. This design enables the printer to achieve a printing accuracy of tens of microns with stable movement, meeting design requirements.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo ensure timely and urgent rescue in remote environments and achieve automatic in situ printing with minimal intervention, the printer must quickly collect comprehensive wound information. Complete and accurate data enables the 3D reconstruction model to better represent the wound's state, facilitating an orderly treatment process. It is essential to integrate a scanning module capable of capturing wound information from multiple angles within the printer's limited space. A small 360-degree servo is fixed beneath the mechanical arm's end effector using a mounting frame and connected to a camera through two rotatable connectors. When the wound needs to be photographed from different angles for scanning, the servo motor can be controlled to rotate a specific angle to obtain the required roll angle, aligning the camera with the wound as needed. The servo's movements are controlled by the printer's main controller, which is compatible with the mechanical arm's movements.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the entire system is powered by a 12V lithium battery, with a voltage regulator chip providing stable voltage due to the varying requirements of different modules. The movement module comprises a three-axis motor for the robotic arm and a linear guide rail for the Z-axis to control the nozzle and camera. The temperature module consists of a constant-temperature box for bioink storage and a system cooling fan. The control module employs a Raspberry Pi to connect input and output devices via USB and High Definition Multimedia Interface (HDMI). When the PC is on the same local area network as the Raspberry Pi, the printer can be controlled through a Virtual Network Console (VNC), enabling remote monitoring and timely interventions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2 3D Model Reconstruction Process\u003c/h2\u003e \u003cp\u003eTo ensure the bioprinter can automatically select the printing system parameters based on the wound and reduce the workload of medical staff, the printer requires an integrated scanning module capable of performing high-precision 3D reconstruction of skin wounds to fill the wound defects. The Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the entire 3D reconstruction process. The actuator at the end of the printer drives the camera to collect wound information from various angles, obtaining the necessary wound images for 3D reconstruction. An algorithm is then used to restore the 3D model of the skin wound. The system allows users to adjust the reconstructed model, mark the wound position, and define printing parameters such as filament distance and layer height. Finally, the user interface provides users with optional printer preset schemes, and upon confirmation, a customized printing program is executed on the wound. The bioink carrier used in this study offers appropriate support for maintaining cell viability, rapid crosslinking, and accurate deposition to promote the formation of multilayer cell-loaded skin structures.\u003c/p\u003e\u003cp\u003eAfter obtaining the wound images, this paper employs the Scale Invariant Feature Transform (SIFT) feature matching algorithm to extract feature points from the image [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. SIFT is an image local feature descriptor based on scale space that maintains image scaling, rotation, and even affine transformation, offering stability, uniqueness, abundance, high speed, and scalability. The algorithm uses an image pyramid to detect features in scale space, determining the position and scale of key points (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). It then uses the gradient of the main direction within the key point field as the directional feature of the point, achieving operator independence from scale and direction.\u003c/p\u003e \u003cp\u003eTo determine the 3D position of a point in a scene, traditional photogrammetry methods require knowledge of the camera's 3D position and orientation, whereas the motion recovery structure method for sparse reconstruction does not. By matching feature points, multiple view images of an object can be connected to calculate the camera pose and 3D coordinates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Multi-view geometry serves as the foundation for sparse reconstruction. The same image undergoes different projections in various views using matrices. Two views are initially selected, with new views added sequentially to calculate camera intrinsic parameters, pose, and other parameters. The spatial coordinates of the matching feature points are then determined through triangulation. During multi-view reconstruction, reprojection may occur (one feature point on one image can match feature points on multiple other images). In such cases, minimizing the weighted square and reprojection error related to the cost function requires bundle adjustment, which greatly benefits the accuracy of subsequent processing. Although sparse reconstruction using the incremental method can be time-consuming, the resulting reconstruction is more robust and the model's accuracy is higher, which is crucial for precise wound printing.