Facet of 4D printing in biomedicine

Owing to the success of three-dimensional (3D) printing in biomedical applications, the latest addition to the technology is four-dimensional (4D) printing, which has gained tremendous interest since 2012. 4D printing is being considered as an upgradation and extension of 3D that includes time as a fourth dimension with the utilization of smart biomaterials, and upon the application of any external stimulus, the shape and size of the printed structure change with time. In this review, we highlight the basic techniques involved in 4D printing, the shape memory effect, and various stimuli like light, temperature, pH, etc., that cause the shape change, leading to the transformation of the structures fabricated. 4D printing using smart materials demonstrates shape memory property and their possible applications in the field of biomedicine and regenerative medicine are discussed in detail. The authors have focused on 4D printing of various tissues, with a special highlight on bone and dental tissue.


Invited Feature Paper-Review
are responsive to external stimuli like pH, heat, temperature, etc., fetches a lot of potential in the area of regenerative medicine ( Fig. 1).
At a TED conference in 2012, Tibbits et al. showcased an object that was printed and its transformation over a period of time [1]. Since then, 4D printing has been evolving and utilized in tissue engineering. The use of time as a fourth dimension helps in reshaping and resizing an object that is preprogrammed to obtain a particular function gradually in the presence of external stimulus [2]. Cost-effectiveness, assembly line, number of components, and failure rate are few of the advantages of 4D printing over the additive printing technique and have a huge influence on biomedical engineering. The technology can minimize the labor, handling, transportation, and processing cost. Post-printing the components of 4D printing gets self-assembled under the influence of the external stimulus, thus minimizing the number of components in the system. The assembly time has been reduced in comparison with conventional processes, for example, processes where the motors, sensors, etc., are assembled post-fabrication [1].
In this review, we discuss the 4D printing techniques and the external stimuli that help in transformation over a period of time. It also includes various smart materials that are printed, including shape memory polymers (SMPs), hydrogels, and shape memory alloys. The potential applications and the presented research on 4D printing in various biomedical fields are also reviewed. Figure 2 represents the effect of SMP on 4D printing. 4D printing is an emerging technology that utilizes the shape memory effect (SME) of materials that can change their geometry, topology, and functionality with time. The external stimuli that can affect the material include pH, light, temperature, magnetic field, and so on. The property of the SME plays a vital role in the 4D printing of structures. When the structure is heated above its required characteristic transformation temperature, it will recover back to its original shape and size, which is the principle behind the SME. Based on the shape recovery ability of the 4D-printed structure, SME is classified into one-way SME, two-way SME, and multiway SME [3]. In one-way SME, the material is designed to change its structure once and cannot recover its original shape [4]. In a two-way SME, the structure can change back to its original shape and undergo a course of transformation and recovery [5]. Two-way SME can occur reversibly when an external stimulus is removed or requires a preprogrammed stimulus. In a multiway SME, multiple smart materials are required for the fabrication of structures, which can undergo shape recovery, obtain their original shape, and change to a designed shape when required (Fig. 3).

Four-dimensional printing techniques
Inkjet printing is one of the non-contact techniques and utilizes tiny ink droplets to reconstruct the structure for 4D printing [6]. Liu et al. studied the self-folding of polymer sheets in the presence of light into a 3D object. An HP Color LaserJet CP3525dn printer was used to produce 2D color ink patterns, and explicit inkjet shrink films were used as polymer sheets. Printed sheets absorbed light (provided by Figure 2: 3D printing of shape memory polymers: (b-d) demonstration of the temporary shapes recovering to their permanent 3D shapes (from left to right) including a cardiovascular stent, an Eiffel Tower, and a bird (reproduced from Ref. [74] copyright with license Id: 5318670525340).

