Highlights on Advancing Frontiers in Tissue Engineering

Abstract

The field of tissue engineering continues to advance, sometimes in exponential leaps forward, but also sometimes at a rate that does not fulfill the promise that the field imagined a few decades ago. This review is in part a catalog of success in an effort to inform the process of innovation. Tissue engineering has recruited new technologies and developed new methods for engineering tissue constructs that can be used to mitigate or model disease states for study. Key to this antecedent statement is that the scientific effort must be anchored in the needs of a disease state and be working toward a functional product in regenerative medicine. It is this focus on the wildly important ideas coupled with partnered research efforts within both academia and industry that have shown most translational potential. The field continues to thrive and among the most important recent developments are the use of three-dimensional bioprinting, organ-on-a-chip, and induced pluripotent stem cell technologies that warrant special attention. Developments in the aforementioned areas as well as future directions are highlighted in this article. Although several early efforts have not come to fruition, there are good examples of commercial profitability that merit continued investment in tissue engineering.

Impact statement

Tissue engineering led to the development of new methods for regenerative medicine and disease models. Among the most important recent developments in tissue engineering are the use of three-dimensional bioprinting, organ-on-a-chip, and induced pluripotent stem cell technologies. These technologies and an understanding of them will have impact on the success of tissue engineering and its translation to regenerative medicine. Continued investment in tissue engineering will yield products and therapeutics, with both commercial importance and simultaneous disease mitigation.

Introduction

Tissue defects and organ loss can result from congenital problems, disease, damage, or surgical removal^1,2 and thus, tissue defects need to be regenerated and repaired. In addition, organ function needs to be regained. This is the promise of tissue engineering. Because of the shortages in organ and tissue supply,³ many patients die every day while waiting for a transplant.^4,5 Tissue engineering and its popular and governmental support in concept was driven by this organ and tissue deficiency. Therefore, methods to develop autograft-like replacement tissues have been explored and demanded by the funding agencies. The major advance has been the development of the tissue engineering concept in the late 1980s and early 1990s.⁶

Tissue engineering aims at producing functional tissue constructs for use in reconstruction or regeneration of damaged or lost tissues and organs,^7,8 such as skin,⁹ spinal cord,¹⁰ and other organs.¹¹ In addition, secondary gain has been the development of models to study function,¹² disease¹³ and test and develop drugs.¹⁴ Tissue engineering can be achieved either ex vivo¹⁵ or in situ¹⁶ by using various molecules, materials, or cells to stimulate local tissue regenerative capacity. It is important to note that this effort of tissue engineering was a fundamental shift in the approach to the treatment of tissue loss. With the end goal being functional organs with a complex interplay of different cell signals and scaffolds, the effort mandated a “system” approach with engineering principles instead of the traditional reductionist methodology of experimentation. The ultimate end goal was beyond the knowledge of a fundamental mechanism, but rather a product to mitigate or cure a disease state.

The field of tissue engineering; however, continues to advance, taking advantage of recent developments in areas such as smart biomaterials,^16–18 induced pluripotent stem cell (iPSC),¹⁹ three-dimensional (3D) bioprinting^20,21 technologies, and dynamic culture methods.^22,23 It is also renewed by new technologies such as genetic engineering, extracellular vesicles (EVs), and artificial intelligence. Literature in the field is vast, and there are excellent reviews of different aspects of tissue engineering.^24–26 Therefore, the purpose of the current article is to highlight only major and recent advances in the field (Fig. 1).

FIG. 1.

Schematic illustration showing overview of major and recent advances in tissue engineering. 3D, three-dimensional; 4D, four-dimensional. Color images are available online.

Advancing Frontiers in Tissue Engineering

Advances in biomaterials and their application in tissue engineering

In tissue engineering, biomaterials are used to provide micro- and nanostructural characteristics, morphology, and surface properties that support cells and can be loaded with appropriate growth factors. Biomaterials used, in the form of matrices or scaffolds for tissue engineering, can be engineered in a way that helps to direct cell growth through specific designs.²⁷ Although biomaterials have been used in tissue engineering since the early 1990s,^6,28 how they have been used and the applications they are used for are constantly evolving. Outlined here are recent advances in the use of biomaterials for tissue engineering (Table 1). It is important to note that the field often has an eye on commercialization, such that the Food and Drug Administration (FDA) approval for a new biomaterial may be a hurdle that shapes the evolution of tissue engineering constructs. It is important to note that this barrier can and has in part limited the imagination of the field, because it is often more expeditious to adapt a natural biomaterial or harness one that has an FDA track record instead of designing de novo a material that will require significant vetting before its clinical use.

Table 1.

