Biomedical applications of three‐dimensional bioprinted craniofacial tissue engineering

Abstract Anatomical complications of the craniofacial regions often present considerable challenges to the surgical repair or replacement of the damaged tissues. Surgical repair has its own set of limitations, including scarcity of the donor tissues, immune rejection, use of immune suppressors followed by the surgery, and restriction in restoring the natural aesthetic appeal. Rapid advancement in the field of biomaterials, cell biology, and engineering has helped scientists to create cellularized skeletal muscle‐like structures. However, the existing method still has limitations in building large, highly vascular tissue with clinical application. With the advance in the three‐dimensional (3D) bioprinting technique, scientists and clinicians now can produce the functional implants of skeletal muscles and bones that are more patient‐specific with the perfect match to the architecture of their craniofacial defects. Craniofacial tissue regeneration using 3D bioprinting can manage and eliminate the restrictions of the surgical transplant from the donor site. The concept of creating the new functional tissue, exactly mimicking the anatomical and physiological function of the damaged tissue, looks highly attractive. This is crucial to reduce the donor site morbidity and retain the esthetics. 3D bioprinting can integrate all three essential components of tissue engineering, that is, rehabilitation, reconstruction, and regeneration of the lost craniofacial tissues. Such integration essentially helps to develop the patient‐specific treatment plans and damage site‐driven creation of the functional implants for the craniofacial defects. This article is the bird's eye view on the latest development and application of 3D bioprinting in the regeneration of the skeletal muscle tissues and their application in restoring the functional abilities of the damaged craniofacial tissue. We also discussed current challenges in craniofacial bone vascularization and gave our view on the future direction, including establishing the interactions between tissue‐engineered skeletal muscle and the peripheral nervous system.

engineering, that is, rehabilitation, reconstruction, and regeneration of the lost craniofacial tissues. Such integration essentially helps to develop the patient-specific treatment plans and damage site-driven creation of the functional implants for the craniofacial defects. This article is the bird's eye view on the latest development and application of 3D bioprinting in the regeneration of the skeletal muscle tissues and their application in restoring the functional abilities of the damaged craniofacial tissue. We also discussed current challenges in craniofacial bone vascularization and gave our view on the future direction, including establishing the interactions between tissue-engineered skeletal muscle and the peripheral nervous system.

K E Y W O R D S
3D bioprinting, bioengineering, biomaterials, craniofacial tissue complex, soft tissues

| INTRODUCTION
The terms regenerative medicine and tissue engineering are often used interchangeably in medicine. However, regenerative medicine is a general term that incorporates tissue engineering and, at the same time, is concerned about the research in self-healing. During selfhealing, the body uses its cellular mechanisms and foreign materials to recreate the cell and its functions and reorganize them in tissues and organs. 1 Tissue engineering, on the other hand, has grown independently from the field of biomaterials. It combines the extracellular matrix (ECM) scaffolds, cells, and physiologically active compounds into functional tissue capable of improving or replacing the damaged one. FDA has already approved the engineered skin and cartilage in clinical use for limited indications. The field of tissue engineering is evolving rapidly, and its application is extended from the replacement of damaged tissues to the research tools to study the pharmacological activity, pharmacokinetics, educational models, etc. 2,3 Before the tissue regeneration and engineering concept, clinical options available to tackle the issue of tissue degeneration or loss of it were limited to organ transplantation, use of prostheses and implants, and transplantation of autologous tissue. Scarcity of organ donors, biocompatibility, and limited supply of autologous tissues (if tissue loss is more, e.g., skin burn) are some of the significant limitations of these approaches. Surgical reconstructions using autologous tissue along with the implants and prostate still have a widespread application when it comes to replacing volume or structural deficits. 4 Complete replacement of the metabolic deficiencies using the surgical reconstructions approach is still an unmet challenge. Autologous tissue transfers have the additional problem of the other surgical site, with the risk of complications and donor site morbidity.
To some extent, organ transplantation has overcome some of the issues of autologous transplant. Organ transplantation can successfully replace the lost or damaged tissue and restore its physiological and metabolic functions. Whole organ transplants like liver and kidney have saved the life of several critically ill patients by restoring their vital functions. 5 However, this approach has inherent limitations, including organ rejection and immunogenic risk, limited availability of donors, and regulatory approvals. Lastly, in the last few decades, prosthetics and implants have become highly advanced and sophisticated but are still limited in their use in replenishing the lost tissue volume or metabolic functions. Immune activation and distortion are still significant challenges that need to be overcome to optimize prosthetics and implants. 6 At the organizational level, the tissue is placed between the cellular and organ level. A group of cells produces the necessary biochemical components and maintains physiological functionality. A particular group of cells also secrete the ECM/scaffold, which supports the structure and cellular growth and helps to transmit the signaling biomolecules essential for the organ's physiological func- rat heart to get the myocardial scaffold, which was later repopulated with the myocardial and endothelial cells to revitalize the heart functions. 7 These findings were extrapolated to the pig heart, confirming the scalability to the bigger organs. 8 Wang et al., on the other hand, focused their attention on making the cardiac patch from the decellularized porcine heart. 9 Studies are now emerged reporting acellular human myocardial scaffolds. 10 Wang et al. also developed a robust protocol to decellularized the porcine heart to obtain the three-dimensional (3D) acellular scaffold with very well preserved cellular gaps and ECM. 9 Several studies confirmed the effectiveness of embryonic stem cells and adult mesenchymal stem cells (bone marrow or cord-derived) in regenerating various tissues and organs; however, the long-term viability of such reconstructs is limited due to the limited ability of cell division. 11 To overcome the hurdle of limited cell division capability, the concept of multipotent progenitors derived from embryonic stem cells (ESC) is getting more popular because of their multipotent nature and proliferativeness, which enables them to recellularized the complex scaffolds. 12 In the recent time, cells like pluripotent human embryonic stem cells (hESCs) have appeared as an attractive candidate stem cell source for obtaining complex tissues (e.g., cardiac cells) because of their remarkable capability for expansion and undisputed potential to differentiate into smooth muscle and endothelial cells including terminally differentiated cardiomyocytes. 13 Identification of the multipotent stem cells, including the induced pluripotent stem cells, has raised new hope in the tissue engineering of complex tissue.
However, the issues like nutrient and oxygen transport in thicker tissues, cell penetration, and toxicity of the degraded products of the scaffold are the major hurdles in its successful clinical application. 14 Although remarkable development has been made in this field, engineered and regenerated tissue has some challenges that must be overcome before their clinical applications, including selecting appropriate cells, biocompatible scaffold, growth factors, low engraftment rate, and durability. To overcome the significant issues, alternative approaches for tissue engineering have emerged during the last decade. 3D bioprinting, which was initially developed for industrial purposes, is the latest approach adapted for tissue engineering, in which cells of interest in bioinks patterned in the desired shapes. The overall bioprinting process is controlled by the programs monitored by the computing systems.
The most critical factor in tissue engineering is the 3D scaffold, which provides a suitable microenvironment for cell proliferation and metabolic functions. A biocompatible material, stem cells, growth factors, and various imaging techniques have significantly supported the advancement in the field. Interdisciplinary research efforts from several areas have contributed immensely to developing the two-dimensional (2D) flat, non-vascular, and tubular organs that are being tested in the preclinical stage, and few are commercially available. On the other hand, solid, more complex tissues, including thick tissues, heart, kidney, and lungs, require innervation and vasculature to support oxygen and nutrient transport. This makes solid-organ engineering much more complicated than flat 2D tissues. Solid 3D organs required more than one cell type with a 3D porous scaffold to support cell division and provide mechanical strength-this requires radial technical advancement to support the vessel growth within the 3D construct. One of the significant challenges in mimicking the natural tissues and organs is accommodating the multiple cell types and their spatial arrangement in 3D-oriented ECM. Overall, the scaffold must be porous, biocompatible, biodegradable, or bioabsorbable for optimum growth and must provide mechanical support to the organ.
3D bioprinting offers precise control over the placement and layering of the cells within the scaffold. Compared to the traditional bioengineering method (Figure 1), 3D bioprinting allows higher precision in the space orientation relationship between the constituent elements of the tissue. In the near future, 3D bioprinting has the potential to overcome all the major issues of traditional tissue engineering.
This review is the birds-eye view on the advances made in 3D printing and its application in tissue engineering of bones and skeletal muscles.
We also propose challenges and future viewpoints in implementing the principles of 3D printing and general tissue engineering to the craniofacial bones and skeletal muscles.