Figure 3:
The various types of Shape memory effect-one-way SME, two-way SME, and multiway SME.
light-emitting diode (LED)) of different wavelengths based on the color of the ink, and this absorption by the ink caused the polymer to heat up, thus inducing self-folding [7].
The fused deposition modeling (FDM) technique forms a layer-by-layer 3D structure by extruding the ink via a nozzle, which is inexpensive and upgradable, but it lacks the ability to produce smooth finished products [8]. FDM employs the use of minimal quantity of materials and can print large structures making them affordable, while it has very low resolution and the finished products have to be improvised via gap filling and vapor smoothening, which leads to increase in production time, thus making FDM not an ideal choice of printing technique. Stereolithography (SLA) and digital light processing printing techniques have better precision, accuracy, and resolution compared to FDM. The polyurethane (PU)-based shape memory material was printed using the FDM 3D printing technique, and the material was able to retain its thermal-responsive characteristics when in water and during voltage experiments [8].
Direct ink writing (DIW) technique is efficient in fabricating scaffolds with respect to structural design with the mechanism of shape transformation [9]. The DIW-based printing required viscoelastic inks that have shear thinning and pseudoelastic behavior that contributes to shape retention of the printed structures [9]. The DIW technique with SMP can help in obtaining stable 4D printed structures. It shows many advantages like achieving high printing resolution, free choices of materials, and the requirement of small number of raw materials. They are used in tissue engineering for printing high-strength biodegradable structures. Wang et al. studied water-based biodegradable shape memory PU as a bioink for fabricating bone scaffolds. Superparamagnetic iron oxide (Fe 3 O 4 ) nanoparticles were added to the ink to promote the induction of mesenchymal stem cells (MSCs) while polyethylene oxide or gelatin was also added for better printability. The scaffolds promoted the osteogenesis of human MSCs (hMSCs) and the scaffolds were used for bone tissue regeneration. The experiment was performed with the purpose of customizing scaffolds for the use of developing minimally invasive surgical procedures and applications in tissue engineering [10].
SLA utilizes a laser to form a 3D structure by polymerizing layers of the material [11]. It covers a large variety of materials and has very high accuracy while printing. The SLA printing technique was utilized to print a photopolymer resin made of tert-Butyl. Acrylate with diethylene glycol diacrylate (tBA-co-DEGDA) network has shape memory properties. Choong et al. successfully used SLA for SMP, which had rapid laser curing. The curing speed, depth, and polymerization shrinkage are taken into account that leads to achieving enhanced curing and high printing features. Additionally, the printed structures were able to withstand high strain deformation when immersed in hot water (open structure) and upon cooling temporary flat structure was obtained. This effect showcased the ability of the SMP to withstand high strain [12].

Stimulus for 4D printing
As 4D printing involves the transition in structure and shape of the material when exposed to a specific external stimulus, researchers have developed materials that are responsive to temperature, water, pH, chemicals, magnetic fields, and light. To enhance the responsiveness of the materials to a particular stimulus, molecular level interaction and its microstructural characteristics need to be optimized. In this section, various stimuli employed for the fabrication of 4D printed materials are discussed (Fig. 4).

Water
Sensitivity to water has been employed in materials via optimizing the sensitivity of molecules toward the aqueous environment. Water has been known to generate various forces involving osmotic pressure and hydrophobic interactions, where the water and hydrophobes do not interact. The nonpolar substances clump together in the presence of water, creating an endothermic reaction as the bond is broken but the molecule does not react with the hydrophobe. These given forces have been employed in materials through optimizing the hydrophilicity or hydrophobicity via substitution. The most common strategy is to fabricate a multilayered material with a significant difference in the sensitivity to water molecules. This difference would implicate a directional force inside the material and cause structural changes. Zhang et al. tuned the hydrophobicity of cellulose films by modifying cellulose with different degrees of stearyl substitution. As shown in Fig. 5(a), lower degrees of stearyl substitution have been observed to show responsiveness to moisture and temperature as well. Further, the non-wetting surfaces of films were made superhydrophobic by employing a layer of cellulose nanoparticles derived from cellulose derivatives with higher degrees of stearyl substitution [13]. Hydrogels are crosslinked materials that exhibit shape memory properties.
Ureidopyrimidinone (Upy) has been employed to crosslink hydrogels due to its ability to form a dimer via hydrogen bonding [14]. Alavijeh et al. demonstrated that hydrogel was formed with Upy and was substituted in gelatin, which can be utilized as a responsive material [15]. Osmotic pressure and swelling require a saturation to generate the force required inducing the change in the shape of materials and due to this reason, the response of water is slower in comparison with other stimuli.

Temperature
Sensitivity to temperature and temperature-dependent phenomena has a wide range of applications. These have been studied at the molecular level for various materials like glass transition and have been substituted in materials to enhance their thermal responsiveness. Swelling and collapsing with temperature change are one of the most common phenomena observed in thermal-sensitive materials, which occurs due to a change in the volume of the material reversibly. Poly (N-isopropyl acrylamide) (PNIPAM) is one of the most studied temperature-sensitive polymers and has been applied for the fabrication of smart sensors and actuators [16]. Folding in polymeric systems has been observed when the temperature of the polymeric solution is raised higher than the lower critical solution temperature (LCST). Recently, multimaterials have been employed for the fabrication of 4D printed polymeric constructs. Researchers have fabricated block copolymers with monomeric units having a significant difference in thermal properties or by blending two SMPs in different proportions. Ge et al. have reported a SMP fabricated by employing benzyl methacrylate for linear chain and multifunctional oligomers-Poly (ethylene glycol) dimethacrylate and Bisphenol A ethoxylate dimethacrylate (Bis-EMA) with Di (ethylene glycol) dimethacrylate (DEGDMA) as a crosslinker. In the given study, a flower-shaped structure was fabricated and blooming effect was observed when the temperature was changed from 20 to 50 and 70 °C as shown in Fig. 5(b) [17].

