Portable hand‐held bioprinters promote in situ tissue regeneration

Abstract Three‐dimensional bioprinting, as a novel technique of fabricating engineered tissues, is positively correlated with the ultimate goal of regenerative medicine, which is the restoration, reconstruction, and repair of lost and/or damaged tissue function. The progressive trend of this technology resulted in developing the portable hand‐held bioprinters, which could be used quite easily by surgeons and physicians. With the advent of portable hand‐held bioprinters, the obstacles and challenges of utilizing statistical bioprinters could be resolved. This review attempts to discuss the advantages and challenges of portable hand‐held bioprinters via in situ tissue regeneration. All the tissues that have been investigated by this approach were reviewed, including skin, cartilage, bone, dental, and skeletal muscle regeneration, while the tissues that could be regenerated via this approach are targeted in the authors' perspective. The design and applications of hand‐held bioprinters were discussed widely, and the marketed printers were introduced. It has been prospected that these facilities could ameliorate translating the regenerative medicine science from the bench to the bedside actively.

beyond the industry in the late 90s. It introduced itself as a revolutionary technology in the world of medicine and medical engineering. 2 Since human organs' have quite a complex geometric structure and are unique to each person, bioprinting technology relies on layer-by-layer printing, would be beneficial in fabricating tissues and in near future organs. It has possibility of producing each person's organ perfectly fit the individual's physiological condition. 2 People have always exposed to a variety of diseases and accidents. 3 They have sometimes plagued by disability obstacles and a variety of diseases, such as cancer, osteoporosis, bone fracture, and skin burns. Physicians typically need testing and experience to diagnose and treat diseases, which usually gained through testing on volunteer patients or laboratory animals. In the event of a limb defect and the need to replace it, there are always long queues of patients waiting for a transplanted organ to arrive from a brain-dead patient. 4 With the advancements in genetics and the ability of stem cells to multiply and differentiate, in vitro, medicinal science has entered a new phase, and it has become possible for humans to get rid of suffering from some diseases and disabilities. 5 Bioprinting technology has become with the help of medical science to promote human health in some related areas, such as drug development, disease modeling, and production of various tissues for transplantation. 6 Already, the cost of medical research is pretty high, and the number of available samples is generally less than required, which has slowed down the research process, which imposes a high cost on research centers economically. 7 This technology has made it possible to print part of an organ needed for research and perform a variety of experiments on it. Also, for patients whose part of the organ is diseased or defective, a suitable replacement for that organ can be made by bioprinting method and implanted into the body of the patient. 8 Ultimately, the main goal of this technology in the near future is to build a living organ and transplant it into patients' body. Also, regarding using bioprinting technology, the costs of drug production and research on diseases would be significantly reduced. 9 Consequently, the development in this area would take a primary tool for research and treatment, from bench to the bedside. 10 The design and manufacture of bioprinters with various applications have considered by research and industrial centers.
Today, more than 30 active companies in the field of bioprinting are operating in developed countries, some of which have proprietary technology and have taken significant steps to print live organ. 11 Massive investment in this field shows the great importance of the issue that three-dimensional (3D) bioprinting could empower the economics of regenerative medicine. 12 Bioprinting, applying the principles of rapid modeling technology, is a more promising solution for tissue engineering (TE). Utilizing this technology, researchers can create a variety of biological materials, including cells and multiple tissue scaffolds, using 3D printing. 13 A new method for body tissue reconstruction emerges from progress in three areas, including rapid prototyping (RP) techniques, smart polymers, and cellular adhesion. 14 Eventually, what results from the ideal combination of these three components is tissue 3D printing. 15 Using the concepts of evolutionary biology, the principles of bioengineering, biomaterial engineering, and computing methods, bioprinting technology seek to create alternative tissues and organs to solve human medical dilemmas. 16 Perhaps it can be said that what is more considered in this TE strategy than other traditional and even modern methods, such as computer-based TE, is attention to the cellular principles. This method tries to rely on what is in the model.
The capture of a natural tissue occurs within the body and, with the utmost effort to comprehensively biologically stimulate the tissues.
Organ printing technology has multiple roots and based on the efforts of several interdisciplinary teams of various disciplines and scientists. Bioprinting utilizes inkjet cartridges loaded with cells and the proper ink, which are dispensed onto a substrate along with biomaterial. 17 In general, the printing procedure consists of three consecutive stages, including preprinting, printing, and postprinting. In the preprinting stage, the initial large-scale design of the target organ designed by computer simulation based on images obtained from the patient's damaged tissues. 18 Digitally reconstructed pictures of that organ from magnetic resonance imaging (MRI) or computed tomography (CT), or mathematical models, can be employed in this way. The printer builds up a 3D structure layer-by-layer until the desired result is achieved. The tissues produced at this stage are not yet functional.
In the postprinting phase, the structures created by the appropriate bioreactors are given nonstop with biomechanical conditions. 19 Although bioprinters have the potential to save several lives in the future, numerous difficulties remain. For example, printed structures may be fragile and unable to maintain their shape when transferred to an appropriate location on the body. Additionally, tissues and organs are complicated and contain a variety of distinct cell types that are tightly controlled. Finally, present approaches are constrained by their reliance on particular materials, a narrow viscosity range, and a lack of precision. Any approach has the risk of causing harm to cells and other printing materials. That is why researchers and engineers are trying to solve issues and design new bioprinters with unique capabilities. 20