\u003c/p\u003e \u003cp\u003eFor flat accuracy, we fabricated a standard chessboard with a 2cm side and utilized the scanning system of the printer for 3D reconstruction. The black and white intersection point set of the checkboard was obtained using the Harris corner detection algorithm [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and was transformed to the X-Y plane through spatial transformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The average error between each point is indicative of the flat reconstruction accuracy of the scanning system. As for the 3D accuracy, we printed an irregular 3D sample using an SLA 3D printer (with an accuracy of 5\u0026micro;m) and also utilized the scanning system for 3D reconstruction. The accuracy of the reconstructed model was obtained using CloudCompare, an open-source 3D point cloud processing software licensed under the GPL, with the initial sample treated as a standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The reconstruction error below the model is relatively large, which can be attributed to the scanning system\u0026rsquo;s inability to fully capture the area due to the influence of the horizontal workbench. However, the accuracy above the model is maintained at a high level (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eScanning accuracy test\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean distance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStd deviation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlat scanning\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3D scanning\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.2\u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e105\u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn order to obtain a processable wound model, it is necessary to generate a dense point cloud. During sparse reconstruction, the 3D coordinates of some feature points have already been determined. According to the principle of multi-view geometry, a point in space will have a projected point in the views of multiple images. When a matching point pair is identified, the 3D depth of the corresponding point can be calculated based on the camera pose information.\u003c/p\u003e \u003cp\u003eThe 3D reconstruction model is stored in the OBJ file format, which is a data format file that includes simple vertex coordinates, texture coordinates, and surface information. The 3D point coordinates are linked to the texture color by reading the information in the OBJ file, enabling the reconstructed 3D model to be visually displayed. The completed model includes both the wound area and non-wound part, making it difficult to accurately distinguish the wound edge from the normal area due to complex factors such as hair, light, stains, and tissue fluid leakage. Rough edge information is displayed in the user interface, and an edge search algorithm is employed to obtain the accurate wound boundary. After simulating the surface morphology, the approximate slope of the original skin morphology is fitted through the skin wound edge points, and the missing skin parts on the wound are reconstructed. For the simulated wound surface, the height of each layer from the lowest point of the wound is obtained by inputting the slice thickness, and the printing trajectory for each slice is autogenerated using a multi-layer path-filling algorithm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). The resulting printed model can fulfill the requirements of skin 3D printing, effectively covering the wound area to promote the healing rate of the injured part.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Bioprinter Validation in Hydrogel Printing\u003c/h2\u003e \u003cp\u003eCurrently, the range of biomaterials used for human skin reconstruction is relatively limited, particularly in the field of 3D bioprinting, which demands exceptional printability [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Alginate, a non-toxic and degradable natural polymer material, is widely utilized in biofabrication research due to its easy crosslinking, excellent biocompatibility, biodegradability, and abundant sources [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The micro-porous environment created by alginate scaffolds can accommodate numerous cells and maintain cell phenotypes, promoting cell adhesion and nutrient penetration. Moreover, the products resulting from enzymatic degradation in the human body exhibit no toxic effects. Gelatin, a polypeptide molecular polymer substance obtained through partial collagen hydrolysis, preserves collagen's specific amino acid composition and displays strong biocompatibility, thanks to the presence of arginine-glycine-aspartic acid (RGD) sequences that enhance cell-material affinity. This attribute makes gelatin a popular choice in tissue engineering research [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. GelMA is a biomaterial synthesized by grafting methacryloyl groups onto gelatin, preserving gelatin's biocompatibility and temperature sensitivity while adding photoreactive crosslinking functionality, making it an ideal bioink for 3D bioprinting [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Considering the characteristics and requirements of skin tissue, this project tested sodium alginate (SA) and GelMA for printing and ultimately selected a blended material as the printing bioink for skin wound repair.