pH
A pH sensitivity has been studied in various molecules, and a significant change in molecular level properties of the materials has been observed. An effect of hydrogen ions on molecular conformation can be observed due to protonation, deprotonation, and tautomerism of many molecules. Researchers have tuned the polymeric properties of hydrogels by tuning the pH of the hydrogels with respect to the pKa. Lee et al. have demonstrated the shrinkage of swelled hydrogels when the pH of the hydrogel was adjusted to less than the pKa of the carboxyl functional group. Protonation of carboxyl groups below pKa takes place, which decreases the swelling in hydrogel and vice versa [ Fig. 5(c)] [18]. In addition to this, the effect of pH on the color of 3D printed structures has been demonstrated. Shanthamma et al. 3D printed sago starch with different proportions of turmeric blends and the pH was modulated by varying the sodium carbonate concentration. The color Figure 4: (a) External stimulus causing the change of temporary shape into permanent shape, (b) different type of changes in shape, size of the structure in response to an external stimulus, (c) various external stimulus that can act on a structure. of the structures changed from yellow to orange-red with an increase in pH and time due to pH-dependent tautomerism demonstrated by cucurmin molecules in turmeric [19]. The pH-induced transformations are not advantageous in in vivo application, but their application in biosensor fabrication is of great potential.

Light
Light-responsive 4D printed materials have the potential to improve the responsiveness of smart optical electronic devices and micro/nano-actuators and can mimic the light-dependent biological processes for biomedical applications. Light responsiveness in 4D printed materials has been substituted through the introduction of light-sensitive materials. Nanomaterials like graphene have been studied to have photothermal effects when exposed to near-infrared (NIR) radiation. Cui et al. reported 4D printed NIR-sensitive nanocomposite with bisphenol (poly (propylene glycol) bis(2-aminopropyl) ether as crosslinking and decylamine as crosslinking modulator [20]. NIR produces a photothermal effect inside the material, which raises the temperature of the material above glass transition, causing it to change its shape [20]. In addition to graphene, carbon nanotubes (CNT) have also been doped into structures to induce photothermal effects. 4D printed structures from CNT-doped methacrylated resins have been reported by Cortes et al. who demonstrated a similar effect [21].

Magnetic field
A magnetic field as a stimulus has been applied to 4D printed materials to change their shape. For the development of magnetic field-responsive 4D printed structures, substitution of the magnetic responsive component in an optimized proportion is significant. Zhao et al. 4D printed a bioinspired tracheal scaffold using poly (lactic acid) (PLA) and Fe 3 O 4 nanoparticles [22]. The fabricated scaffold changed shape when exposed to a magnetic field as shown in Fig. 5(e) [22]. 4D printed scaffolds for tissue engineering applications have been developed by Wan et al. using poly (D, L-lactide-co-trimethylene carbonate) (PLMC), poly (trimethylene carbonate) (PTMC), and Fe 3 O 4. Fabricated scaffold demonstrated magnetic field and temperature responsiveness, as well as SME and biocompatibility [23].

Polymers are used in 4D printing
Polymers used in 4D printing are active materials that can change shape when exposed to an external stimulus and are thus referred to as smart materials. Based on external stimuli, polymers are distinguished into thermo-responsive polymers, hydroactive polymers, pH-active polymers, and light-responsive polymers [24]. The most commonly used materials are SMPs and hydrogels. (Table 1).

Polymers with shape memory
SMPs are active materials that have many mechanical properties and are more robust [25]. They are used in 4D printing due to their capability to transform the shape in response to external stimuli. Researchers are studying these smart materials because they showcase similar behavior as seen in 4D printed materials. The stimuli-triggered responses of SMPs are similar to the time-dependent shape-changing of 4D printed material. Many properties of SMPs are investigated by using conventional and unconventional techniques like uniaxial testing, bulging, etc. The advantages of SMPs include low density, flexibility, enhanced shape manipulation, and high strain recovery. Neuss et al. studied (epsilon-caprolactone) dimethacrylate as a biodegradable polymer network to observe cell behavior and suitability of fibroblasts and mesenchymal stem cells. The SMP supported cell proliferation and viability and was completely cytocompatible with most of the cells [26]. When exposed to stimuli, swing to and fro in two primary shapes, but there are some SMPs that can acquire a tertiary stage, which are known as triple SMPs (TSMPs). Bodhagi et al. studied TSMPs made of PU-based filaments involving hot-cold programming of the SMP and used the New Creator printer, which is based on FDM printing technology [27]. The SMP, when exposed to high and low temperatures, showcased hyper-elasticity and elastoplastic behavior, respectively. TSMPs have great potential in the biomedical field, like tightening/self-shrinking staples and self-bending stents. SMPs have been classified into three types based on the response to the external stimulus, namely thermalresponsive SMPs, photochromic SMPs, and chemical induction SMPs.
Thermally responsive SMPs have thermally induced shape memory constituted of two partially compatible phases-the stationary phase and the reversible phase. The stationary phase is responsible for restoring the original shape, while the reversible Human MSCs [75] phase changes the shape of the molded product. Photochromic SMPs have a chromophore containing a group known as a photochromic chromophore group, which is responsible for the isomerization process when exposed to light, leading to molecular changes. The material still has the potential to be restored to its original form, but the process is slow. The chemical induction SMPs are materials that are deformed and recovered under the influence of chemicals. The chemical induction can happen by pH change or equilibrium ion displacement, wherein in the pH change method, the material is soaked in hydrochloric acid solution and sodium hydroxide solution consecutively. As a result, the material expands in the acidic solution due to mutual exclusion between the hydrogen ions, while, when soaked in sodium hydroxide solution, the acid-base neutralization occurs, leading to the shrinkage of the material into its original shape.