| Tissue regeneration
In acute wounds, the wound healing process naturally heals, while in chronic or incurable wounds, due to an internal or external wound agent's intervention, it stops at one of the healing stages, and the wound healing process slows down or disrupted. 26 As soon as the wound created, the body has the task of starting and continuing several reactions simultaneously, including preventing bleeding, preventing the invasion of bacteria and microorganisms, removing dead tissue and foreign bodies from the wound site, and producing new tissue in the wound area. 27 TE could create an artificial environment for cell growth and stimulate tissue regeneration. 28 A tissue is made up of 3D units that are repeated at a size of 100-1000 nm in vivo environment. 29 In order to coordinate multicellular activities, generate mechanical qualities, and integrate with other organ systems via microcirculation, this repeating tissue's 3D structure is essential. Structural support is provided by the scaffolds during tissue restoration in order to promote 3D cell evolution. 29 Tissues are also influenced by the local cellular environment. Biochemical, cellular, and physical catalytic signaling pathways involved in cellular destiny events such as differentiation, proliferation, migration, and death are all dependent on the microbiological environment (10 m). Micro-and macro-scale levels should be addressed in order to fabricate functional scaffolds successfully.
Today, researchers are trying to design scaffolds with multiple capabilities, since scaffolding, must be able to guide tissue formation and even prevent possible damage, in addition to providing temporary structural support to the tissue being formed. 30 Optimal scaffolding design and fabrication methods should create porous structures that allow cell activity, material translocation and angiogenesis. These porous structures must have pores that are first sized and second interconnected to each other.
In conventional scaffolding methods such as solvent casting and salt washing, gas foaming, freeze-drying, and fuzzy separation, it is impossible to precisely control the shape, size, and relationship of pores to each other. Also, the widespread use of organic solvents in many of these methods can lead to adverse effects on biological tests' results in vitro and inflammatory responses in the body. On the other hand, it is impossible to reproduce the scaffolds in the mentioned methods so that all samples' structures and properties are the same.
The conventional and RP methods of 3D scaffold production are classified into two categories, each creating a different type of scaffold. Standard scaffolding fabrication techniques include the production of porous polymer structures such as cell adhesion substrates; however, complex structures with tunable microscale and macroscale are challenging to create using conventional procedures.
Traditional scaffold fabrication methods such as solvent casting/ particulate leaching are intended to define the shape and pore size of the scaffold. However, they are mostly limited to the prior scaffold internal design or connectivity of the void space in 3D cell cultures.
Using a solvent and salt particles of a certain size, the polymer solution is dissolved in the salt matrix, which is then submerged in water, and the salt leaches away to form a structure with high porosity. Solvent casting with particle leaching is only suitable for thin membranes of thin-walled 3D specimens; otherwise, the soluble particles cannot be separated from the salt matrix. 31 This approach produces scaffolds with a porosity of between 50% and 90%. The high porosity and capacity to fine-tune the pore size of the scaffold created by this method make it ideal for the formation and proliferation of 3D cells.
The number of pore networks between layers of porous sheets is limited, limiting its suitability for an application. Hazardous solvents that take a long time (days or weeks) to evaporate are used in this method. 31 Moreover, freeze-drying or lyophilization is a method in which a synthetic polymer that has been dissolved in a suitable solvent is used to dry a sample. 32 A solid solvent is evaporated by sublimation after dissolving a polymer solution. This leaves a solid scaffold with multiple linked pores. A fence of matter surrounds the ice in the ice phase, allowing the solutes to be isolated from one another. The scaffolds are created after subsequent drying, and the macroporosity is accomplished by simply dissolving and freeze-drying the initial empty area occupied by ice crystals. The advantage of this technology is that it does not use high temperatures, which could reduce the activity of biological factors that are incorporated. In addition, the freezing process can be modified to alter the pore size. Experts claim that this technology is flourishing in manufacturing scaffolds for a wide range of applications. 33 However, despite the fact that this method is frequently used in producing the scaffolds, it still has several drawbacks, including high-energy consumption, a protracted timescale, and the use of cytotoxic solvents. As a solution to these issues, the researchers experimented with different freezing temperatures (ranging from 10 to 70 Fahrenheit) and the addition of an additional annealing stage. 33 The use of water and ice crystals rather than an organic solvent during scaffold creation makes freeze-drying a more appropriate method for biomedical applications; however, this method is challenged in the fabrication of hierarchically organized scaffolds in biomedicine, such as vascular systems. The constructed scaffold must also be rinsed numerous times to eliminate the cytotoxic solvents used to mix the polymer and reduce cell death.
In order to deal with the use of high temperatures and organic cytotoxic solvents, the gas foaming technique has been developed.
This technique uses comparatively inert gas foaming agents such as carbon dioxide and nitrogen to press a biologically degradable polymer model with water until it is saturated or full of gas bubbles. 34 A sponge-like structure with pore sizes ranging from 30 to 700 μm and porosity of up to 85% can be created using this method. 34 In some cases, the product produced may have a solid polymeric skin or a closed pore structure.
In nanofibrous scaffold development, electrospinning is crucial for producing fibers from a solution using electricity. 35 Utilizing electrospinning, a high-voltage charge causes droplets on the spinneret to erupt, causing them to stretch and elongate. The homogenous distribution of pores is challenging to achieve using electrospinning, despite the fact that it is an easy and quick approach for making nanofibrous scaffolds. As a result, only limited uses in biomedicine have been found for this technique. 35 In contrast to traditional scaffolding methods, RP systems use powder, liquid, and sheet materials to create items. Layer-by-layer, an RP machine can create objects made of wood, ceramic, plastic, or metal from a computer-generated model with tiny horizontal cross sections. 36 The RP scaffold construction methodology allows for fine spatial control over the polymer structure to overcome some of the difficulties inherent in standard production methods. These approaches allow for the creation of personalized and patientspecific scaffolds that are suited for the tissues and organs in question.
TE has a wide range of possible applications for RP scaffold creation. Micro-and macro-scale features can be controlled independently to create multicellular structures that are necessary for complicated tissue functions. To begin with, the construction of 3D circulatory beds will provide support for much larger tissue formations than would otherwise be conceivable. Combining clinical imaging data with 3D fabrication processes allows for the creation of personalized scaffolds and mass production of the scaffold designs.
Some of the most common RP processes include 3D printing, fused deposition modeling (FDM), selective laser sintering (SLS), and stereolithography. Stereolithography is a 3D printing technique that uses a thin layer of UV-curable material to build up a solid object layer-by-layer. 37 A stereolithography system consists of four essential parts, including a tank with a photosensitive liquid resin, a transportable constructed platform, a UV laser for radiating resin, and a dynamic mirror system. 37 The process begins with a UV laser by depositing a layer of photosensitive liquid resin on the platform. The platform is then lowered vertically once the first layer has been set.
Afterward, the process is repeated until a 3D scaffold is formed. It is then cleaned and postcured under UV light to remove any remaining resin. As a result, the waste associated with subtractive production methods is eliminated by this technology. Stereolithography is a robust biofabrication method that can be used in a wide range of fields, including biosensing, environmental remediation, drug discovery, and energy harvesting. 37 Using computer-controlled extrusion and deposition procedures, a solid polymer is melted and extruded onto the surface of a 3D object using the FDM technology. 38 The scaffold is constructed from numerous layers of neighboring microfilaments. Thermoplastic biopolymers have been processed using FDM. Also, SLS uses a laser as a power source to sinter powdered materials defined by a 3D model. This method has been used to create a wide range of materials, including polymers, metals, and ceramics. Tools and practical prototype features can be built directly from computer models using 3D Printing. The powdered substance is applied in layers and selectively fused via inkjet printing, where the adhesive is produced. The unbound powder is removed from the layers, resulting in a complex 3D shape. 39 This procedure can be used to manufacture ceramic, metal, and metal/ceramic composite parts.
3D polymer deposition is a novel TE fabrication process that enables scaffold structure microlevel precision control. 40 Even though the scaffolds created by the 3D Printing technology are able to replicate the shape of the natural tissue and the mechanical features of the scaffold, they lack the ability to mimic the nanoscale ECM properties that are essential to the success of the procedure.
Using material transfer techniques for producing a biological pattern and assembly of relevant materials, cells, molecules, tissues, or biodegradable biomaterials with a predetermined structure to perform some biological activities, bioprinting is a 3D printing technology.
Using solvent-free, aqueous-based printing methods, it is now possible to print 3D scaffolds for transplantation with or without seeded cells.
Furthermore, bioprinting is a technique that uses cell development to enable individualized medication. 3D bioprinting technologies can currently be divided into acellular and cellular. It is possible to print a scaffold and biomaterial without a cell using acellular bioprinting technology. Compared to cell-based bioprinting, acellular bioprinting provides a higher level of accuracy and greater shape complexity because it does not require the maintenance of cell viability during construction. It is possible to create living tissue constructs by integrating cells and other bioagents into the material during manufacture. This means that in order to build these structures, circumstances, and parameters must be optimized based on the presence or absence of living cells and biological components.
Autonomous self-assembly, biomimicry, and mini-tissue building blocks are only a few examples of 3D bioprinting methods. It is currently the most commonly used approach to deposit and pattern biological materials using micro-extrusion, laser-assisted printing, or inkjet printing. For example, inkjet bioprinting uses picolitre droplets of bioink to build 2D or 3D objects on a substrate without the need for touch. These approaches, for example, are ideal for a wide range of applications, including biomedical research, due to their low cost, rapid speed, and high cell viability (80%-90%). In addition, the approach has narrow material selectivity, frequent print head clogging, and difficulty retaining biological material in liquid conditions for droplet production. 41 The capacity to precisely insert cells, biomaterials, and chemicals for tissue regeneration has increased interest in bioprinting technology. 42 Additionally, this technique has been utilized to perform trials for 3D printing customized skin transplants for patients with extensive wound regions, muscle, and even micro stereolithography 3D printing to repair damaged nerve connections. 43 The delivery of nutrients and oxygen to cells is a critical aspect of cell viability.
Angiogenesis is often the most critical and challenging part of a TE process. So far, various methods have used to supply tissue oxygen to facilitate angiogenesis, which has not been as effective because the rate of vascular formation within tissues is naturally prolonged. 44 Due to the need to create branched vascular structures in printed tissues, the first and most crucial step is to print tubular structures. In practice, it is possible to create vascular tubes by placing the masses of endothelial cells together in three dimensions and connecting them in two directions, plane and vertical. 45 Reproducibility and scalability of 3D bioprinter technologies allow us to build structures. Recently, the use of bioprinters has provided new possibilities for the development and application of vascular systems in the rehabilitation of different tissues. 46