| SOFT FACIAL TISSUES
In anatomy, at the organizational level, tissues are the structure between cells and the complete organ. A functional tissue is a complex of similar cells and ECM secreted by the participating cells. Tissues combine together to form the physiologically active organdifferent tissues combine to serve a common function of the organs.
In Vertebrate, tissues are grouped into connective, muscle, nervous, and epithelial tissues. 15 The appearance of all the tissues varies depending on the type of the organism, organs, and precursor cells.
For example, during embryonic development, endoderm and ectoderm give rise to the epithelium layers. A minor contribution from the mesoderm gives rise to the specialized epithelium that creates the vasculature. 16 A typical epithelial tissue is covered by a single layer of cells with tight junctions having selective permeability. 17 Epithelial tissues cover all the tissue surfaces that come in direct contact with the external environment, such as the digestive system, oral cavity, breathing tracks, and skin. Its location explains its functions of selective absorption, secretion, protection, and separation from the adjoining organs. 17 Similarly, neural ectoderm gives rise to neural tissues like the brain, motor nerves, retina, etc., and non-neural ectoderm gives rise to epidermal tissues like nails, hair, feathers, breaks. On the other hand, mesoderm gives rise to skeletal tissues (e.g., bone and cartilage F I G U R E 1 The classical tissue engineering approach of skull), connective tissue (e.g., dermis, fat), muscular tissues (e.g., voluntary muscles), and vascular and hemato tissues (e.g., vessels, osteoclasts). 18 Whereas endoderm developed to form pharyngeal tissue (e.g., Auditory tube) and glandular tissues like thyroid, thymus, parathyroids. During embryonic development, the neural crest also gives rise to skeletal tissues (bone and cartilage, dentin), neural tissues (Neurons, sensory ganglia, glia), connective tissues (dermis, fat), and vascular tissues (pericytes, smooth muscles). 17 The skeletal muscles of the head are known as craniofacial muscles. 19 Superficial epidermis and dermis layers are innervated with nerves by the peripheral nervous system. A little deeper, the muscles that control the expression (smile, wink, superficial muscles of expression) are placed. Deeper, craniofacial complex has the muscle of chewing (pterygoid, temporalis, masseter, digastric, mylohyoid, etc.), which helps close or open the jaws. The craniofacial complex also has the eyes' muscles that move them in orbit, for example, extrinsic ocular muscles. 19 Blood vessels that bring oxygenated blood and remove deoxygenated blood (part of the circulatory system) are the crucial component of the complex. Different tissues like skeletal, vascular, muscular, nervous, and connective tissue contribute to the head complex. 20 During embryonic development, the neural crest gives rise to skeletal tissues (bone and cartilage, dentin), neural tissues (Neurons, sensory ganglia, glia), connective tissues (dermis, fat), and vascular tissues (pericytes, smooth muscles). All these tissues do not form in isolation but rather interact with each other to form so that each cell knows where to connect to blood vessels and bones. 20 In parallel to tissue engineering, the present research is also focused on how all these muscles come together and form a complex system. The current research focuses on identifying the molecular and cellular mechanisms and processes that control head and craniofacial muscle development and the structural integration of all these parts to form the complex. 21 How this muscle knows where to attach to bones and move in relation to other structure and how the blood vessels and connective tissues come together with hard and soft tissues to build craniofacial complex is also the unresolved mystery. For craniofacial tissue engineering, it is crucial to know how the precursor cells learn where and when to differentiate into appropriately patterned head components.
The craniofacial system is the most affected system in terms of congenital disabilities, somewhere around 1 out of 300-500 live birth. 22 Study of the origin of this tissue, differentiation, and integration could be useful to regenerate these tissues in disease, congenital disabilities, or in cases of trauma.