Hydrogels
Hydrogels are hydrophilic polymeric biocompatible materials with good biological affinity that are frequently used in 4D printing. They respond to the stimulus by changing their volume or by reversibly deforming. Although there are certain setbacks while printing hydrogels, like their fragile and brittle nature, the mechanical strength of hydrogels can be tuned by infusing a secondary polymeric network. Crosslinking density, hydrophilicity, and microstructural anisotropy are some of the internal properties that affect the material swelling degree [28]. A composite mixture containing agarose, acrylamide, and laponite hydrogels was studied by Guo et al. as a potential ink for 4D printing. They observed that laponite played an essential role in improving the printability and easily extruding while maintaining the shape and stability post printing [29]. The highly crosslinked agarose and polyacrylamide networks lead to a stable, rigid, and highstrength 4D gel. This study demonstrated the utilization of 4D ink that could be used in the biomedical field to generate scaffolds, sensors, medical devices, etc. [29]. Hydrogels respond to different stimuli like thermal, light, and pH. Thermally responsive hydrogels are materials whose volume changes with temperature, of which PNIPAM is the best studied material belonging to this classification. Bakarich et al. prepared a hydrogel consisting of alginate and PNIPAM and printed a mechanical valve [30]. The smart valve printed was able to control the water flow. Whenever it is exposed to hot water, it closes automatically and opens up in cold water. It was able to reduce the flow rate by 99%. Alginate worked as an enhancer for the mechanical properties, and PNIPAM was supported as a heat-sensitive material. This combination of hydrogel and ink can lead to fabricating substances in the biomedical field [31]. The light-responsive hydrogels do not require to be in direct contact with the stimulus and have UVresponsive activity due to the high energy of short-wavelength light [32]. The light-responsive hydrogel was fabricated by Schiphorst et al. using spiropyran photoswitches to make valves for microfluidic devices. The pH-responsive hydrogels have their volume changed according to the concentration of hydrogen ions present, which is affected by the pH. The swelling behavior of acrylic acid-based hydrogels was studied by Hu et al. femtosecond laser direct writing is used for the fabrication of hydrogels [33,34]. The pH, when greater than 9, leads to the release of protons and ionizing of the sample and the expansion of polymer grid size, whereas, when pH is less than 9, the carboxylic group accepts protons and deionizes the sample. When placed in acidic and alkaline solutions, the hydrogel exhibits swell and strong swelling, respectively [35].

Shape memory alloy
Shape memory alloys (SMAs) are smart materials that are metal-alloy systems, which, when exposed to temperature or load change, exist in multiple phases. They have the capability to return to their original shape after several deformations, the characteristic property of SME. SME is used in a variety of industries, such as civil engineering where iron-manganese-silicon (Fe-Mn-Si) alloy systems are used; aerospace and automotive applications where nickel-titanium (NiTiHf) alloy systems are ideal due to their ability to operate at high temperatures; and actuators and sensors where nickel-manganese (NiMn)-based SMA materials are used due to their ability to be activated by magnetic systems. The most studied SMA system for biomedical applications is the titanium/nickel (Ni-Ti) SMA system. Many studies have been dedicated to studying the SME and the thermomechanical properties of nitinol. Kuribayashi et al. fabricated a new type of stent known as an origami stem graft by using single-foldable Ni-Ti SMA foil, unlike the conventional use of wire mesh. It was observed that the Ni-Ti SMA stent was easily deployed at near body temperature or by superelasticity. This type of origami stent has shown promise in vascular surgery and minimally invasive surgery [35].

Application in regenerative medicine
4D printing has many applications in regenerative medicine targeting specific organs [36].