\u003c/p\u003e \u003cp\u003eThe printability of hydrogels was observed at the nozzle outlet at room temperature (25℃), and the extrusion conditions for different concentrations of mixed bioink were categorized into three types:\u003c/p\u003e \u003cp\u003e1)Unextrudable: The bioink cannot be extruded through the needle or forms wrinkled filaments at the needle outlet, resulting in discontinuous printed fibers.\u003c/p\u003e \u003cp\u003e2)Printable: The bioink forms regular and smooth filaments through the needle, enabling stable fiber printing on the platform.\u003c/p\u003e \u003cp\u003e3)Overextrudable: The bioink is extruded as spindle-shaped filaments, sometimes breaking into droplets, leading to stacked printed fibers.\u003c/p\u003e \u003cp\u003eThe printability experiment results are illustrated in the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. At low concentrations of GelMA and SA (0\u0026thinsp;~\u0026thinsp;4% GelMA and 0\u0026thinsp;~\u0026thinsp;1% SA), the solution's viscosity is too low to form regular bioink filaments, indicating poor printability for skin printing. However, when the GelMA/SA solution concentration (\u0026gt;\u0026thinsp;20% GelMA and \u0026gt;\u0026thinsp;4% SA) is too high, the bioink exhibits excessive viscosity, preventing smooth extrusion at the nozzle outlet and formation of a continuous tissue scaffold. Consequently, the GelMA and SA concentrations in the red and yellow areas of the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea are unsuitable for printing bioink. In this experiment, we determined that a specific range of mixed GelMA/SA hydrogel solution concentration (green area in the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) offers suitable viscosity to form smooth and uniform filaments upon extrusion, as SA provides high viscosity for extrusion and GelMA enables easy photo-crosslinking to maintain a stable structure in the body.\u003c/p\u003e \u003cp\u003eFor this project, a 2/5% SA/GelMA hydrogel was selected as the printing bioink. Various nozzle moving speeds and constant extrusion speeds were tested to form hydrogel filaments with different needles. By adjusting the printing parameters, hydrogel lines with a diameter of 200\u0026ndash;1000 \u0026micro;m were obtained, as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, which were deemed suitable for tissue reconstruction.\u003c/p\u003e \u003cp\u003eThe portable bioprinter was employed for cell printing and experimental research, which has been verified by different cell types. 2/5% SA / GelMA mixed with murine L929 and HFF-1 were used as printing bioink and printed to test cell viability. The experimental results, displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, demonstrated that the survival rates of L929 and HFF-1 cells were (94.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.02%) and (89.3% \u0026plusmn; 5.85%), respectively. These findings were quantitatively analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Bioprinter Validation in Rat Wound Models\u003c/h2\u003e \u003cp\u003e Animal models serve as essential experimental tools in modern medicine, facilitating our understanding of the objective laws governing life sciences. In this study, we employed SD rats in skin damage repair experiments using dorsal skin reconstruction models. Artificially controlling the shape, size, and depth of wounds on rats allows for a highly versatile approach in animal experiments focused on skin defect repair [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSD rats were divided into three groups: in situ bioprinted skin containing primary fibroblasts, commercial skin dressing (Jihao, China), and an untreated group. A 2x2 cm dorsal wound was created on each rat's back, with three rats per group to ensure reliable results. The rat experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University and were performed according to the set regulations for animal experiments. We used our in situ skin bioprinter to print GelMA hydrogel containing primary fibroblasts from rats' dorsal tissues onto the experimental group's wounds, applied commercial wound treatment dressing to the control group, and left the untreated group as a blank comparison. We monitored the wounds and recorded data weekly. No inflammation occurred during the entire process, and most rats recovered within 28 days. On day 28, we evaluated skin slices stained with hematoxylin and eosin (H\u0026amp;E) and immunohistochemistry staining of CD31 to compare the healing effects among the three groups.