Advantages, Disadvantages, or Limitations of Different Biomaterials Used for Tissue Engineering

No.	Biomaterial	Advantages	Disadvantages/limitations	Types of tissue engineering products	Refs.
A	Polymers
1	Natural polymers	• Biocompatibility • Cell adhesion motifs • High processability • Elasticity • Degradability	• Limited mechanical properties	• Various tissues such as heart, bone, liver, and cartilage	27–31
2	ECM	• Mimicking native tissue		• Tissues such as bone, skin, meniscus, and kidney	32–36
3	Synthetic polymers	• Can be bioresorbable and processed in a controlled way • High mechanical properties	• Inflammation • No cell adhesion molecules	• Tissues such as bone, cartilage, nerve, and brain	37–40
4	Hydrogels	• Cells, drugs, and biomolecule delivery • Minimally invasive techniques	• Mechanical properties • Adhesive strength • Cell adhesion	• 3D bioprinting • Injectable materials and drug delivery vehicles for regeneration • Minimally invasive regenerative therapeutics • Cartilage regeneration	41–43
5	Smart and functional polymers—composites	• Biological properties • Antibacterial activity • Physical properties, e.g., self-healing, shape-memory, stimuli-responsiveness	• Controllability of responsiveness may be affected by environment	• Injectable regenerative therapeutics for treating bone defects	8,44,45
B	Bioceramics	• Bioactive • Biocompatible • High compression strength • 3D printed scaffolds with mechanical characteristics comparable to human cortical bone	• Low tensile strength • Brittleness • Weak under cyclic or high loads	• Hard tissue engineering such as bone, cartilage, and tooth	46–51
C	Ceramic-polymer composites	• Cell incorporation • Enhanced tissue infiltration	• Brtittleness	• Injectable or 3D-printed composites for dental and cartilage tissue engineering	52–55
D	Metals	• Biocompatibility • Degradable metal alloys • Improved mechanical properties	• Uncontrolled corrosion	• Absorbable implants for bone repair • 3D porous scaffolds	56,57

3D, three-dimensional; ECM, extracellular matrixes.

Advances in polymeric biomaterials

Natural polymers

Natural polymers that are most commonly used in tissue engineering are collagen,²⁹ gelatin,³⁰ chitosan,³¹ alginate,³² hyaluronic acid (HA),³³ and polyhydroxyalkanoates (PHAs),³⁴ because of their availability and biocompatibility.^35,36 Peptides present on some of them, such as collagen, help cell attachment, migration, and function.²⁹ Silk, a natural polymer,³⁷ has been increasingly popular in many tissue engineering applications due to its high processability, strength, and elasticity.^38,39 The PHAs comprise another group of natural polymers with special interest, as they are characterized by degradation through surface erosion that helps to maintain their general structure.⁴⁰

Guided growth of neuronal cells was observed in vitro following the use of highly aligned electrospun fibers of a blend of the poly(3-hydroxybutyrate) [p(3HB)], and poly(3-hydroxyoctanoate) [p(3HO)].⁴¹ In another application, p(3HO) was used to produce cardiac patches, which exhibited favorable mechanical properties closely matching those of native cardiac muscle, and surface topography that enabled efficient cell adhesion and proliferation.⁴² To produce new constructs with improved properties, polymers can also be used in combination, for example, electrospun fibers made from a combination of chitosan and gelatin were found to enhance bone regeneration capability.⁴³ When PHAs were blended with the synthetic polymer polycaprolactone (PCL) to produce a scaffold that delivers seeded cardiac progenitor cells and implanted in the postmortem murine heart, the implants enabled the adhesion of cardiac progenitor cells, stem cell proliferation, and retention.⁴⁴

Natural extracellular matrixes

Natural extracellular matrixes (ECM) have been used in a wide range of tissue engineering applications.^45,46 The ECM provides a natural structure that maintains some of the biological cues of the native tissues. The ECM chemical cues also help with cell attachment, differentiation, and function. There are various methods that have been used for the preparation of mammalian-tissue-based decellularized matrices, including chemical, biological, and physical methods and their combinations.⁴⁷ The majority of research, though, has been focused on the decellularization of tissues or organs.⁴⁸ It was shown that seeding decellularized hearts can result in obtaining contractile hearts by day 4 after keeping them in a bioreactor.⁴⁹ Using electrical stimulation and physiological load, constructs pump function was achieved by day 8. This represents an interesting area for the application of decellularized ECM in the tissue engineering of various organs.

Kusuma et al. made a major advance by demonstrating that immortalized cell lines can produce high-quality ECM from a single cell source.⁵⁰ Moreover, processing steps such as homogenization, pepsin digestion, or urea extraction have been used to create solutions that can be used to create surface coatings that retain some of the key properties of the native ECM. The ECM is proposed for numerous applications, due also to its versatile processing characteristics that have already allowed its use in 3D printing⁵¹ and electrospinning.⁵² For example, Kim et al.⁵³ used skin-derived ECM bioink for the 3D printing of skin tissue, with some success. Further, Carvalho et al.⁵⁴ combined cell-derived ECM with PCL and electrospun the solution to create microfibrous scaffolds for bone tissue engineering. The incorporation of ECM in the fibers enhanced cell proliferation and osteogenic differentiation, maintaining similar mechanical properties to PCL alone. The regulatory requirements of the field allow the strategy for efficient decellularization to appear to be one of the most viable pathways toward a product in short order.⁴⁹