Brain and nervous system
Atoufi et al. fabricated hydrogels from agarose and alginate tetramer, which was stimuli-responsive as well as conducting [37]. The fabrication process started with grafting aniline on sodium alginate, and then agarose was added to the hydrogel as a biocompatible polysaccharide. Combination of alginate-aniline tetramer acted as compound that responded to the electric field. The hydrogels were characterized for proton nuclear magnetic resonance (H 1 NMR) and Fourier transform infrared spectroscopy (FTIR), and the highest ionic conductivity was observed in the grafted hydrogel with 10% aniline tetramer. These hydrogels were conducting with potential application in neurodegenerative medicine [37]. Miao et al. developed a 4D reprogrammable conduit of soyabean oil epoxidized acrylate with 0.8% graphene, using an SLA printing technique for a nerve guidance system. hMSCs were seeded in the conduit, and it was seen that they arranged in highly directional form and showed neurogenic differentiation. The in vitro studies confirmed that the conduit has promising applications in treating central and peripheral nerve injury [38].

Heart and cardiovascular system
Montgomery et al. designed a scaffold for delivering functional tissues via an injection to avoid open-heart surgery, which is generally used to implant cardiac patches to replace scar tissue [39]. Poly [octamethylene maleate (anhydride) citrate], a biodegradable polymer, was used to fabricate elastic scaffold and it was observed that cell viability and function were not compromised while injecting the microfabricated scaffold to engineer the cardiac patches. Polymer stents and homologous tissue-engineered heart valves have been developed to address the lack of self-repair and growth abilities in pediatric patients. Cabrera et al. used the FDM technique to print polymer stents with growth potential (which was observed as the internal diameter of the valves increased significantly) to minimally invasively implant the stent and grow according to the development of the patient. When put in a water bath (37℃), the stent pushed out of the implant and self-expanded. There were some cracks observed during degradation in the layer surface of the self-expandable stents to accommodate the growth of the patient [40]. For treatment of myocardial infarction, bone marrow-derived stem cells reduce revascularization and infarct size, although the reports suggest that there is loss of cells in this technique. Pedron et al. fabricated thermally responsive and biodegradable polymer via photolithographic techniques to reduce cell loss during transport. The polymer consists of polyethylene glycol (PEG) and polylactic acid (PLA) that make up the nonreactive diacrylate triblock copolymer layer and another layer made up of PIPAAm (poly(N-isopropylacrylamide), which constitutes the stimulus-responsive layer [41].

Skin
Skin is susceptible to many pathogens and harmful microorganisms, and being a physical barrier, it is more prone to infection in case of any injury. The pH value ranges from 5.5 to 6.5 in case of uninfected wounds, and upon infection, the raise in pH above 6.5 is observed. Many studies have been ongoing to fabricate a memory material that can reduce the healing time of a wound. GelDerm is a hydrogel-based dressing developed by Mirani et al., which is a pH-responsive hydrogel that changes the color when the pH of the skin changes due to infection [42] (Fig. 6). A closed-loop patch was developed in another study for the treatment of chronic skin wounds. The smart patch was developed with PNIPAM-based particles that were placed on an alginate hydrogel sheet. The Figure 6: A diagrammatic representation of multifunctional wound dressing GelDerm which is a hydrogel-based dressing, pH-responsive hydrogel that changes the color of the hydrogel when the pH of the skin changes due to infection also releases antibiotics (reproduced from Ref. [74] copyright with license Id: 5318671130053). particles and the hydrogel sheet were attached to medical tape about 3 mm thick to make a wearable platform [43]. The patch had many components, including sensors, microheater, drug carriers on hydrogel patch, and wireless electronics system. The application of this patch could be of detection of healing marker and targeting treatment of conditions.

Stents
Stents are medical devices used to narrow the passages of arteries and veins, etc. Stents can be made of metal or plastic and are a tiny tube that can be expandable. They are used to treat many diseases, like coronary heart disease, renal arterial disease, deep vein thrombosis, etc.
Many researchers have used 4D bioprinting methods to fabricate stents that self-deform according to size and shape. Morrison et al. successfully developed a personalized medical device using polycaprolactone (PCL) for treating tracheobronchomalacia, in which there is an excessive collapse of the airway that leads to cardiopulmonary arrest [44]. The device was designed such that it could accommodate air growth while preventing external compression. It was successfully implanted with no reported adverse reactions or complications attributable to the splint. Mearian et al. developed medical stents for patients with bronchomalacia, a disease in which the walls of bronchial tissues have weak cartilage. The stent was made using PCL, which was printed in vivo like a trachea using Formiga P100. The developed stent was biocompatible with slow degradation ability and was successfully implanted in young patients with defective bronchomalacia. The stents started degrading after the planned six months and were excreted from the body once absorbed. Around 200 splints can be printed at a time, and they are radially expanding, flexible, and able to withstand pressure from surrounding tissues [45].