\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, the three groups exhibited distinct healing trends. By day 7, the in situ bioprinting group's wound closure rate (33.02\u0026thinsp;\u0026plusmn;\u0026thinsp;10.11%) was comparable to the commercial dressing group (26.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82%), but significantly different from the untreated group (13.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01%). By day 14, the in situ bioprinting group's wound closure rate (81.99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89%) substantially surpassed that of the commercial dressing group (60.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19%) and the untreated group (25.27\u0026thinsp;\u0026plusmn;\u0026thinsp;4.12%). This trend persisted until day 21, with closure rates of 95.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18%, 87.76\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33%, and 76.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56%, respectively. By day 28, the wounds in the in situ bioprinting group and the commercial dressing group had completely healed, while the untreated group's wounds maintained a closure rate of 90.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16%. According to other research on skin healing, skin scaffolds provide extracellular matrix (ECM) structures, functional cellular components, and other biological factors to enable and accelerate skin regrowth [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR38\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Fibroblasts secrete growth factors for skin reconstruction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e20\u003c/span\u003e], which may have contributed to the rapid healing observed in the in situ bioprinting group.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed presents the H\u0026amp;E staining results, which were evaluated to compare the reconstructed skin following different treatments. Native healthy rat skin tissue served as a control group to assess the healing effects of each experimental group. Morphological analysis revealed that the recovered skin in the in situ bioprinting group exhibited \"ridge\" patterns in the epidermis, similar to native skin. In the commercial dressing group, fewer \"ridge\" patterns were observed in the regenerated skin, while the untreated group displayed unreconstructed portions, indicating superior healing effects in the in situ bioprinting group. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee presents the epidermal thickness of different groups, demonstrating that the regenerated skin in all three groups was considerably thicker than the native rat skin. The in situ bioprinting group had the thickest epidermis, while the commercial dressing group exhibited more uniform epidermis compared to the untreated group. During the skin wound recovery process, autologous cells migrate to the wound area and accelerate healing through cell proliferation, which may result in the increased thickness of the epidermis. In situ bioprinted skin may provide a better bioscaffold that fully covers the wound area, facilitating cell migration. Additionally, growth factors secreted by the cells contained within the in situ bioprinted skin may contribute to faster healing by attracting autologous cells.\u003c/p\u003e \u003cp\u003eThe immunohistochemical staining results of CD31 are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, illustrating the neovascularization within the regenerated skin. The number of microvessels in the in situ bioprinted skin group was significantly higher than in the commercial dressing group, while no obvious microvascular regeneration was observed in the untreated group. These findings were numerically analyzed using ImageJ software. Upon closer examination of the enlarged image, a distinct hollow vessel structure was evident in the in situ bioprinted group, with vessel diameters reaching 30\u0026ndash;40 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). The experimental results demonstrate that our in situ bioprinted method can promote wound epithelialization.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we analyzed the risks associated with various skin injuries in harsh outdoor environments and proposed different treatment methods based on the severity of the injury. To address the challenges of treating skin wounds in the field, we developed a portable 3D skin printer that can capable to harsh environments and meet the needs of individuals quickly. Traditional bioprinters use gantry structures that are typically large in size and require strict operating conditions, making them either immobile or only mobile within the laboratory [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In contrast, our printer utilizes the flexibility of a mechanical arm, providing a larger printing area and smaller size that can be easily collapsed into a suitcase-sized package when not in use. This allows the printer to be moved and operated outside of laboratory settings. We also designed a portable bio-ink compatible with this printer and conducted a comparative test. The experimental results demonstrate that the portable printer offers significant advantages over traditional skin injury treatment methods.