| In situ bioprinting
In situ bioprinting is a novel technology that merges with portable bioprinting, so it has some facilities to improve the disease and accelerate healing. 47 When compared to standard in vitro bioprinting, in situ bioprinting may be favored since de novo tissues are produced immediately on the predicted anatomical site in the real organism. 48 In situ bioprinting may be described as the direct printing of bioinks to build or repair living tissues or organs at a fault location in a clinical context. 49 Alternatively, the anatomical area where regeneration is needed in the body might be used as a printing site. Conventional 3D printing methods are often limited to flat surfaces, but this methodology intends to repair and replace damaged tissues with curved surfaces or more complex geometries. 48 However, the safety and sterility of this technology must be thoroughly validated before it can be implemented in clinical settings. Because of the lack of expertise necessary for in situ bioprinting in the clinical environment, clinicians prefer to use off-the-shelf alternatives, such as implanting a prefabricated construct at the defect location. 50 However, recent research has proven the enormous promise of this method, notably in the areas of skin, bone, and cartilage regeneration.
In general, in situ bioprinters are divided into two categories, including the robotic arm approach and hand-held approach. There is less human intervention in the former one, and the system includes a three-axis moving bioprinter. This device may have one or more print heads and may utilize a variety of bio-inks and cross-linking agents.
Hand-held bioprinters are a portable device equipped with a printing mechanism that enables the direct printing of biological materials and cells at the site of damage. 43 Among the advantages of this type of bioprinters, convenient transportation, easy access to the damaged area due to the small size of the device, and the low cost of device manufacturing are the most significant parameters. 51 Various studies were performed on the use of hand-held bioprinters. 52,53 Due to the lack of the establishment of an artificial microenvironment, the in situ TE technique has an advantage over the conventional TE approach. A functioning tissue or organ can only be created in the absence of these biochemical and biophysical factors, which is why ex vivo techniques rely primarily on mimicking the original tissue milieu (which includes the requisite biochemical and biophysical signals). As a result, surgeons are able to use in situ bioprinting techniques, which entail either robotic arms or a portable device with a separate bioprinting unit that may be inserted into a patient's body to repair injured tissue. 54 An in vivo bioreactor governs the development and maturity of the printed construct under this method. However, it is essential to keep the recipient organ immobilized during the printing process (e.g., the musculoskeletal system and craniofacial skeletal abnormalities, etc.). While in situ bioprinting has yet to reach its full potential, it is understandable to develop the technique for superficial organs. It is possible to print not just on horizontal surfaces but also to cover irregularly shaped tissue defects with a contour deposition approach using special robotic hands.
Additionally, in situ bioprinters have a practical application in the treatment of chronic wounds such as diabetic wounds, pressure ulcers, and burn wounds. The skin bio-printer employs a cartridgebased delivery method coupled to a portable XYZ plotting device.
Cartridges are identical to those used in conventional inkjet printing.
Each cell type is housed within a separate cartridge, much as different color inks are held within separate cartridges. The cartridgebased architecture of the skin bio-printer enables the direct printing of almost any cell type, macromolecule, or biomaterial in a wound region. 55 Aside from the additional stages and equipment required, the in vitro printing of scaffolds also increases the danger of infection.
The incision must be more significant for surgical reasons than the proposed procedure. The implantation location may also require the use of another adhesive media. This is not a crucial issue with current technology because printing takes place on-site, there is no need for transportation, and the printed scaffold can cure right where it is placed. On-site printing becomes inherently stable because the printer can construct an inherently stable form configuration at implantation.
As a result, patients can be released from the hospital sooner, allowing for faster recovery, and the hospital saves money since fewer beds are needed due to lower bed occupancy.