| LIMITATIONS OF CURRENT SURGICAL OPTIONS
One of the most devastating, frequent, and expensive problems in healthcare is the partial or complete functional loss of tissue or organs. The most widely used treatment option is surgical repair, mechanical devices, or tissue transplantation from a different site or the matching donor. Unlike modern tissue engineering techniques, which are still in their initial development phase, the classical surgical option is still preferred. Modern tissue engineering aims to replace the damaged tissues with implants or constructs that could maintain and restore the natural function of the damaged tissues or organs.
Although artificial skin and cartilage are some of the engineered tissue that has been approved by the FDA, they still have limited application.
Although surgeries are still one of the most widely used options to restore the function of the damaged tissues, they still have their inherent problems. On the other hand, tissue engineering is based on the principles of engineering, molecular biology, and material science to develop functional substitutes to replace, restore, and improve the lost functions of tissues and organs.
Surgical approaches evolved over time, including replacing the damaged tissue with the tissue of the unaffected site in the same individual and organ transplantation from the donor individual. Surgical strategies like replacing damaged tissues and organs with artificial devices such as joints, heart valves, and bones have evolved over time. Surgical procedures often are not enough to recapture the original physiological functions of the tissues and hence require supplements to support the lost metabolic function. This often required the use of growth hormones, calcium, proteins, etc. Surgeries are benefited by the significant advances in the field of medicines, but they have several limitations of their own, which includes (1) hormonal supplements are most widely used in case of the loss or damage of the endocrine glands, for example, insulin is chronically given to lose of pancreatic gland. Such hormonal replacement therapy to normalized the metabolic functions often leads to hormonal imbalance, for example, insulin imbalance could lead to the hypo glycemic or hyperglycemic situation and several other physiological complications.
(2) Implants often require repeated surgeries; this is crucial in pediatric patients because, in such patients, they are required to replace because of the growth and anatomical changes.  , cells could be genetically modified to repair the genetic   fault before implanting the developed genetically modified tissue on   the required site. This approach not only eliminates the need for   donors and the long waiting list for the patients, but surgeons can adapt with the advances in tissue engineering to carry out minimally invasive surgery. Tissue engineering is now an established technique, and promising results are coming worldwide. Hopes are very high; the need is massive, and potential benefits are endless. However, much work still needs to be done, and many questions remain unanswered. One of the most fundamental questions is the expansion of the target cells and the generation of the growth signals, which could direct the cells to form functional 3D organs with proper vasculature for oxygen and nutrient supply. Knowledge of the basic physiology of the tissue and individual cells is complimenting the growth of tissue engineering. However, signaling to govern the tissue growth and cell migration in culture and in vivo needs better understanding. For example, understanding why skeletal muscle satellite cells multiply rapidly while cardiac myocytes do not divide at all in culture is essential. Cardiomayocytes are terminally differentiated and cannot replace the damaged site due to the infraction. Understanding the signaling that governs the rapid division of the skeletal muscle satellite cells could be useful to modify cardiomyocytes to replace the infracted site genetically. Similarly, liver cells rapidly regenerate in vivo however grow poorly in culture. Remarkable progress has been made in identifying the organ-specific stem cells and their ability to differentiate into required cells types. Stem cells hold the ability to provide a limitless supply of cells. However, it is necessary first to identify the protocol and standardize it to isolate the stem cells and confirm their ability to differentiate into required cell types. At the same time, a detailed investigation of the signaling and growth factors is required, which leads to differentiation. One major task is identifying and isolating the subpopulation and investigating their characteristic features that contribute to their division and chemotactic migration to form the specific organ. To overcome the challenges of tissue engineering, close collaboration between clinicians, biologist, chemist, material scientists, engineers are required. In addition, the adaption of 3D bioprinting, which has the highest feasibility toward the synthesis of living tissues, is essential to meet the inherent challenges of tissue engineering.

| 3D PRINTING: THE CURRENT STATE OF THE ART
3D printing, an additive manufacturing technique, is widely used for the precise fabrication of tissues. One essential requirement for the bioprinting of tissues and bones is the materials compatibility for the bioprinting process. 23 3D fabrication using printers requires printable biomaterials compatible with the live cells. Other crucial factors are non-toxicity, crosslinking ability, biocompatibility, sufficient loadbearing ability, shear-thinning properties, and support for cell proliferation and adhesion with adequate plasticity.
Like noncraniofacial bones and tissues, the craniofacial complex is composed of nerves, blood vessels, bones, cartilages, muscles, and ligaments. Together, these complex components perform several face functions like speech, smile, mastication, and esthetics. Irreparable damage to the craniofacial complex could have a long-term psychosocial impact highlighting the requirement of precise restructuring of the damaged part. 24 For restructuring, if a transplant is required, the autologous source is considered a gold standard. However, in the case of significant damage, the autologous source could not be sufficient to fulfill the volume. This makes tissue engineering a potential source of bones and tissues for transplantation. As esthetics are an important feature, precession in craniofacial tissue engineering is crucial. In general, the craniofacial complex has several similarities with the other organs and tissues. Hence, the concepts of surgeries, therapies, tissue culture, transplantation, and 3D bioprinting are also applicable to the craniofacial complex. However, due to the complex geometry, craniofacial bones and tissues engineering face unique hurdles.
3D bioprinting involved fabricating the structure similar to the one that needs replacement by depositing biomaterials loaded with the cells (bioinks) or without cells (mostly for scaffold) at the micrometer scale. 3D printing takes place with the help of an extruder move along three axes oriented in space. 25 The movement of the extruder along all the axes is controlled by design developed using an image program and shaved in file format (e.g., g.code) that is followed by the printer. Due to the potential of 3D printers in tissue engineering, their application has increased over the last few years, and various printable bioinks with printing properties like printability, flexibility, and printing fidelity have been developed.
In order to create a bioprinter that is capable of producing com- Among the other 3D printing principles, two-photon polymerization-based 3D printing offers the best spatial resolution because of its nonlinear light-induced effects in the photosensitive material. During the process of two-photon polymerization, the oxygen present in the surrounding quenches the radicals up to some extent. This ultimately helps the process to take size down to around 100 nm. Another distinct advantage of this process is that many polymers have almost nonlinear absorption in the near infra-red region of the spectrum, which help the laser to penetrate deep inside the material. This feature helps creating nano structures that are otherwise difficult to build.
The first commercial two-photon polymerization-based 3D   30 The group also confirmed that the cell adopted the elongated morphology when cell are attached to the 3D microstructure surface. Both observations indicate that the 3D microstructures fabricate using two-photon polymerization could be the tool to study cellular interaction, cell signaling, migration, cancer metastasis, and tissue engineering. 30 Overall, two-photon polymerization-based 3D bioprinting is an excellent approach for the development of the nanoscale structures.
Its application in the biomedical and tissue engineering is not only limited to the fabrication of the 3D bioprinted tissues and drug delivery vehicles but now extended to the test the effects of the geometric topographies on the stem cell differentiation. In future, two-photon polymerization could be the catalyst for the development of the selfhealing and self-regenerating 3D tissues where stem cells could be patterned in the two photon polymerized nanoscale scaffold, which then depending on the spatial arrangement, and clues will differentiate into the required cell type. 32