Engineering of bone tissue
4D printing has many applications in bone tissue engineering and has showcased a lot of potential in defect repair with minor or irregular bone defects. The characteristic properties of 4D printed structures like self-remodeling and functional maturation work in favor of the fabrication of structures similar to native bone tissue (Fig. 7).
4D printing of bone tissue based on shape memory scaffolds Hard tissue constructs have been printed using the 4D printing technique. Senatov et al. studied porous scaffolds made of PLA as bone implants prepared by fused filament fabrication. It was also combined with hydroxyapatite to form a30% porosity and studied the mechanical, structural, and SME properties. The scaffolds underwent compression-heating-compression cycles, and it was observed that ceramic particles inhibited the growth and were able to withstand the three compression cycles. The scaffolds showed 98% shape recovery and can replace minor bone defects as self-filling implants [46]. This demonstrated the use of shape memory scaffolds for fabricating 4D printed constructs for personalized bone defect repair. Miao et al. developed a biocompatible and temperature-responsive scaffold [38]. The scaffold consisted of epoxidized acrylate and was printed using a 3D laser printing technique. The hMSCs from bone marrow were seeded and proliferation and attachment were observed. From this study, it is observed that renewable resources are of great utilization in the fabrication of biomedical scaffolds and can help in developing advanced 3D constructs.

4D printing of bone tissue based on injectable stimuli-responsive hydrogels
Various stimuli-responsive hydrogels have been explored for bone tissue engineering and the characteristic property of such thermo-responsive hydrogels is that they exhibit a lower critical solution temperature. Wu et al. prepared an injectable hydrogel for cell-free bone repair. The scaffold was made of chitosan/silk fibroin to which coppercontaining bioactive glass nanoparticles were added. It was observed that the gel was porous, exhibited good injectability, and underwent gelation quickly, supporting cell proliferation of a clonal murine cell line of immature osteoblasts derived from mice (MC3T3-E1 cells). Furthermore, when placed in rats with calvarial bone defects, the gel completely restored the bone defect by forming vascularized bone tissue and mineralized collagen deposition [47].

4D printing of bone constructs along with blood vessels and nervous network
The development of blood vessels and nervous network around the construct is very important and is one of the challenges that are faced in fabricating the bone construct. Many methods have been developed to overcome this challenge, which includes the formation of hollow self-folding tubes that were fabricated from methacrylate alginate and hyaluronic acid hydrogels. Kirillova et al. 4D printed hollow tubes which had a diameter as low as 20 m. The mouse MSCs were seeded along with the construct, and they were viable. The construct supported cell survival for 7 days without loss of cell viability [48]. Devillard et al. focused on reproducing the biological functions that helped in fabricating vascular alveolar bone constructs [49]. They used alkaline phosphatase and thrombin that were printed along with the bone construct and showed multiple activities, including calcification and fibrin deposition. This study demonstrated that 4D printing of constructs with multiactivity has many potential applications in bone tissue engineering [50].

Dentistry
4D printing has been an emerging technology in the field of dentistry. Many studies have been focused on the fabrication of structures using smart materials in orthodontics, prosthodontics, endodontics, and implantology.
Orthodontics This field deals with conditions in which teeth are not properly aligned. Many arch wires have been developed using nickel-titanium (Ni-Ti), PU, and polyethyl methacrylatebased materials. Jung et al. fabricated a wire using PU block copolymer via melt spinning. The wire showcased high shape retention and shape recovery, and during the orthodontic test, it was observed that the wire was able to correct the teeth that were not aligned properly. The PU wire has potential in the orthodontics field [51]. Masuda et al. prepared an orthodontic elastic material consisting of ethyl methacrylate (EMA)-based resin and 1-butanol. The material showed SME and increased strain, which has the potential for treating orthodontic problems [52].
Endodontics This area of dentistry is devoted to the root canal system, its diagnosis, treatment, and prevention. It is important to prevent the microorganisms contaminating the dental pulp and the surrounding area by fabricating a three-dimensional seal that can be achieved by 4D printing. Tsukada et al. observed the thermal properties of SMP-2 (Kuraray Corp, Kashima, Japan), a commercially available crosslinked trans-1,4-polyisoprene [TPI] also known as pure gutta-percha. Many properties were studied, including shape recovery, recovery stress, and relaxation modulus, and it was observed that the SMP-2 was able to generate recovery stress that was able to seal the internal space of the root canal [53].
Prosthodontics It is one of the branches that takes care of the replacement of missing teeth and the areas of soft and hard tissues. The associated tissues can be retained by implants or can be fixed or removed. Denture relining has a number of disadvantages, including bad odor, heat generation, and oral mucosa irritation. An alternative for direct relining is dentures that have SMP lining, which helps in overcoming a few of the drawbacks of relining dentures. These dentures require fewer periodic recalls from patients, reduce discomfort, and can undergo reversible changes that stay stable for a longer period of time [54]. Another application of 4D printing and smart materials in prosthodontics could be the use of SMPs in removable partial dentures (RPDs).

Implantology
The area focuses on the implantation of artificial teeth in the jaw, wherein titanium and its alloys are most commonly employed [55]. They are biocompatible, resistant to corrosion, and lead to osteolysis and hypersensitivity reactions. A dental device with SMP (Cu-Zn-Al or Ti-Ni alloy) for artificial roots was fabricated and implanted in alveolar bone. On expansion of the dental implant, SMP activated from a deformed to a relaxed state and tightly fitted into the cavity. It was observed that the osteointegration with alveolar bone was rapidly achieved with instant fixation.