| DESIGN AND APPLICATIONS OF PORTABLE BIOPRINTERS
The ultimate purpose of emerging and introducing portable bioprinters is the ease of use and fast transportation in clinical conditions without the need for complex operator training. This technology is evolving at an incredible rate, which various institutes and companies are developing this technology with some innovations, with the common goal of accelerating the device's repair process and portability.
Regarding the wound dressings in treating acute and chronic wounds, they suffer from tremendous challenges, including the need to be changed at multiple times, scar tissue, the possibility of infection at the wound site, and the reduction of angiogenesis for wound healing. To deal with these issues, new methods of treating via handheld bioprinters have entered the market, which has solved these issues considerably. Multiple features along with several challenges could be considered utilizing portable hand-held bioprinters to regenerate multiple tissues ( Figure 1).

| The bioink flow-rate in bioprinting
The physical properties of bioinks could be altered based on the concentrations of hydrogel and cellular components in the bioink. Extrusion printing aims to produce stable, stackable filaments in a repeatable and predictable process, as well as to understand the connections between bioink qualities and extrusion parameters; hence, the desired output may be achieved. Hydrogels made from alginate, alginate/gelatin, and other materials that give repeatable and dependable outcomes with excellent form fidelity are commonly used to represent extrusion dynamics. However, while these hydrogels have improved our knowledge of the elements that affect bioprinting, they have limitations that do not applied to other materials.
Also, several key features are needed for the extrusion bioprinting process, including dynamic viscosity, loss modulus, and storage modality. 56 When shear stress and applied pressure are kept under control, the viability of cells is sustained. As a result, it is critical to know how flow rate, shear stress, applied pressure, moduli, and viscosity are all and practicality by hand. 60 The most prominent advantage of handheld bioprinters is that the operator can hold the bio-printer to print bio-ink directly at the wound site, without needing a computer system and defect scanning. Various natural and synthetic biomaterials have been prepared and used as bio-ink in portable bio-printer that have applications in wound dressings to treat acute and chronic wounds, bone and cartilage disease, skeletal muscle injury and dental trauma.
Also, gelatin methacryloyl (GelMA) and chitosan methacryloyl hydrogels are biocompatible and biodegradable hydrogels that have recently been widely used in various fields of medical engineering.
These hydrogels with a concentration range of 5-20 wt% can be mixed with cells and extruded by an improved printer into an X-Z robotic system portable bioprinter. 61 An affordable and versatile tool for creating functional materials at the point-of-carry (POC) has been developed by BioPen. 62  The two components immediately mix together during extrusion to produce a liquid droplet. (g,h) Co-axial filaments (dyed PBS in the core, GelMA: Gel: HA in the shell) in stereomicrographs with light on during extrusion. The shape of the filament is preserved, and it can be deposited as a continuous thread. (i) Stereo-micrograph of co-axial filaments extruded without light exposure and subsequently photo-crosslinked (GelMA 10% in the core, GelMA: Gel: HA in the shell). Rather than stacking, two crossed filaments blend together. (j) Confocal microscopy shows a line printed without light exposure, demonstrating the mixing of the core (red) and shell (green). (k) Stereo-micrograph of co-axial filaments with the light on during extrusion (GelMA 10% in the core, GelMA: Gel: HA in the shell), producing two stacked filaments. (l) Confocal microscopy of a line printed with light on during extrusion, displaying the core (red) and shell (green). Source: All pictures were reprinted with permission from reference 46 deposited biomaterial. Third, BioPen is able to write on curved surfaces, which is challenging for current printing methods. BioPen holds particular promise for low-cost POC diagnostics, which is an urgent requirement. 62

| The ergonomics of the portable bioprinter
In addition to their obvious importance in health care, medical gadgets also play a significant role in the product development. One of the essential design components of medical devices is the product's ergonomics, which have been designed, engineered, and prototyped for over 20 years. 63 Ergonomics is a branch of applied science that focuses on the design features of an object that humans can use effi- This process is done by installing a start and stop button. During the extrusion process, the curing lamp is set to a low-power setting to allow for a precure that may be tailored to any pattern or percentage of full power. Intermittent use of the curing lamp during extrusion can be used to impart desired mechanical qualities to the extruded product.
Hand-held and hand-intensive products are fundamentally influenced by the hand's inherent geometry. Three palmar arches, known as the grip axis, appear when a user shuts his or her fingers and grasps an instrument. The location of fingertip grip surfaces and controls on a device should be defined in terms of precision, control, and comfort based on hand geometry and size and strength.
Hands are extremely sensitive to touch that a raised dot on a piece of glass that is only 3 mm high may be detected by a fingertip, Right-handed and left-handed users alike should be able to operate the same ergonomic control surfaces with equal ease. Textured surfaces are common on ergonomic control surfaces because they help grip and control. 65 It is pivotal to know what kind of traction the gadget needs before designing a texture that would allow the user's fingers to bite in and give that traction. Hand-held medical equipment's control surfaces are heavily influenced by the ergonomic design idea of dynamic grip security. 66 Hand-held gadgets with dynamic grip security allow users to safely grasp an instrument while using fingertip controls on the same hand. This action is usually required in medical equipment that requires the user to hold it. Creating product forms that allow the user to apply a combination of precision and power grips is a challenge for the designers. Using an ergonomic approach based on the natural hand geometry, the ring and baby fingers can be combined into one powerful super finger.
Because of this, the thumb, index, and middle fingers may be used in a more dexterous and precise trilateral precision grip in conjunction with the control surface that is specifically created for them. 66 For portable medical devices, ergonomic design is not limited to these five factors: user focus, hand architecture/dynamics, attention to detail, complicated grip solutions, and tactile control surfaces. No hand-held or hand-intensive device can function properly without them, though. In order to create the best devices, ergonomics is at the center of the design process.