| BIOINKS FOR 3D BIOPRINTING OF SOFT SKELETAL TISSUES AND BONES
Bioprinting is an excellent opportunity to engineer 3D tissues and organs that match and mimic anatomical and physiological functions.
ECM, which governs many physiological functions of the cells apart from giving the structural features, is difficult to replicate artificially because of its complexity. The success of bioprinting depends on the survival and proliferation of cells in the constructs. In recent times to enhance cellular viability, several innovations have taken place in design and materials development (e,g biopolymer, hydrogels,). The formulation of live cells in biomaterial, which facilitates the task of bioprinting, is called bioink. They must meet certain characteristics like biocompatibility, physio-chemical, and rheological properties to be effective. Bioink is considered the most advanced innovation in bioengineering as it provides higher reproducibility with accurate control over the anatomical features. At the same time, it offers flexibility and can be extruded out as filaments or droplets from the nozzles.
Irrespective of the various advantages, its overall adaptability depends on how sensitive the biomaterials is to the bioprinting process. 33 Fundamentally, bioinks must copy the functions of cell support, proliferation, differentiation, and cell-adhesion from ECM of the target tissue. Further, to be printable, bioinks should have the optimum rheological properties. Bioinks with think vicious constancy are mostly suitable for extrusion-based bioprinting, whereas less viscous bioinks are suitable for inkjet bioprinting. The gelling time along with the viscosity of the bioinks determines the resolution of the fabricated construct. 34 As high polymer in the bioink is not suitable for cell migration and proliferation, a recent trend is toward using less polymers in bioinks to support better cell growth. 35 The development of suitable bioink is a dynamic area of research, especially for soft skeletal tissue engineering.
F I G U R E 2 Schematic representation of the TPP experimental setup 30 Most of the bioink characters match with the hydrogel. However, hydrogels intrinsically do not have the printable filament formation property. Hydrogels are often developed into pippeitable and filament-forming forms, which could be cast into the molds. Transformation of the hydrogels into printable filament formation form is often done before printing or sometime after the deposition. Classically, bioprinting involves the continuous deposition of cell-laden biomaterials onto the support. As higher shear stress could damage the cell and affect its viability, reducing it nozzle diameter and pressure need to be taken into account while printing. Overall the parameters that influence the printing process include (1) viscosity of the bioink, (2) temperature at the nozzle (to reduce the cell damage), (3) feasible crosslinking process, (4) uniformity in filament formation, (5) optimum pressure (to reduce the cell damage), and (6) gellation. 33 Based on the functions, bioinks are classified into: Structural bioink: These bioinks are mainly used to create the frame of the structure. The most commonly used polymer/biomaterials to make this bioink included gelatins, alginate, cellulose, decellularized, and demineralized ECM, etc. The selection of materials for bioinks depends upon required properties, including cell viability, shape, and size. 36 Sacrificial bioink: As the name indicates, these bioinks are generally removed from the fabricated structure to give rise to the desired geometry within the structure. Most widely, such bioinks are used to make channels to mimic the natural vasculature. To conveniently remove from the structure, the properties of such bioink need to be different from the surrounding material. These bioinks are crucial to make the thick, functional tissues and organs with proper arrangement for the transport of oxygen and nutrients. Carbohydrates, sugars, pluronic, and uncrossed gelatins are the most commonly used sacrificial materials. 37 Functional bioink: These bioinks are very crucial for the success of the final constructs. They are not only associated with the structural integrity and functions of the constructs, but primarily it is associated with the differentiation of the cell. Other than the biomaterials like polymers, these inks often contain the growth factors to stimulate stem cell differentiation. 38 Support bioink: as the name indicates, these bioinks are widely used to offer support to the final construct. These bioinks are meant to grow the construct up to the desired points, after which the construct supports themselves. These bioinks could also be removed from the fabricated structure once they start to support themselves ( Figure 3). 39 Synthetic and natural polymers are widely explored for their utility in the bioprinting of skeletal muscles. Natural polymers like fibrin, alginate, collagen, and gelatin calcium alginate have been used widely for skeletal muscle fabrication for better crosslinking and cellsupportive properties. 40 44 Cells like MCF-7, NIH 3T3, and HUVECs were found to survive and perform well when presented in a bioink composed of 1.5%-2% gelatin methacryloyl hydrogels then 1% GelMA. 45 Jia et al. to prepare vasculature smooth muscle used bioink composed of gelatin methacryloyl, sodium alginate, and 4-arm poly(ethylene glycol)-tetra-acrylate bioprinted with two-layered coaxial extrusion 3D bioprinting system. 46 Endothelial cells and mesenchymal stem cells, which were present in the bioink, differentiated into the smooth muscle cells in the presence of TGF-β1. 46 Decellularized ECM (DeECM), which could be obtained by discarding the native cell to leave behind the ECM scaffold, is also explored as an option for bioink. As such materials are obtained from F I G U R E 3 Different types of bioinks the tissue itself, it has the advantage of being close to the natural tissue and hence is considered as the best choice for tissue and organ regenerations. DeECM is also found to contain the cytokines, various proteins, and proteoglycans that could assist the stem cell in differentiation, proliferation, and adhesion. 47 To date, various tissues are regenerated using the DeECM based bioinks; the most prominent among them are bone, spinal cord, brain tissue, vasculature, adipose tissue, heart, liver, kidney, and skeletal muscles. 48  Natural polymers like cellulose and alginate were derivatized using methacrylic anhydride, and synthetic polymers poly(ethylene glycol) diacrylate were mixed with gelatin methacryloyl to obtain photopolymerizable hydrogel composites. 54 To form multiple microfilaments fibers or droplets, microfluidic heads were developed. The microfluidic head technique allows a fast switch between different bioinks to form the fibers of different bioinks. 35 Costantini et al. developed a new 3D bioprinting method to construct artificial skeletal muscle tissues. The group combined two different cell-laden (C2C12 and BALB/3T3 fibroblasts) bioinks made up of PEG-fibrinogen/alginate using the microfluidic head. Myotube formation was noted on the side seeded with C2C12 cell-laden bioink. 55 Cameron J. Ferris et al. developed the specialized bioinks suitable for the drop-on-demand type of printing. To prepare the bioink Dubelcco's Modified Eagles Medium mixed with Poloxamer 188 was used to hydrate the gellan gum. The ink was found to be suitable for a drop of demand printing, with reproducibility and without cell precipitation. 56 Stimulus responsive character was exploited for the development of the smart bioink. Polymer like poly(N-isopropylacrylamide) has a low critical solution temperature of 32 C and allows the phase transition at 32 C from liquid to gel phase (above 32 C). 57 This property enables the bioink to be in the liquid phase during the printing process. It turns to gel when it comes to the surface, having a temperature more than the critical solution temperature. This quick conversion from liquid to gel allows cell-laden bioinks to maintain the shape of the bioprinted structure. Shear stress was also used to prepare the stimuli-responsive smart bioink. Bioinks prepared from such materials loosed their viscosity under a high shear rate, allowing better printing under high pressure. 58 Kim et al. too fabricated the human skeletal muscle prepared the cell-laden functional and sacrificial bioink. Functional human primary muscle progenitor cells-laden bioink was prepared using fibrinogen, gelatin, hyaluronic acid, glycerol. The sacrificial bioink to generate the vasculature for the muscle was prepared using gelatin, HA, and glycerol. The bioprinted skeletal muscle preparation using these bioinks has shown that the bundle of muscle was composed of a tightly packed myofiber-like structure. 59 Seyedmahmoud et al. prepared the hierarchical skeletal muscle to match the function of native tissues.
They used C2C12 myoblasts cell-laden gelatin methacryloyl (GelMA)alginate bioinks. The observations confirmed that the 10% (w/v) GelMA-8% (w/v) alginate crosslinked using UV light and 0.1 M CaCl 2 delivered the optimum condition to stimulate muscle tissue formation compared to other hydrogel compositions. Moreover, the improved metabolic function was seen with the addition of oxygen-generating particles to the bioinks. 60  Additionally, less PAX7 is expressed by the satellite cells of craniofacial muscles, but they still express a crucial transcription factor called Pitx2, which is essential for embryonic development. 66 For example, the regenerative capacity of extraocular muscle is maintained irrespective of age and disease. 67 Since satellite cells of craniofacial muscle are implicated in the regeneration of crucial facial muscles, they have the potential to become the vital target for craniofacial tissue regeneration.