\u003c/p\u003e \u003cp\u003eOur bioprinter system can rapidly and precisely deposit materials and cells onto specific areas of the wound with high accuracy using an extrusion method. In an animal study using a rat skin defect model, we demonstrated that our system effectively covers the wound with a skin structure composed of rat primary fibroblasts, contributing to expedited skin wound recovery. Wound area assessments over a four-week period showed that wounds treated with in situ bioprinted skin closed more quickly than those treated with commercial dressings.\u003c/p\u003e \u003cp\u003eCompared to the use of robotic arms, another trend in portable printers is handheld devices that allow for direct writing of bio-materials containing specific living cells. These handheld bioprinters are smaller than robotic devices, allowing for adjustment of the printing process according to changes in the wound conditions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, the extracellular matrix used for tissue repair must be carefully considered, including shear-thinning property, hydrogel pore-size and rapid gelation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Meanwhile, handheld bioprinters have lower precision and are difficult to use for manufacturing complex structures. They can only be used to repair simple structures, and require highly skilled operators, making them unsuitable in harsh field environments.\u003c/p\u003e \u003cp\u003eIn the future, we plan to use the printing system to investigate the printing properties of other biological materials and cell types to further accelerate wound healing. This technology has broad application prospects, including the potential to address the vast global demand for skin wound repair, reduce the secondary injuries and psychological pain experienced by skin wound patients due to skin transplantation, and find applications in drug testing and screening, as well as medical aesthetics. Furthermore, this technology could be applied to battlefield wound repair in even more challenging conditions, providing military personnel with essential equipment for emergency combat injury treatment and enhancing their ability to address sudden casualties during field operations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYCL and XZ wrote the manuscript; YZG designed the experiments; XHZ and QL assisted with experimental setup; JEM and HYY jointly supervised this work; BZ was in charge of the whole trial. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been supported by the National Key Research and Development Program of China (Grant No. 2018YFA0703000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants, however, animal model was used. The study protocol for the experimental use of the animals was approved by the ethics committee of Zhejiang University according to the standard operating procedures (SOPs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creativecommons.org/ licenses/ by/4. 0/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePeck M D (2012) Epidemiology of burns throughout the World. Part II: Intentional burns in adults 38(5):630-637. https://doi.org/10.1016/j.burns.2011.12.028\u003c/li\u003e\n\u003cli\u003eJiang Y F, Huang S, Fu X B, Liu H W, Ran X W, Lu S L, Hu D H, Li Q A, Zhang H W, Li Y, Wang R X, Xie T, Cheng B A, Wang L F, Liu Y, Ye X B, Han C M, Chen H D (2011) Epidemiology of chronic cutaneous wounds in China 19(2):181-188. https://doi.org/10.1111/j.1524-475X.2010.00666.x\u003c/li\u003e\n\u003cli\u003eSinger, Adam J, Clark, Richard A F (1999) Cutaneous Wound Healing \u003c/li\u003e\n\u003cli\u003eMin S J, Lee J S, Nah H, Moon H J, Lee S J, Kang H J, Hwang Y S, Kwon I K, Heo D N (2023) Degradable and Tunable Keratin-fibrinogen Hydrogel as Controlled Release System for Skin Tissue Regeneration 20(3):1049-1059.\u003c/li\u003e\n\u003cli\u003eLiu T, Qiu C, Ben C, Li H H, Zhu S H (2019) One-step approach for full-thickness skin defect reconstruction in rats using minced split-thickness skin grafts with Pelnac overlay 7(1) https://doi.org/10.1186/s41038-019-0157-0\u003c/li\u003e\n\u003cli\u003eMohamed Haflah N H, Ng M H, Mohd Yunus M H, Naicker A S, Htwe O, Abdul Razak K A, Idrus R (2018) Massive Traumatic Skin Defect Successfully Treated with Autologous, Bilayered, Tissue-Engineered MyDerm Skin Substitute: A Case Report 8(2):e38. https://doi.org/10.2106/jbjs.Cc.17.00250\u003c/li\u003e\n\u003cli\u003eMartin-Piedra M A, Santisteban-Espejo A, Moral-Munoz J A, Campos F, Chato-Astrain J, Garcia-Garcia O D, Sanchez-Porras D, Campos A (2020) An Evolutive and Scientometric Research on Tissue Engineering Reviews 26(9-10):569-577. https://doi.org/10.1089/ten.tea.2019.0247\u003c/li\u003e\n\u003cli\u003eLesher A, Curry R H, Evans J, Smith V A, Fitzgerald M T, Cina R A, Streck C J, Hebra A (2011) Effectiveness of Biobrane for treatment of partial-thickness burns in children 46(9):1759-1763.