| Considerations for cross-linking of printed bioinks
Bioinks used in extrusion-based printing must maintain the survival, motility, and proper metabolism of the cells integrated into the extrusion-based printing process. Printing sturdy, thick, multilayer constructions at high resolution typically necessitates additional processing due to the fragile and delicate nature of the materials that meet these characteristics. However, it is possible to print more substantial materials with greater mechanical endurance that can crosslink at longer intervals, provided that the cross-linker can permeate the printed material efficiently. Depending on the printing procedure, most bioinks require a stabilizing cross-linking step before or after printing. The molecular structure of the printed material should be strengthened in order to keep the extruded bioink's form, avoid collapsing, and build firm structures that can withstand the conditions of in vitro culture and/or implantation. Incorporating cross-links between the polymer molecules is one way to do this, which results in a material that is both self-supported and noticeably stiffer. In order to maintain the bioink's cytocompatibility, this change must be light enough.
A bioink's mechanical qualities, its ability to retain its form after exiting a nozzle, and the composition of a cross-linker all influence the timing of the crosslinking process. 3 When pressure is exerted from the upper layers, weak hydrogels tend to spread over the substrate and distort. As a result, when using hydrogels, cross-linking must be done simultaneously as extrusion or before adding a fresh layer of bioink.
This also applies to cross-linking agents that cannot penetrate deep into multi-layered structures. There are several ways that the cross-linker can be introduced, such as by coaxial extrusion of the bioink; spraying, deposition, or projection of the cross-linker onto new layers; or printing into a crosslinking-inciting environment. 3 The most prevalent methods for reinforcing printed bioinks are based on ionic, enzymatic, light irradiation, and thermal energy-induced cross-linking ( Figure 3). 67

| Effective factors on the survival of printed cells
The printing process relies on a thorough knowledge of the effects of shear stress, compression, cavitation, and other external stresses, such F I G U R E 3 Various bioink cross-linking strategies that could be used in bioprinting through portable hand-held bioprinters. (a) Ionic crosslinking: A simplified schematic depicting the formation of electrostatic bonds among ions (green) and polymer chains with opposite charges, resulting in a branched network. (b) Enzymatic cross-linking: A simplified schematic depicting the enzyme-catalyzed formation of covalent bonds among polymeric biomolecules. (c) Light-induced cross-linking: A simplified representation of the cross-linking process in the presence of a photo initiator (yellow). When exposed to UV light, the photo initiator produces free radicals, which start a polymerization process between the functionalized chains. In addition to the nozzle design, the diameter of the distribution orifice also impacts cell survival. 70 Figure 4 illustrates the impact of the nozzle design and diameter on the cell's interactions and survival.
Shear stresses grow with increasing viscosity or storage modulus, which requires more enormous pressures to extrude it.
In cell printing, the shear field at the nozzle, which is considered the principal source of cell damage and loss, is of particular importance. Through shear, mechanical cell disruption occurs during bioink extrusion from syringe nozzles. Fluid near the nozzle walls is thinned while still flowing laminarly. Cells are immediately exposed to a velocity gradient profile that varies depending on the nozzle used throughout the printing process, typically a maximum intensity at the center of the nozzle, leaving a static field around the nozzle wall. 71 Shear-thinning and nonshear-thinning biomaterials are employed in bioinks. During the application of shear force, shear-thinning materials can be injected and have the potential to swiftly self-heal. When physical or chemical stimuli are applied to nonshear thinning materials, gelation occurs. In extrusion, shear stress is maximized at the nozzle walls. Equation (2) shows the relation among the shear stress (τ wall ) The effect of cell seeding density and shear rate on 3D cell-laden biomaterial strands and nozzle extrusion. (a) Extruded cell-laden filaments may contain a number of cells proportionate to the density of cell seeding. The concentration and distribution of polymeric chains (dark blue) have a direct impact on cell proliferation potential. Poor cell seeding density causes poor cell dispersion within the printed strand, resulting in low cell-to-cell contact and a restricted growth rate. Physical pressures exerted by neighboring cells restrict proliferation and survival. Cell density may be adjusted to provide a uniform distribution of cells enclosed inside the printed filament, allowing cells to retain the necessary contact with other cells in order to stay mitotically active and grow. (b) Keeping the number of cells in a printing syringe constant but altering the nozzle aperture will impact cell printability. Large nozzles (>800 m) provide limited cell-nozzle barriers and cell-to-cell contacts, resulting in a wider dispersion of suspended cells inside the bioink. These parameters may assure good cell survival after extrusion but result in poor overall construct resolution. Narrower nozzles (250 m) with a lower surface area for the same amount of cells, on the other hand, compel paste encapsulated cells to contact with one another, resulting in high density at the nozzle aperture. Upon printing, a tiny orifice may generate excellent resolution as well as significant cell death. Medium size conical nozzles (250-800 m) provide excellent cell dispersion inside the nozzle and an improvement in print resolution without affecting cell survival significantly; Source: reproduced with permission from reference 69(c) Shear stress's effect on cell viability during extrusion-based bioprinting (the system used in portable hand-held bioprinters). Cells along the wall are exposed to greater shear stress intensity than cells in the middle of the nozzle, resulting in damage. Cell viability diminishes exponentially as shear stress rises and the distance along the nozzle radius (R). 72 Also, ΔP presents the pressure drop in the cylindrical nozzle, while the total length of the cylindrical nozzle is showed by L.
As a result, printing settings must be adjusted in accordance with the intended bioink. Bioinks may be printed accurately using blunt Increased ATPase activity and calcium deposits were seen after stem cells were subjected to higher shear stress levels. 74 Overall, two key elements, material qualities and printing settings govern the effect of process-induced shear stress in extrusion-based bioprinting. 75