| Cell-laden bioinks for craniofacial tissue regeneration
In addition to this, recently, Michael R. Hicks confirmed that Human pluripotent stem cells could be induced to differentiate into skeletal muscle progenitor cells. 68 Kim et al. successfully generated craniofacial myogenic progenitor cells from human induced pluripotent stem cells. 69 The application of human pluripotent stem cells to create progenitor cells for skeletal tissue regeneration is valuable information to future research about its use in whole cranial tissue regeneration. Induce pluripotent stem cells derived craniofacial muscles could be used as an autologous source for craniofacial tissue engineering or reconstruction surgery. 70 Further research is required to formulate induce pluripotent stem cell-laden bioinks along with the factor that could induce the differentiation of the iPSC to skeletal muscle progenitor cells. At present, no standardize protocol is available for generating craniofacial myogenic precursor cells from human iPSCs. The immediate requirement is the standardization of such protocol by analyzing the crucial signaling pathways mechanisms during craniofacial embryonic myogenesis. 69

| CRANIOFACIAL TISSUE ENGINEERING
Although the ex vivo skeletal muscle tissue culture was developed around a century ago, the reconstruction of tissues from progenitors began in the early 60s of the last century when cross-striated muscle cells were developed in the petri dish from chick embryonic muscle cells. 71 The importance of extracellular materials for cell survival and proliferation was also pointed out by Konigsberg, which led to its widespread use in modern tissue engineering. 72 Several materials of natural and synthetic origin (polycaprolactone-based polymers, fiber, alginate) were identified and developed to fabricate the skeletal muscle tissues in the lab.
Further, to enhance the differentiation to the skeletal muscle, skeleton/scaffold of specific functionalities like support to the cell growth, mechanical strength, chemical, and electrical conductivity, soluble growth factors were developed. For more complex tissue engineering, which required more than one type of cell (e.g., tissues with the vasculature) co-culture technique was developed, for example, skeletal muscle cells with fibroblasts to engineer the myotendinous junction or endothelial cells to vascularize muscle or with neural cells to obtain neuromuscular junctions. 73 Despite the advance in skeletal muscle tissue engineering, the fabrication of fully functional skeletal muscle tissue is a distinct task. Specifically, the engineered skeletal muscle tissues are lacking in strength when matched with their natural equivalent. 74  Craniofacial tissue complex is involved in many critical functions, including mastication, speech, smile, and has high aesthetic importance. 75 Craniofacial bones, skeletal muscles, ligaments, blood vessels, nerves complex, and teeth are the critical component of the craniofacial complex. Damage to the craniofacial complex could not only severely affect the overall functions of the face, but because of the aesthetic appeal, it could take a toll on psychosocial behavior. In a broad sense, damage to the craniofacial tissues has a physical and social impact, and hence accurate reconstruction to restore the functional and aesthetic appeal is urgently required.
During embryonic development, mesenchymal cells (MSc) originate from the neural crest, which then subsequently differentiate into almost all the craniofacial tissues, including bone, ligaments, tendons, cartilages, teeth, etc. 76 The neural crest is the intermediate group of cells native to the vertebrates, originates from the ectoderm germ layer which later differentiates into smooth muscle, neurons, glia, melanocytes, and craniofacial bones and cartilages. For the formation of the craniofacial structure, MCs work with mesodermal cells. 77 During the development of craniofacial tissues, MC bifurcates into two lineages, one into the terminally differentiate stage and the other linage give rise to the off-spring mesenchymal cells. 78 After complete morphological development of the craniofacial tissues, off-spring MC continues to reside inside in all the cranial tissues as stem cells, which letter called as MC stem cells. In adults, MS stem cells help to maintain a constant turnover of the cells to keep the physiological function intact during injury; MC stem cells differentiate to regenerate the tissues of the craniofacial complex. 79 Irrespective of the very high potential, the inherent natural ability of MC stem cells to differentiate or regenerate into craniofacial tissues is not yet studied. Hence, there is substantial scope to its utility in craniofacial tissue regeneration.
Despite the several advantages of the MS stem cells, craniofacial tissue generation turns out to be a difficult task because of the complexity of the craniofacial complex. It requires the combined effort of remotely similar disciplines like robotics, polymer chemistry, mechanical engineering, cell biology, genetics, and material science. In the engineered tissue, cells must know their place with respect to the other cells, must participate in the coordinated cell signaling pathways, and must differentiate and synthesize ECM. In this respect, craniofacial structures are very complex and offer several hurdles in their artificial tissue engineering. Initially, craniofacial tissue engineering was based on the principles of classical methods. The focus was on the isolation of the stem cells and using them for tissue engineering.
Several human-shaped craniofacial tissues, including bones and cartilages prototypes, were prepared using the MC stem cells. Adipose tissue was also fabricated from the MC stem cells to be used in facial tissue restructuring. 79 Craniofacial muscles share several similar issues with noncraniofacial tissue engineering. This leads to use many of the tissue engineering concepts from noncraniofacial tissue engineering. But there are still enough differences that present unique hurdles, most of which are concerned with 3D orientation, complexity, and vascularization.
Apart from the congenital disabilities, the most common causes of craniofacial abnormality and damages are surgeries, trauma, cancers, sports injuries, etc. 80 Out of these, craniomaxillofacial injuries contribute to major deformities, and congenital disabilities are the primary cause of concerns among the infants. 81 In craniofacial muscle reconstruction, craniofacial bone plays a crucial role because they provide the anchoring platform for soft tissues and teeth. As craniofacial bones are the platform for the soft tissues of the craniofacial complex, accurate craniofacial bone reconstruction is essential to reinstate the regular functions of the complex. 82 For example, Gaihre et al. 83 addressed the defects of craniomaxillofacial bone by restoring it using biocompatible polymers like chitosan, alginate, cellulose, collagen, fibrin, and silk. The synthetic polymers used for scaffold preparation were poly (L-lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, and poly(propylene fumarate). 83 Tu et al. managed to resolve the craniomaxillofacial bone defects by employing prosthesis composed of the hand-made customized prosthesis of hydroxyapatite /epoxide acrylate maleic. After implantation, none of the patient has shown any complications. 84 In a separate study conducted by Nunes et al. on nine patients with hydroxyapatite implants, bone ingrowth was observed with no indication of inflammatory reactions in the surrounding tissues. 85 Goetz et al. used 3D printed scaffold made up of tricalcium phosphate to repair the defects of craniofacial bone. 86 Rotaru et al. rehabilitate the craniofacial bone defects using customized 3D implants made up of autologous or alloplastic materials. In one radically revolutionary example, full-face transplantation was done from a cadaver in 2005 in Italy. 87 The full face transplant is reserved for the situation where the person has unrecoverable injuries to the face. 87 In recent times, another important example of craniofacial restructuring using a complete full face transplant was conducted by a team of Spanish doctors. 88  An alternative approach to rehabilitate craniofacial tissue damage is the use of prostheses. Conventionally prostheses used face several challenges; the most critical one is matching the prostheses appearance with the patients in terms of color, stiffness, size, and shape.
Such matching is a tedious and time-consuming process. 3D printing could help match the shape and size of the prostheses as per the patient's requirement, and the overall process is not labor-intensive.
Rehabilitation using prostates is commissioned only when surgical restructuring is not possible. A few of the major advantages of prosthetic rehabilitation is lower costs with shorter treatment time than surgical reconstruction. 93 In craniofacial restructuring, typically, the prostheses are required to reconstruct the dental, oral, orbital, and nasal regions, and polysimethylsiloxane is the most widely used material for the fabrication. 94 With the advance in 3D imaging and 3D printing techniques, the fabrication of such prostheses has changed considerably in terms of shape and time. At present, very limited prostheses fabricated using 3D printing technology used in clinical settings are available. However, the availability of advanced additive technology can be used in complex craniofacial engineering to enhance the quality and outcomes of prosthetic restoration. In the future, PDMS prosthetics printed directly using advance 3D printers could considerably improve the quality at a lower cost for the craniofacial application.