\u003c/li\u003e\n\u003cli\u003eLal S, Barrow R E, Wolf S E, Chinkes D L, Hart D W, Heggers J P, Herndon D N (2000) Biobrane (R) improves wound healing in burned children without increased risk of infection 14(3):314-318. https://doi.org/10.1097/00024382-200014030-00013\u003c/li\u003e\n\u003cli\u003eHayes D W, Webb G E, Mandracchia V J, John K J (2001) Full-thickness burn of the foot: successful treatment with Apligraf. A case report 18(1):179-188.\u003c/li\u003e\n\u003cli\u003eKirchmajer D M, Gorkin R, Panhuis M I H (2015) An overview of the suitability of hydrogel-forming polymers for extrusion-based 3D-printing 3(20):4105-4117. https://doi.org/10.1039/c5tb00393h\u003c/li\u003e\n\u003cli\u003eOzbolat I T (2015) Bioprinting scale-up tissue and organ constructs for transplantation 33(7):395-400. https://doi.org/10.1016/j.tibtech.2015.04.005\u003c/li\u003e\n\u003cli\u003eLi X, Lian Q, Li D, Xin H, Jia S (2017) Development of a Robotic Arm Based Hydrogel Additive Manufacturing System for In-Situ Printing 7(1):73.\u003c/li\u003e\n\u003cli\u003eLv J, Yu X, Shen Q, Zhu Y (2013) The Application Progress of Stem Cells in Construction and Regeneration of Engineering Tissue 32(1):99.\u003c/li\u003e\n\u003cli\u003eCalvert P (2007) Printing cells 318(5848):208-209. https://doi.org/10.1126/science.1144212\u003c/li\u003e\n\u003cli\u003eKeriquel V, Guillemot F, Arnault I, Guillotin B, Miraux S, Amedee J, Fricain J C, Catros S (2010) In vivo bioprinting for computer- and robotic-assisted medical intervention: preliminary study in mice 2(1) https://doi.org/Artn 014101 10.1088/1758-5082/2/1/014101\u003c/li\u003e\n\u003cli\u003eSkardal A, Mack D, Kapetanovic E, Atala A, Jackson J D, Yoo J, Soker S (2012) Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Healing of Large Skin Wounds 1(11):792-802. https://doi.org/10.5966/sctm.2012-0088\u003c/li\u003e\n\u003cli\u003eKeriquel V, Oliveira H, Remy M, Ziane S, Delmond S, Rousseau B, Rey S, Catros S, Amedee J, Guillemot F, Fricain J C (2017) In situ printing of mesenchymal stromal cells, by laser-assisted bioprinting, for in vivo bone regeneration applications 7 https://doi.org/ARTN 1778 10.1038/s41598-017-01914-x\u003c/li\u003e\n\u003cli\u003eHakimi N, Cheng R, Leng L, Sotoudehfar M, Ba P Q, Bakhtyar N, Amini-Nik S, Jeschke M G, Gunther A (2018) Handheld skin printer: in situ formation of planar biomaterials and tissues 18(10):1440-1451. https://doi.org/10.1039/c7lc01236e\u003c/li\u003e\n\u003cli\u003eAlbanna M, Binder K W, Murphy S V, Kim J, Qasem S A, Zhao W, Tan J, El-Amin I B, Dice D D, Marco J, Green J, Xu T, Skardal A, Holmes J H, Jackson J D, Atala A, Yoo J J (2019) In Situ Bioprinting of Autologous Skin Cells Accelerates Wound Healing of Extensive Excisional Full-Thickness Wounds 9(1):1856. https://doi.org/10.1038/s41598-018-38366-w\u003c/li\u003e\n\u003cli\u003eOconnell C D, Bella C D, Thompson F W, Augustine C, Beirne S, Cornock R, Richards C J, Chung J H Y, Gambhir S, Yue Z (2016) Development of the Biopen: a handheld device for surgical printing of adipose stem cells at a chondral wound site 8(1):015019-015019.\u003c/li\u003e\n\u003cli\u003eDuchi S, Onofrillo C, Oconnell C D, Blanchard R, Augustine C, Quigley A F, Kapsa R M I, Pivonka P, Wallace G G, Bella C D (2017) Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair 7(1):5837.\u003c/li\u003e\n\u003cli\u003eSingh S, Choudhury D, Yu F, Mironov V, Naing M W (2020) In situ bioprinting - Bioprinting from benchside to bedside? 101:14-25. https://doi.org/10.1016/j.actbio.2019.08.045\u003c/li\u003e\n\u003cli\u003eChoudhury D, Anand S, Naing M W (2018) The arrival of commercial bioprinters - Towards 3D bioprinting revolution! 4(2) https://doi.org/10.18063/IJB.v4i2.139\u003c/li\u003e\n\u003cli\u003eDuan B, Hockaday L A, Kang K H, Butcher J T (2013) 3D Bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels 101(5):1255-1264. https://doi.org/10.1002/jbm.a.34420\u003c/li\u003e\n\u003cli\u003eLee C H, Cook J L, Mendelson A, Moioli E K, Yao H, Mao J J (2010) Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study 376(9739):440-448. https://doi.org/10.1016/s0140-6736(10)60668-x\u003c/li\u003e\n\u003cli\u003eAllevi (2023) Allevi 3. https://www.allevi3d.com/allevi-3/. Accessed on 7th Nov, 2023\u003c/li\u003e\n\u003cli\u003eBIOTECH S (2023) SUNP BIOMAKER 2i. http://www.sunpbiotech.cn/Printer-system/8.html. Accessed on 7th Nov, 2023\u003c/li\u003e\n\u003cli\u003eLowe D G (1999) Object recognition from local scale-invariant features\u003c/li\u003e\n\u003cli\u003eLowe D G (2004) Distinctive Image Features from Scale-Invariant Keypoints 60(2):91-110.