| TISSUE REGENERATION VIA PORTABLE BIOPRINTERS
A variety of biomaterials were printed through utilizing hand-held por-  sheets onto a burning bed to improve wound healing results. 78 Their study suggest that cell-containing biomaterial sheets are deposited on the wound like a paint roller, covering the surface with a uniform sheet of skin, stripe by stripe, as well as in vivo trials on full-thickness wounds. 78 With the advent of next-generation safe cell and biomaterial delivery made possible by this automated technology, therapeutic applications that go beyond full-thickness burn injuries will arise in the future.
Recently, Albanna et al. 50 presented a new in situ skin bioprinting system's design and proof-of-concept validation. This device is capable of combining imaging technologies to determine the topography of wounds with precise cell administration to meet the specific demands of each patient (Figure 8a). The authors claimed that the developed bioprinting system has advantages, including hand-held and capable of being easily transported, identifying and measuring the wound sizes and topologies, delivering various cell types, easy sterilizing, and a low cost.
Wound scanner to accurately assess wound architecture and to allow accurate distribution of appropriate cell types to particular wound surfaces have been integrated in this in situ bioprinter, which marks a significant breakthrough in customized wound treatment.
Portable 3D wound scanner and a print-head with the XYZ movement system, each driven by an independent dispensing motor, are the primary components of the bioprinter. In order to move around the operation area, everything is mounted on a tiny frame (Figure 8b).
Skin wounds can be reconstructed by combining the wound scanning system with the cartridge-based delivery system (Figure 8c). An in vivo study by the mice and porcine models demonstrated that this in situ bioprinter provided instant and suitable coverage of wound beds by delivering autologous or allogeneic fibroblasts and keratinocytes with the biological hydrogel, such as fibrinogen and collagen. 79 Thus, wound therapy with our in situ bioprinting method produced quick and appropriate covering of critical wounds for maintaining homeostasis, re-epithelialization, and scar prevention. 79

| Skeletal muscle regeneration
Volumetric muscle loss (VML) is caused by skeletal muscle injury as a result of trauma or surgery. 93 VML is mostly caused by trauma of a F I G U R E 5 Skin printer on a hand-held device (a) A schematic illustration depicting the operation of a hand-held bioprinter. A cross-linker solution (blue color) and one or more bio-ink solutions (green color) comprising premixed biomaterials and cells are produced. In a culture dish or at a wound site, a hand-held bioprinter transforms bio-inks into homogeneous or architected biomaterial sheets or tissues. (b) Hand-held bioprinter rendered picture. A handle (1) allows you to place yourself above the target surface or wound. The deposition speed, V, is determined by a stepper motor, pulley, and driving mechanism (2). The dispensing flow rates for bioink (4) and cross-linker solution are controlled by two onboard syringe pump modules (3). (5). For spatial organization of solutions and sheet production, a 3D manufactured microfluidic cartridge (6)   In addition to the 3D wound scanner, the primary components of the system include 260 m diameter nozzles operated by up to 8 independently dispensing systems coupled to a print-head with an XYZ movement system. All of the components are installed on a compact frame that may be moved about the operating area. (c) The idea of skin bioprinting. Wounds are scanned first to get accurate information on wound topography, which is then used to direct printheads to deposit specific materials and cell types in the correct areas. Source: All pictures were reprinted with permission from reference 79 severe enough nature to leave victims in a state of social and economic hardship. 94 Organized and self-renewing, skeletal muscle is an important part of the human body Because of intrinsic soft tissue damage, a lack of adequate regeneration, and fibrosis, VML injuries have limited ability to recover. 96 However, conventional reconstructive therapy for such injuries prevents amputation, but they are limited in returning muscle strength and movement. 97 VML therapy procedures include prosthetic bracing and autogenic muscle flap transplantation, while these treatments have some limitations. 98 Owing to a lack of definitive therapy, VML leads to constant pain and disability. 99 The minimal bioavailability of biological growth factors or low cell engraftment has limited the efficiency of regenerative medicine techniques such stem cell and growth factor administration. 93 Because it produces functioning tissue constructions that can restore damaged muscle function and structure, TE has considerable potential as an alternative therapy. 100 Portable automated bioprinters capable of printing scaffolds in situ have recently emerged as a potential solution to many of the problems associated with VML damage. 100 Hydrogel-based scaffolds were printed into the defect region of mice with VML damage in a study conducted by Russell et al., 100

| Cartilage regeneration
Cartilage is an aneural, avascular, and alymphatic tissue that its injuries' primary symptom could not be sensed 102,103 due to the fact that it has a sparse population of a chondrocyte, possessing the ability to regenerate post injury is prevented. 104  MSCs, which was covered with fibrin glue spray before being filled in. They engineered a hand-held bioprinter (Biopen) to co-axially print the two different bioink in a core/shell distribution (Figure 11d-g).
They reported that the Biopen could be used in multiple applications, such as wound skin regeneration and pulp dental disease treatment.
Using the nozzle's unique design, the researchers were able to perform single-session surgery on sheep's knee joints, depositing cells and the scaffold directly into the cartilage lesion. The results were promising. Two bioinks extruded from separate cartridges in a coreshell method during coaxial bioprinting, so the core bioink may contain stem cells and the shell bioink is cured by the photo-initiator to process mechanical strength in the bioink. The outcomes investigated that the 3D-printed scaffold had better characteristics than control groups and showed a novel formation of hyaline-like cartilage via an in vivo study (Figure 11h-j).
Li et al. 85 combined 3D scanning and in situ bioprinting technologies to regenerate cartilage and bone defects. In their research, they modeled three flaws, such as substantial long-bone segmental defects, free-form femoral condyle fracture, and ICRS-grade IV chondral lesion ( Figure 12). It was discovered that using bioink for the treatment of cartilage disease, a tiny molecular molecule was effective. Since HA has superior features of biocompatibility, biodegradability, viscoelasticity, and nonimonogenesity compared to other potential cartilage printing bioinks, it was chosen suitably.
They utilized UV-polymerized sodium alginate and polyethylene (glycol) diacrylate (PEGDA) to mend the bone deficiency. Hydrogels such as alginate are frequently utilized in 3D bioprinting as bioink because of its exceptional performance, such as appropriate cell attachment, printability, biocompatibility, biodegradability, low toxicity, and moderate gelation. PEGDA is also utilized in TE and targeted drug release as a drug carrier and small molecule due to its low degradation rate, noncellular adhesion characteristics and tunable mechanical properties. 84 To make bone and cartilage scaffolds stronger, PEGDA has been utilized to carry tiny molecules including bone-morphogenetic protein 2, collagen, and oligosaccharides. This increases the scaffold's flexibility as well. 106 F I G U R E 1 1 (a-c) In situ 3D bio-printing applied to a rabbit's knee joint. Source: reprinted with permission from reference 59. (d) The Biopen's design, which exhibits two distinct chambers controlled by a motor. The printing nozzle (insert) is linked to the two chambers, allowing for coaxial printing of the two distinct bioinks in a core/shell distribution. (e) A picture of the Biopen. (f) Core/shell distribution representation. The photoinitiator VA086 for UV photocuring is included in the GelMa and hyaluronic acid methacrylate hydrogel (HAGelMa) in the shell, which provides mechanical support to the printed biomaterial while also protecting the inner core. Photo-initiator is not present in the HAGelMa in the core, but it does contain mesenchymal stem cells. Generally, the core/shell portable 3D bioprinting strategy could rapidly produce high modulus scaffolds with high cell survivability and have a potential for in situ surgical cartilage engineering use.
In addition, the authors pointed out that the fabricated portable 3D printer might be used to implant co-axial cell-containing scaffolds in surgery, particularly for the restoration of musculoskeletal tissue. 82 Xu et al. 81