| Craniofacial bone regeneration
Autogenous bone grafts, especially from the iliac crest and rib bones, are considered as the most trusted source for craniofacial bone regeneration. 95 However, the origin of craniofacial and other long bone is from the different germ layers, which need to be considered for grafting. Another primary concern is similar to all the bone grafts, for example, inadequate supply and donor site morbidity. One critical post graft concern is the limited vascularization, which could lead to graft resorption and loss of structural features. Conservation of the periosteum layer and environment at the site of implantation was found to stimulate the revascularization process on the grafted bone. 96 One major issue with the craniofacial bones is their complex 3D structure compared to the long bones, which make bones like iliac, fibula, or ribs difficult to restructure to fit into craniofacial bones morphology. To overcome the challenge of limited availability and complex 3D structure, tissue and bone engineering has promised the concrete approach to treat the defect of craniofacial bone defects by synchronizing active constituents, cells, and growth inducers. 97 Craniomaxillofacial defects were successfully resolved in a clinical trial by using a stem cell-mediated bone repair method. A similar approach was used to slow down the degeneration of bones in osteonecrosis of the femoral head and for prophylactic management of distal tibial fractures. 80 The use of the stem cells for bone engineering is based on the original work carried out by Friedenstein et al., who reported the osteogenic differentiation from multipotent-stromal-cell and mesenchymal-stem-cell. 98 Friedenstein was the first who reported that the bone marrow contains the specialized cells known as melanocytes, which are not only essential for the osteogenesis but also essential for the development of the native microenvironment. 98 Due to their favorable osteogenic potential, MSC is considered an important cell source for facial bone tissue engineering. 99  The recovery was confirmed with the expression of protein bone morphogenetic protein (BMP)-2, which is essential for bone repair mechanism and homeostasis. 105 Similarly, Azevedo-Neto et al. transplanted subcutaneous adipose tissue to repair the craniofacial damage. Adipose tissue was found to stimulate craniofacial bone damage and confirmed by the expression of adipolactin (expression specific to adipose tissue). 106 Craniofacial bone regeneration capacity of cells like amniotic epithelial cells, umbilical cord-derived mesenchymal stem cells, and amniotic fluid mesenchymal cells was also evaluated. One major advantage of such cells is their ability to assist blood capillaries formation during bone healing. 107 Craniofacial bone marrow cells were also studied for their bone regeneration capacity. In a recent rat study, mandible-derived BMSCs showed higher bone mineralization as compared to the BMSCs.
BMSCs from the marrow of mandibular or maxillary bones have shown better osteogenesis and stimulated higher expression of osteoblastic markers than the bone marrow extracted from the long bones of the same patients. 108 On a similar line of research, BMSC obtained from the calvarial bones was found to stimulate bone regeneration. 109 Despite the positive results obtained from the craniofacial bone marrow cells, limited availability is the major hurdle in its widespread application. In several studies, growth stimulators have been found to play a crucial role in stimulating the progenitor cells to osteogenesis.
In recent times, the use of growth factors has received wider acceptance. One of the most commonly used growth stimulators used for craniofacial bone tissue engineering is the bone morphogenic protein. 110 Despite the in vitro and ex-in vivo success of craniofacial bone tissue engineering, its clinical application has several hurdles to overcome. This includes a limited supply of the autogenous progenitor cells, long-term viability of the transplants, isolation, selection, storage of the stem cells, lack of proper 3D microenvironment for differentiation, and loss of multipotentiality character after six or seven cell cycles in vitro cell culture. 111 One of the most potent remedies to such issues is the use of a 3D scaffold of ECM and seeding them with the progenitor cells or creating the 3D cell culture to match with the natural microenvironment. Table 1  Somites lead the path to the axial skeleton, mesoderm from the limb bones, whereas the neural crest gives rise to the craniofacial bones and cartilages. 119 Bone formation is the conversion of mesenchymal tissue to calcified bone, which is the target process needed to be  Al 2 O 3 was used first and is still popular because of its non-toxic nature, durability over a long period, biocompatibility, and inertness toward the tissues. 123 Zirconia is equally popular as Al 2 O 3 because of its toughness among the available oxide ceramics. 124 Other novel bioinert ceramics, which are gaining importance, include titanium dioxide (TiO 2 ), silicon carbide (SiC), and carbon materials. 125 For better mechanical strength, the incorporation of other material into Al 2 O 3 or composites of hydroxyapatite was also proposed. 126 For example, Glass/Alumina composite was used to make the complex structure using 3D printing technology. 126 The CT scans and CAD created the prototypes of the complex structure, which is essential to disclose the minute details of the complicated craniofacial complex. 126   Polymer-like polylactic acid, which is linear aliphatic polyester, is also widely used for bone tissue engineering. 135 Other than preserving its mechanical strength in physiological conditions, it is less viscous, biocompatible and its degradation products are non-toxic.  DMB, on the other hand, is the allogenic bone graft regularly used for filling the gaps and healing the defects. They are usually prepared by first removing the sift tissue, fats and blood followed by the acidbased deminerilization and freez drying. 143 The final product usually contains the collagen and BMPs and transforming growth factor-beta 1, 2, and 3 granting it the osteogenesis properties. 143  It's worth noting that 3D bioprinting's uses are not restricted to organ printing. It also has a lot of potential in less-explored areas including drug delivery via scaffolds, investigating disease causes, and developing tailored therapies. 161 Bioprinting of rifampicin-loaded PCL scaffold for possible osteomyelitis treatment, 162 paracetamolcontaining PVA tablets with three different geometries, 163 5D additive manufacturing techniques to create personalized models of patients' pathology, 164 and 3D bioprinting of GelMA-based models to investigate the trophoblast cell invasion phenomenon, allowing studies of key placental functions. 165 Rhodamine B was delivered using the 3D printed poly(ethylene glycol) dimethacrylate (PEGDMA) delivery system fabricated using a two-photon polymerization. 166  oped the phosphate-based glass to be useful for craniofacial skeletal muscle engineering. 168 The scaffold developed from the glass was found to release the non-toxic ions while retaining its sustained degradation property. The glass was also tuned into the fibers, which offers a high surface area to volume ratio. This ensures more surface area for cell adhesion. 168 Skeletal muscle cells are heavily influenced by surface topography, and several studies have confirmed the topographical effects on the cellular response. 169 Among the structural features, parallel grooves are the pattern that is studied widely for skeletal muscle development and direction. In recent times, nanotopography has been the focus of research for its influence on skeletal muscle development and direction. Hydrogella is also widely studied to offer a similar environment for the 3D engineering of skeletal muscle. The primary research focus is on its ability to act as a topological surface to direct the skeletal tissue growth and allows the cell to adhere to it for growth and development. As discussed earlier, hydrogels could be prepare from the natural or synthetic polymers or the decellularized ECM. In skeletal muscle tissue engineering, myoblasts must migrate, align, and proliferate to develop the end structure. Fibrils are present in the hydrogel prepared from the ECM. Such fibrils, which are proteins, offer the cues to the cells to develop into the 3D structure. 170 Landeret et al. used the same principle to develop the type 1 collagen hydrogel in which collagen fibers influence the myotube assembly in skeletal muscles. 171 The hydrogel was also developed into the mold to guide the myoblast depending upon the shape and size. A scaffold made up of the composite of glass fibers in a collagen gel was also found to be useful for the differentiation of primary human masseter muscle-derived cells. 172 Similarly, photocrosslinkable acrylated gelatine was studied for its role in directing and aligning the myoblast in a 3D environment. 173 Progenitor cells have the indigenous character to differentiate into the different cell types. Progenitor cells extracted from the craniofacial skeletal muscle have a very high potential of being differentiated into the respective muscle cells, making them the most useful cells to restore facial functions. 174 In addition to the anatomical complications, the craniofacial skeletal muscle is different in origin from the other skeletal muscle; hence the proper selection of the progenitor cells is required, which must be from the facial muscle. In craniofacial tissue engineering, the progenitor cell is differentiated and expanded into the facial tissues. 174 One of the well-known progenitor cells with high myogenic differentiation potential is the mesenchymal stem cell. The characteristics like the formation of myotubes, development into muscle fibers, high proliferation, and synthesis of own ECM make them the most suitable progenitors for craniofacial skeletal muscle engineering. 175 Mesenchymal stem cells (MSC) obtained from the bone marrow have been shown to have multilineage differentiation properties and hence are the suitable source for craniofacial muscle engineering. However, this source has the drawback of a deep invention surgical procedure and requires additional differentiation. 176 Alternatively, skeletal muscle has its MSC known as satellite cells. The satellite has the proven myogenic differentiation property and hence is more suitable for skeletal muscle engineering as compared to the progenitor's cell obtained from the bone marrow. 176 One added advantage of the satellite cells is its ability to migrate through the basal lamina sheets and differentiate to muscle cells immediately after the local trauma. 176 Once differentiated to myoblast, they get easily attached to the preexisting cell of the damaged site. 175 Facial satellite cells are also resistant to apoptosis, which makes them the most suitable cell type for tissue engineering. 177 Craniofacial tissues are anatomically complex, and hence the scaffold required to tissue-engineered such muscles is a design challenge.