\u003c/li\u003e\n\u003cli\u003eHarris C G, Stephens M J (1988) A combined corner and edge detector\u003c/li\u003e\n\u003cli\u003eAgrawal P, Soni S, Mittal G, Bhatnagar A (2014) Role of Polymeric Biomaterials as Wound Healing Agents 13(3):180-190. https://doi.org/10.1177/1534734614544523\u003c/li\u003e\n\u003cli\u003eDaly A C, Prendergast M E, Hughes A J, Burdick J A (2021) Bioprinting for the Biologist 184(1):18-32. https://doi.org/10.1016/j.cell.2020.12.002\u003c/li\u003e\n\u003cli\u003eXiao W Q, He J K, Nichol J W, Wang L Y, Hutson C B, Wang B, Du Y A, Fan H S, Khademhosseini A (2011) Synthesis and characterization of photocrosslinkable gelatin and silk fibroin interpenetrating polymer network hydrogels 7(6):2384-2393. https://doi.org/10.1016/j.actbio.2011.01.016\u003c/li\u003e\n\u003cli\u003eDi Bella C, Duchi S, O\u0026apos;Connell C D, Blanchard R, Augustine C, Yue Z L, Thompson F, Richards C, Beirne S, Onofrillo C, Bauquier S H, Ryan S D, Pivonka P, Wallace G G, Choong P F (2018) In situ handheld three-dimensional bioprinting for cartilage regeneration 12(3):611-621. https://doi.org/10.1002/term.2476\u003c/li\u003e\n\u003cli\u003eJin G R, Prabhakaran M P, Ramakrishna S (2014) Photosensitive and Biomimetic Core-Shell Nanofibrous Scaffolds as Wound Dressing 90(3):673-681. https://doi.org/10.1111/php.12238\u003c/li\u003e\n\u003cli\u003eBhardwaj N, Sow W T, Devi D, Ng K W, Mandal B B, Cho N J (2015) Silk fibroin-keratin based 3D scaffolds as a dermal substitute for skin tissue engineering (vol 7, pg 53, 2015) 7(1) https://doi.org/10.1039/c4ib90045f\u003c/li\u003e\n\u003cli\u003eZhong S P, Zhang Y Z, Lim C T (2010) Tissue scaffolds for skin wound healing and dermal reconstruction 2(5):510-525. https://doi.org/10.1002/wnan.100\u003c/li\u003e\n\u003cli\u003eWang M, He J, Liu Y, Meng L, Li D, Jin Z, Jin Z The trend towards in vivo bioprinting \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"sn-applied-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"SN Applied Sciences","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Wound repair, In situ 3D bioprinting, Mechanical arm control","lastPublishedDoi":"10.21203/rs.3.rs-3575253/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3575253/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrent treatments for skin wounds typically involve multiple surgical procedures that require complex processes and expensive costs, making it difficult to achieve timely treatment in field environments. We developed an innovative in situ printing method, utilizing robotic arm control, to address the significant challenges of large-scale skin wound repair resulting from natural disasters such as earthquakes, fires, and explosions during relief efforts. Our portable 3D printing equipment, which integrates debridement, precise 3D scanning and modeling of wounds, and compatibility with cell-loaded bioink, facilitates rapid repair of large-area skin wounds in specialized field environments. Compared with traditional methods, this in situ printing method has significant advantages, including the ability to customize treatment according to the unique needs of the wound, achieve rapid healing, and the potential to reduce the total cost. We conducted experiments on rats with full-thickness dorsal skin defects and compared the performance of in situ bioprinting method with commercial skin defect repair dressings. Our results demonstrate that the in situ bioprinted skin achieved faster wound healing and more uniform re-epithelialization than the commercial dressing treatment. This study demonstrates the potential of in situ bioprinting method as a promising and effective strategy for rapid skin wound healing, especially for patients in remote environments where traditional wound treatment methods may not be readily available or practical.\u003c/p\u003e","manuscriptTitle":"Development and Application of a Mechanical Arm-Based In Situ 3D Bioprinting Method for the Repair of Skin Wounds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-29 16:13:22","doi":"10.21203/rs.3.rs-3575253/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-08T15:30:32+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"5943ca9d-8536-4e35-b913-d034b9d3e913","date":"2023-12-22T14:11:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-12-12T10:08:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38740bac-f4e7-4d92-b567-58ecd37762d3","date":"2023-12-03T14:51:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-02T09:49:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-29T11:31:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-11-25T16:43:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"SN Applied Sciences","date":"2023-11-07T15:37:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"sn-applied-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"SN Applied Sciences","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8c767333-485f-4e2d-b2d9-4626030652d4","owner":[],"postedDate":"November 29th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-18T18:23:00+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-29 16:13:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3575253","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3575253","identity":"rs-3575253","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}