Imaging techniques
Scanning techniques like computed tomography (CT) scan and magnetic resonance imaging (MRI) are essential for diagnosing 3D internal images of a patient's body like shape, size, texture, color, and skin-surface area [56]. 3D scanners help design implants, prosthetics, anatomical models, surface texture, and restore shape, whereas CT scans help see the tissues inside a solid organ at different density levels. In addition, it provides detailed information about the human body, such as the eyes, lungs, neck, spine, brain, vessels, and other internal organs with a high-resolution image. A powerful magnetic field and radiofrequency pulses are used in the MRI technique to achieve a detailed image of the internal structure as well as help in differentiating between the abnormal and normal tissues of the body [57]. 3D scanning can detect even the minute details of the whole body by providing the flexibility to scan different shapes and sizes by changing the field of view.
4D CT and 4D MRI scans are advanced imaging diagnostic technologies in which the scanned images of the body are recorded with respect to time [58]. 4D printing builds a layerby-layer structure with the help of CAD software data, which helps in handling the challenging situation of printing medical requirements for procedures like complex surgery, smart implant manufacturing, the printing of an organ, and grafting of the skin with utmost accuracy. The 4D CT/MRI scan provides minute details required by the physical model to know about the dimensional pre-requisites of the actual printing model and then provide it with information about how much it should get expanded and change its shape [59,60]. Using the input information from the imaging records obtained by the 4D CT and MRI scan, a customized 4D printed structure can be made, which the surgeons use to execute a mock surgery that helps improve the success rate of the operative procedure. To start this process, scanning and imaging via CT and MRI scans are done in order to capture a medical image and to detect the defect. Then, software like 3D slicer, Mimics Magics, and InVesalius transforms scanned data into a 3D model. Further patientspecific modification of the implants and devices is carried out and employs chemical simulation processes to analyze specific body parts to perform the required function. Final 4D printing happens, where smart material is explicitly modified according to the shape and size of the patient's anatomy. This technology manufactures medical devices with complex shapes and models efficiently, and it provides a brilliant ability to fabricate an internal structure with higher flexibility. The implementation of 4D printed structures is monitored in the patient's body.
In the field of orthopedics, scanned images from 4D CT and MRI help in determining any adjustments that are required, related to the problem or defect that is detected, and then provide information regarding the changes in the shape of the implants that are made according to such variations [61]. Furthermore, 4D printing has an unsettling effect in orthopedics by constructing implants with high quality and performance, thus making them flexible for the manufacturing process, causing the individual patient's data to be captured by these innovative technologies and creating endless opportunities like the potential to develop self-bending stents and other models that help in the treatment in the respective field.
In a research study, 4D printing is described as a technology where the length, width, and height comprise the three dimensions, whereas the fourth dimension is time, which is represented as a change in the position of the bones [62]. 4D CT scan technology is characterized by multiple haptic models depicting the movement of metacarpals during thumb movement, and it was performed using single volume acquisition technology to lower the risk of exposure to radiation. Thumb movements like abduction, key pinch, and opposition were scanned and fabricated using a 3D printer. Validation of the data about the angle between the first and second metacarpals was collected via 4D imaging and 4D printing, and later, it was recorded and compared. The transition of carpal and metacarpal bones during the opposition was observed through the 4D printing reconstruction method. This experiment demonstrates a strong correlation between the 4D printed models and 4D CT scan data regarding spatiotemporal anatomy. The 3D and 4D printing technologies help print a 3D physical model, which is helpful for educational purposes and for the surgeon to understand the specific problems related to the patient before deciding on the treatment or developing a model for the surgery. Oversampled 3D CT scan data are 3D reconstructed retrospectively by the patient's respiratory motion in the 4D CT scan technology. The technology reduces motion artifacts and enables precise radiotherapy to be delivered to tumors in the lungs and breast cancers.
4D printed implants that can grow in vivo have self-deforming components. 4D CT and MRI techniques help contribute extensively to the process by capturing the data where smart and intelligent materials act as the pillar of resolving the problems associated with humans in future.