| Craniofacial muscle engineering
The scaffold must match the design requirement of the damaged tissue, and it must fit into the damaged part. Clinical imaging techniques like CT and MRI help to create the image of the size and shape of the scaffold to fit into the affected area. Mechanical stress, load bearding ability, and porosity are other crucial aspects of the scaffold that need to be considered during its design. 178 3D Scaffold is complex to fabricate.
Hence the materials used to make it should have flexible physical and chemical properties. 179  Irrespective of the techniques used for the scaffold fabrication, it must be capable of handling the bioresorbable and biodegradable materials and must produce the porous scaffold with large surface areas. One modern technique which has the potential to fabricate the complex scaffold is fused deposition modeling based on 3D printers.
3D printing and FDM offer the rapid fabrication of the porous scaffold and can copy the complex structure of natural tissues. 182 The FDM technique is nearly the same as the solid free form process, with the addition of bioink extrusion heads that work on a platform working in all three directions to develop the 3D construct. 183 Scaffolds composed of biodegradable materials are considered better than nonbiodegradable materials. Degradation of the scaffold after the tissue fabrication allows the simultaneous formation of ECM from the seeded cells, which further supports the cellular interactions and proliferation.
However, the disadvantage of the degradable scaffold is that they are not easy to handle and are highly fragile. Irrespective of the drawbacks, natural biodegradable scaffolds allow higher cell adhesion when made with the fibrin and growth medium. When used together, thrombin and fibrin form the fibrin gel, replaced by the ECM proteins produced by the muscles progenitor cells in around 4 weeks. 184 This is crucial because it is observed that the myoblast was found to grow faster in the degrading gels. 184 Taken together, the ideal scaffold made up of the degradable or nondegradable polymers for craniofacial skeletal muscles can be fabricated by seeding the proginator cells into it. 185 In craniofacial bone and soft-tissue engineering, scaffolds are required to act as a template of the ECM and possess characters similar to the natural ECM. In addition, a major challenge in scaffolds engineering is its ability to form a bond with cells and support their proliferation. Previous reports have confirmed that the cell adhesion is directly linked with the surface area and, in turn, with the porosity.
Interconnects pores channels are essential for the oxygen and nutrient movement to and fro. The scaffold should also possess mechanical strength matching with the natural tissues and organs and should degrade into non-toxic metabolites, allowing natural ECM growth.
Therefore, an ongoing need to identify novel scaffold platforms capable of facilitating bone engineering is required.
In the end, for craniofacial and non-craniofacial bone and tissues,