| Bone regeneration
Although surgical procedures have improved significantly in recent years, bone tissue regeneration after injury is still difficult to achieve structurally and functionally. 108 The conventional treatments of critical-sized bone defects included culturing bone tissues from autografts or allografts and implanting the cultured bone tissue into the defected area are going on. 109 Autograft approaches are considered the gold standard in bone fracture treatment. In spite of the fact that autografts have been around for a long time, they have a number of disadvantages, including donor site morbidity, sensitivity, deformity, muscle weakening, infection, and persistent discomfort. 86 Foreignbody implants, which fill in the bone deficiency and offer structural support and mechanical strength, are an additional treatment option for problems. 110 However, foreign-body implants do not offer permanent treatment for children and middle-aged individuals since they are associated with poor functional and esthetic consequences. 111 Bone tissue engineering (BTE) provides an alternative approach for generating new bone tissues and treating bone diseases. 112 The major difficulty of BTE is to produce natural bone tissue-like mechanical strength and sufficient porosity for vascularization, which is categorized as hard tissue.
Bioprinting research has looked at bone tissue. 90 Open fractures in the skeletal system can be treated using 3D scanning and 3D bioprinting. 89  Furthermore, they demonstrated that LAB with in situ bioprinting technology is suitable for bone repair and random-shaped cartilage defects. 12 An in situ extrusion bioprinting for cartilage and bone deformities from the Cohen et al. 113 work was successfully reported. These matrices were printed on an osteochondral defect in order to assess the potential for bone and cartilage tissue regeneration in their study.
Their study demonstrated the feasibility of the in situ reconstruction system with great precision due to the geometric feedback and also progressive path planning techniques, while biomaterial selection for scaffolding and its applicability in the human body are significant challenges yet.
Furthermore, as reported previously, Li et al. 85 showed 3D scanning and bioprinting to have the ability to fix skeletal and cartilaginous problems. Using in situ printing to quickly and accurately obtain a 3D digital model, a new treatment method for osteochondral injuries has Mostafavi et al. 86 were able to design fused portable printers called Pen Bone and insert the material ink directly into the cavity ( Figure 14). In their work, scaffolds were prepared from a polycaprolactone (PCL)-based ink compound with zinc oxide nanoparticles and hydroxyapatite (HAp) microparticles. HAp, which has a significant contribution to hydrophobicity, has been used to improve protein absorption. Also, zinc oxide nanoparticles were used to inhibit bacterial growth on the scaffold surface. Printed scaffolds showed that they have an appealing support structure for MSCs.

| Dental regeneration
The advent of novel portable bioprinting technology has attracted many scientists' attention in various scientific fields, including dentistry. 114 Campos et al. 87 prepared a printable agarose (AG), collagen type I (COL1), and fibrin hydrogels in 0.5 wt% FIB, 0.3 wt% COL1, 0.2 wt% COL1, and 0.5 wt% AG amounts. They stimulated root canal vascularization using human primary dental pulp cells and endothelial cells ( Figure 15).  Although, the implantation of fabricated structures has faced to several issues associated to printed tissues integrations with environmental tissues, namely, production of fitted construct for injured area, surgical removal of dead tissues from defect location and risk of contamination. To tackle these limitations, in situ bioprinting technology emerged to directly print and deposit constructs at the patient's defect site. 115,116 Campbell et al. 117  Current investigation has reported various external cues for gelation of bioinks that are not biocompatible, as these studied cues cannot be applied within the patient body. Another limitation in the maturation of printed tissues is an insufficient vascular network to provide nutrient molecules and oxygen to deep tissues. 123 However, it is proposed to solve vascularization limits before the clinical development of in situ bioprinting fabrication.
Additionally, biocompatibility between expanded stem cells and biomaterials, adequate cell proliferation, and correct differentiation, which supplies the capacity to stem cells, restore the natural ECM.
The biodegradation rate of printed biomaterials should match with the ECM production rate by seeded stem cells. The biodegradation rate control in vivo has multiple problems. Other post-printing challenges, including rapid solidification, combining environmental healthy tissues, and make sterile conditions are vital considerations in tissue biofabrication and maturation processes. 48,124 The improvement and successful therapeutic interventions are expected to provide more cost-effective and user-friendly treatment. Therefore, in situ bioprinting integration with robotic system technology prepares a more accessible technique for surgical operations. Besides, various advanced imaging instruments would be combined with robotic systems to evaluate the damaged area to fabricate printed tissues in vivo.
All these progress will accelerate in situ bioprinting technology translation from the bench to the bedside. 9,125