Future prospective and conclusion
The 4D bioprinting technology holds countless potential in the fields of tissue regeneration and biomedicine and overcomes the limitations of 3D printing [63]. 4D printing has many applications, including the fabrication of site and shape-specific stents and targeted drug delivery. This printing technology is expected to become a next-generation technology to produce transformable constructs for biomedicine [64]. With the utilization of smart multi-print models, it is easier for healthcare workers to get in vivo information related to blood clots, spread of infection, and tumor growth. It is beneficial in the manufacturing of 4D medical devices such as splints, stents, and orthodontic devices as per the requirements of the patient. These devices can also be bioprinted at any point in time, which was previously not possible.
The biofabrication of dynamic cellular constructions for biomedical applications is one of the greatest potentials of 4D bioprinting [65]. To fabricate the constructs that mimic the intrinsic microenvironment of the body, a stimuli-responsive material is used, which interacts with the host tissue for superficial and robust integration. The constructs have the ability to respond to different stimuli, which include temperature, pH, swelling, humidity, calcium concentrations, electric field, and magnetic field, which will lead to a new era in tissue engineering, biactuation, biorobotics, and biosensing [66]. The 4D biofabrication process, in addition to stimuli-responsive biomaterials, also uses cell traction forces in order to create a static and dynamic structure. The inability to provide enough strength to support the pressure of the fluids becomes a major challenge in the case of 3D printing [67]. In contrast, the 4D printing technique approach helps to more accurately and physiologically mimics the microenvironment of native tissues, creating hope for the bio fabrication of alveolar and tubular structural tissues such as glands, skeletal muscles, vessels, and cardiac tissues.
Due to inflammation, the local tissues in body get affected and the printed tissues get surrounded by the immune system of the body, studies could be done in order to investigate the interactions between the printed tissues and the intrinsic tissues in an uncontainable manner [65]. Usage of smart material in 4D printing technology helps to design and transform the structural function, which is very helpful in increasing the medical areas' ability to produce patient-specific devices that require a specific shape and organization [66]. Materials such as metals and polymers are used in the biomedical field and the blend of materials and response of a biomedical device creates a 4D print, which turns out to be a very beneficial, dynamic, practical, and responsive system in the tissue engineering application.
4D printing is a better technology as compared to the traditional manufacturing processes in terms of product quality and performance, as it allows to develop dynamically reconfigurable structures using smart materials and provides with useful, responsive systems, which can make a significant difference in the fields of medicine, engineering, and some other possible areas. Development of various materials with multi-sensitivities for use in enhancing the dynamic nature of devices is still a challenging issue and is an objective for future experimentation [65]. The additive manufacturing process can provide many future perspectives, which include a series of endless possibilities such as enhancement of the stimulus-responsive performance; research on self-growing and reacting functions; improvement in the life span of the printed product; preprogrammed cycle capability and recycling time; exploring the structural complexity of the product; and using the mature technology in which the implantable medical application is applied. The mechanical properties of printed objects deplete over time after the repeated execution of the folding and unfolding of the procedures, due to which the scaffolds are not able to recover their original shape, and because of this, it is imperative to design the mechanics of stimuli-responsive bioinks [66]. Multiple folding is required in order to achieve full functionality, especially for the ones that undergo development and biorobotics, which might be needed on demand to render the desired utilities. For this, we need to advance the innovation in 4D printing by incorporating rational computer designs of sophisticated multiple stimulus-responsive procedures to secure the dissemination of building complex selfmorphing objects for widespread applications in biomedicine such as tissue engineering, soft robotics, and biomedical devices.
4D bioprinted constructs demand printing objects for a wide spectrum of applications. Most of the materials used in non-biomedical applications result in providing a harsh environment for the tissues, organs, and their functions [68]. For the further expansion of 4D printing, it is important to produce a novel and multifunctional ink that is highly desirable. Also, it would require advances in technology in the areas of software, modeling, mechanics, and chemistry. Fabrication of artificial tissues and organs can be done by doing the cell culturing or implantation subsequently, whereas the biological constructs that encapsulate the cells and bioactive agents can be printed directly using ink that contains cells. The trending form of 4D bioprinting is through polyjet and syringe printing, and in future, for this technology, various platforms of 3D printing can be of the utmost potential. As the development of medicines takes place, it is a major focus of study that these medicines have the ability to release drugs at a suitable time and anatomical location [64]. The spatial and temporal delivery of therapeutic agents can be optimized with the help of 4D bioprinting technology where precise control can be taken over the encapsulation and release of drugs through machinery that can self-fold and unfold in a programmable manner. Progression in the characteristics of 4D printing has been anticipated in creating a plethora of unexplored research areas and development in a wide range of multiple-dimension printing, not only in tissue and organ regeneration but also covering a full spectrum of science and technology [69]. Table 2 compares the 4D and 3D printing technologies.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Open Access
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Properties
3D Printing 4D printing Fabrication process A layer-by-layer formation of 2D sections The fabrication process is similar to 3D printing but there is an inclusion of change in shape/size after exposure to external stimulus Materials Nylon, resin wax, polycarbonate Smart materials-hydrogels, shape memory alloy, shape memory polymers Physical properties Stable after printing Can be changed when exposed to external stimulus Usage Ready to use after print Not completely ready for use even after printing Shape memory programming No programming step There is a programming step to introduce shape memory effect External stimulus No effect of external stimulus Heat, temperature, pH, and magnetic field play a role in changing the shape/size