| OVERLOOK
Several natural and synthetic polymers have been investigated as a scaffold for bone tissue engineering, which includes collagen, chitosan, poly(caprolactones), poly(propylene fumarate), and polyesters such as polylactide, polyglycolide, and their copolymer poly(lactide-co-glycolide). 187,188 In addition to the polymers, ceramics and their composites have been used for craniofacial and noncraniofacial bones. Tricalcium phosphate, which is a biodegradable derivative, has been widely used in research and clinical application. 189 Tricalcium phosphate, due to high crystallinity, is more fragile, and its remolding features are different from the natural bone. The issue of crystallinity was overcome by developing the microsphere composite scaffold made up of PLAGA and low crystalline calcium phosphate.
The scaffold was found to be highly porous, which is interconnected for cell migration, differentiation, and proliferation. 190 Due to the recent progress in bone tissue engineering, the design, However, the limited supply of such tissues has restricted their wider clinical use. 116 Moreover, donor site morbidity like pain and infections demands a persistence search of the alternate approach and materials and methods for craniofacial bones and tissue repair. The ultimate goal of craniofacial reconstruction is to restore shape, size, functions, and esthetics with proper consideration of a change in anatomy in the case of younger patients whose structure is meant to change over the period. 199 To date, rib bone, iliac rest, and scapula are considered as the suitable option in case of non-availability of cranium bone. 200 In general, an autologous transplant from the cranium is considered the gold standard because it can easily be integrated into the existing craniofacial skeleton.  The porous surface of the scaffold not only offers higher surface area, which is essential for the cell adhesion, but also acts as a mechanical platform to the surrounding tissues and improves its mechanical stability. 217 In addition to the 3D network structure, pores of the scaffold assist in guiding the new tissue formation. As discussed earlier, growth factors and specific ions essential for the cellular function are delivered via scaffold; porosity is such case helps to entrap more of such materials by offering higher surface area. The scaffold material could also be fine-tuned to release such factors over a period of time, ensuring the sustained availability of growth factors. Porosity, although it has several advantages, the compromised mechanical property is also the function of highly porous scaffolds. 218 Hence the optimum balance should exist between the mechanical and interconnected channels of the scaffold. Rapid prototyping fabrication techniques use 3D computer-aided designing data, which enables fast fabrication with improving precision and mechanical properties. 220 Rapid prototyping also offers precise control over the complex 3D architecture, design repeatability, and consistency. 221 Although prototyping has several advantages, it is compatible with limited numbers of biomaterials as compared to the conventional tissue culture. 222 Alternative approach is 3D printing, which involves placing continuous layers or droplets of biomaterials to form the 3D scaffolds. 3D printing offers high resolution and precise control over the pores as compared to the conventional methods. Various 3D printing techniques are discussed earlier in this review. 218  signaling. 224 Hence, in craniofacial bone tissue regeneration, a proper balance between mechanical strength and porosity, is required. 225 For bone tissue engineering, the porosity of 20 to 1500 μm has been reported. 226 In one of the reports, the pore size of 40 μm was found to be more populated than the 100 μm pores, which, however, was found to support the cell migration. 227 In another study, the optimum cell migration and proliferation was found in the pore size of 300 μm. 228 However, it should be noted that the cell proliferation and differentiation is also the function of the type of the cells, effects of growth factors, scaffold material, and overall conditions. 229 One another consideration that is very crucial is angiogenesis. Scaffolds planted with the stem cells or endothelial cells along with VEGF and other growth factors have positively affected angiogenesis. 230 Minimum porosity of 30-40 μm was found to support the growth of endothelial cells. 231 The pore size of 160-270 μm was also found to support the process of angiogenesis. 232 Fixed untunable scaffolds restrict the growth up to the size of scaffolds. The conventional scaffold made up of the nondegradable materials restricts the migration of cells and nutrients and can lead to deficiencies in the growing tissues.
On the other hand, a fast degrading scaffold compromised the overall strength of the tissue and could risk the structural integrity of the construct before it could get strong enough to stand on its own.
Overall, due to the immense importance of "smart" scaffolds, which could be autotuned with the cell differentiation, migration and strength are the future requirements.

| Bone forms only within a vascularized site
Although much progress has been made in bone and skeletal muscle tissue engineering, however, lack of vascularization is holding back its Recent reports suggest that in bone tissue engineering, bone formation takes place at the surface. In contrast, due to the limited supply of oxygen, its growth is limited at the center. 233 It is noted that the osteocytes or their precursor cells are found to close to the blood vessels. 234 To overcome the vascularization problem, Warnke et al. However, complete maturation and stabilization of the vessel before transplantation is the major hurdle in the clinical application of pervascularized tissues. Immature microvessels in the prevascularized tissues due to insufficient biological clues cause limited anastomosing with the host vessels. Such tissues also have a high tendency to undergo deterioration because of imperfect fusion. 237 Immature microvessels are also more fragile, and higher gaps between the cells can cause oedema after the grafting. 238   organs, which is meant to substitute the damaged part, will ensure the success of tissue engineering. In the future, it is essential to ensuring that tissue engineering will successfully face the challenge of innervation of engineered tissues using biomaterials and tissue engineering techniques. Although innervation is part of a complex set of challenges in tissue engineering, artificial tissues will significantly benefit from embedded neural cells that will ensure proper development and function.

| CLINICAL APPLICATION OF THE CRANIOFACIAL TISSUE ENGINEERING
3D bioprinting technology is becoming increasingly important in medicine, with promising applications in bone restoration, rehabilitation, and regeneration, as well as expanding therapy choices in a variety of fields. [243][244][245] It is a novel technology that poses a difficulty in both human and animal studies.
In terms of 3D printing in cranial bone regeneration, recent findings presented a comparison between human and animal investigations. 246 Between 2021 and 2017, six human research were published, including two prospective clinical trials and four retrospective case reports. [247][248][249][250][251] Studies included 81 patients (16 from the clinical trials and 62 from the case series). Mandibular bone abnormalities were the most often implanted 3D printed biomaterials, followed by calvarial, maxillary, and nasal deficiencies. The great majority of the abnormalities occurred as a result of tumor removal or trauma. 246 For the length of observation, the immediate and long-term bone regeneration was effective, and only one research reported one incidence of failure. 249 Three investigations showed biomaterial infection and/or exposure, as well as fibrous invasion of the scaffold rather than bone penetration. 251 However, these issues were effectively resolved, and the scaffold's long-term viability was not jeopardized.
Reported research evaluated animal studies as well. 246 The 36 animal studies were published during the period 2007-2017 and they included 614 animals where the most common used ones were rabbits, followed by rats, mice, pigs, sheep, and dogs. The majority of defects included calvarial, mandibular and maxillary. Histological, biochemical, histomorphometric, and microcomputed tomographic data in animal investigations showed that rapid and long-term bone healing was successful during the time of observation. Some studies, however, only found bone growth around the scaffold structure, not on the interior.
Only two studies revealed scaffold-related problems. [252][253][254][255][256][257][258] The human trials had a high rate of success with few major issues, but they were all found to have a high risk of bias, and the quality evaluation indicated that none of them met the criteria for a high-quality research design. qualities. 246 Table 2 summarizes the current clinical and preclinical application of the 3D printing in craniofacial tissue engineering.

| CONCLUSION
Tissue engineering is a very complicated process, but this review presents an optimistic picture that with innovations in biomaterials, genetics, chemistry, and regenerative medicine, engineered tissues will have real clinical application in the coming days. In the days to come, 3D printing may make the process of tissue engineering more appealing. Looking at the present research, it seems that vascularization is the biggest hurdle that is holding back the trials of 3D-