| Perspectives
As mentioned previously, multiple 3D bioprinting technologies have been appeared and applied to diverse medical applications, ranging from evaluating of the cellular process to regenerating tissues, organoids, and organs for in vivo implantation. 126 The remarkable ability of 3D bioprinter technologies makes a promising strategy for tissue damages, even in situ and has been displayed to transfer cells, biomaterial, and biomolecules to target damaged locations in a site-specific manner. 127 3D bioprinting technology can be integrated with other advanced approaches such as cell therapy, and microfluidics to fabricate automatic manageable printed devices to reconstruct the diverse biological structures and essential composition of targeted tissues as well as increasing drug discovery procedures and developing an effective treatment for genetic disorders. 128,129 3D printing strategies, which are commonly dependent on the printed biodegradable cell-free constructs, are eventually applied for implantation in the TE field. In this top-down method, the cells as key factor of tissue regeneration is only seeded at the end of 3D printing procedure, some hurdles, such as inaccurate cell distribution over the printed grafts occurred, which led to failing appropriately regeneration due to the incorrect ECM ultrastructure with loss sufficient cell-ECM interactions. Therefore, 3D bioprinting appeared as bottom-up approach to create artificial printed tissues or organs in a layer-bylayer manner through critical tools, including proper biomaterials, various cell types, and growth factor molecules. 2,130 Recent studies show the great potential of in situ 3D bioprinting technology as a feasible regenerative method in TE. 131 It has been suggested that the natural cellular microenvironment in the patient body can promote maturation and merging of printed grafts to injured tissues or cells due to different and huge chemical and physical signals. 121 Moreover, in situ printing procedures with less invasive operating procedures can be introduced as a user-friendly technology for surgeons that increase outcomes therapeutically. 132 Also, some successful efforts reveal the future perspectives of the portable hand-held bioprinters' applications. Researchers created an easy-to-use syringe for the hydrogel that could chill the gel quickly before application on the front lines. 133 An ice pack-like chamber in the syringe that contains calcium ammonium nitrate crystals was used.
Within 30 s of introducing water to the chamber, the crystals begin to activate and chill the hydrogel to its operational temperatures. As a result, they could modify the hydrogel delivery device so that it not only swiftly cools the hydrogel but also retains it at that temperature, providing a 10-min window to fill penetrations in the eye. This is like caulking a bathroom seal in terms of simplicity. Hence, injuries to the eyes caused by piercing objects will heal faster with the new seal and delivery system. 133 The portable hand-held bioprinters could promote this healing process with the homogenous extrusion of the material from the nozzle.
Moreover, surgical sealants have been used for sealing or reconnecting ruptured tissues but often have low adhesion, inappropriate mechanical strength, cytotoxicity concerns, and poor performance in biological environments. 133 To address these challenges, researchers The practical idea of sealing or reconnecting ruptured tissues through employing the portable hand-held bioprinters is another perspective that could promote surgical and suturing operations. Highpressure bleeding in pigs' hearts can be stopped with a novel bio-glue, an experimental adhesive gel activated by a flash of light. According to the World Health Organization (WHO), more than 234 million procedures are performed each year around the world. 135 According to a previous study, surgical suturing is particularly challenging when dealing with sick, damaged, or tiny blood arteries. 136 Fibrin Glue and Surgiflo, two commonly used surgical materials that effectively stop bleeding during surgery, take minutes to solidify and may necessitate additional stitching in some circumstances. 137 Wounds and punctures of hearts, among the most difficult of surgical challenges, could also be sealed using only the bio-glue, with no stitches.
Meanwhile, combining the portable bioprinters with other instruments could be applicable. Zhao et al. 138 designed and fabricated an in situ bioprinting device that can print inside the human body utilizing a micro bioprinting platform attached to an endoscope. The platform was designed and built using printed circuit micro-electro-mechanicalsystem techniques. The platform's viability was tested via a highprecision control system and a suite of performance tests. Applying a stomach model, two-layer tissue scaffolds were created, and human gastric epithelial cells along with human gastric smooth muscle cells were employed as bioinks in gelatin-alginate hydrogels to imitate the stomach's architectural shape. Cell viability and proliferation on printed tissue scaffolds were shown to be high after 10 days of cell culture, indicating that the cells were performing well biologically. The authors used an endoscope to insert the device into a transparent stomach model for the experiment. 138 The prototype was ready to begin bioprinting on the stomach wall with epithelial and muscle cellladen gels, on arrival. It is possible that these 3D-printed tissues could someday repair genuine ulcers in the digestive system because they retained their vitality and proliferated for 10 days. A significant step forward in treating stomach wounds could be made with this in situ bioprinting technology, as conventional therapies are typically slow and not consistently effective. The authors prospected that microrobots with cameras and other sensors could be developed in the future, allowing them to carry out more sophisticated tasks. The bioink kind of portable bioprinter is one of the most complex issues.
Choosing a bioinject that can gel at 37 C is critical since multiple biomaterials liquefy at body temperature.

| Commercialized hand-held bioprinters
The promising results of the bioprinters and the countless advantages compared to conventional methods of fabricating TE scaffolds have attracted considerable attention. 139 Of the foremost obstacles and challenges of bioprinters' commercialization is their high manufacturing cost. Over time, with innovative designs and bioprinters classification, some difficulties have been solved depending on their application. 140 A $10.8 billion market is predicted for bioprinting, but it still faces significant commercial challenges due to the bioprinting process' optimization, which is currently not automated and requires manual operations in various steps, resulting in a slow processing speed and increasing the risk of error. 19 Regulatory approval, insurance, hospital and medical regulations, and logistics are just a few of the major hurdles that must be overcome in addition to manufacturing that may be scaled up. 19 Today, portable bioprinters have mostly removed this industry's previous limitations. 48 As recent surgeries are being robotically and intelligently established, portable bioprinters can be a trustworthy option for minimally invasive and cost-effective surgeries. 48 Pereira et al. 141 designed the first commercial bio-printer in Germany and later commercialized it successfully. Commercially available bioprinting in situ printers have yet to be developed. Even so, a few commercially available robotic surgical systems, such as the MAKOplasty ® knee replacement system and the da Vinci ® surgical system, are now in use in the field of orthopedics (such as cardiac, thoracic, colorectal, and many others). 142 They have demonstrated its efficacy in conducting the procedure with reproducibility and accuracy. In its infancy, in situ bioprinting may not completely replace traditional TE methods, but it will certainly enhance them.
Commercially available portable bioprinters are manually controlled and do not have a computerized system, whereas, they are cost-effective and portable. 11

CONFLICT OF INTERESTS
The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.