Despite the many positive attributes of the decellularized matrix for use in the field of tissue engineering, it does also come with limitations. One such limitation is its degradation rate, and this is a property that often needs consideration when using biomaterials for tissue engineering. For optimal regeneration, the degradation rate of decellularized matrix should be closely matched with the regeneration rate of the target tissue, and in many of applications this means that the degradation rate needs to be reduced.⁵⁵ Current decellularization methods and processes achieve both a thorough removal of all cells and retention of other nonantigenic parts of the original tissue composition that can aid/guide in tissue regeneration.⁵⁶

Decellularization is also not a “one-size-fits-all” approach, and the protocols must be adapted for different tissue types while integrating factors such as their density and the matrix components. Decellularized matrix produced from tissues, which have specific mechanical properties, must maintain structural matrix components such as collagen fibers and many proteins that are necessary as endoskeleton and thus decellularization protocols need to be tuned to preserving these components. Increased preservation of active factors and structural components would also increase the bioactivity of decellularized matrix, making it an even better natural guidance material for tissue engineering.⁵⁷

It is important to note why this strategy is imperative and that it is linked to the “systems approach” already mentioned in contrast to precedent scientific reductionist work. In a system approach, the “principle” is that the ECM or the scaffold is imperative to drive and maintain differentiation. One can ask the fundamental question as to whether an osteoblast is an osteoblast when it is not surrounded by its ECM. Many in the stem cell field would argue that the cells and the ECM are intrinsic to one another and that molecular flexibility in differentiation and dedifferentiation occur without the union of the cell and the ECM. With this principle in mind, the strategies of decellularized matrices are rational because we do not have all the cues that are both physical and chronologic to the complex interplay between the cell and its ECM. Certainly, with further study and insight, smart or rational designs will incorporate the natural cues found in the natural ECM and allow synthetic polymers to support cell differentiation with similar efficiency to natural polymers.

Synthetic polymers

Synthetic polymers have been widely used in tissue engineering because they are widely available and inexpensive; can be bioresorbable; and can be processed in a controlled and multitude of ways to make them suitable for different applications. Commonly used synthetic polymers include polylactide (PLA),^58–60 polyglycolide (PGA),⁵⁸ poly(lactide-co-glycolide) (PLGA),^59,61,62 PCL,^60,63,64 poly(glycerol sebacate) (PGS),⁶⁴ and polydimethylsiloxane (PDMS). The PDMS has unique applications in tissue engineering among synthetic polymers due to high oxygen (O₂) diffusivity, ease of fabrication, biocompatibility, and flexibility.⁶⁵ It has been explored for engineering of cell sheets,⁶⁶ and it is widely used for the development of 3D organ-on-a-chip (OoC) cultures⁶⁷ that helped advancing the field of engineering tissue models tremendously (see the Advances in Microfluidic Culture Systems section). The PDMS is a nonbiodegradable polymer, and it was therefore used more commonly in ex vivo rather than implantable tissue engineering constructs.⁶⁷

Blends of synthetic polymers were also explored to combine the properties of different materials.⁶⁸ Synthetic polymers have been also used in combination to build different phases in the resulting structure. For example, Fang et al. used PLA to produce shell and PGA to produce core in electrospun nanofibres.⁵⁸ The materials were found to accelerate wound healing in vivo. Synthetic polymers can also be combined with natural biomaterials to form semi-synthetic polymers. For example, Jiao et al.⁶³ melt-blended PCL and HA to 3D print scaffolds for bone tissue engineering. Constructs had improved mechanical characteristics as compared with those that were made from PCL alone.

The bioresorbability of many synthetic polymers poses advantages in many tissue engineering applications. However, controlling the rate at which degradation occurs can be a clinical challenge. Xu et al.⁶² experimented with adding magnesium to PLGA to make composite films with low ranges of magnesium weight percentages, and they found that magnesium extended the duration of degradation and also improved the tensile strength of the films.

Hydrogels

Another advancing recent frontier has been in the area of hydrogels. Hydrogels have been extensively used for 3D bioprinting, which has been a very active area of research in the past few years.⁶⁹ Hydrogels can be made from various natural or synthetic polymers and have been used for the engineering of different tissues, because of their ability to encapsulate cells,^70,71 while having the permeability required for the diffusion of O₂ and nutrients across the material. An aspect in which previously they have fallen short is their mechanical properties⁷² and the lack of adhesiveness.⁷³ Recently, however, these problems were largely addressed. For example, Shirzaei Sani et al.⁷⁴ produced an adhesive HA/elastin-like polypeptide hybrid hydrogel, which is characterized by remarkable adhesive, antimicrobial activity, and tunable physical properties. This enhances the translation of the hydrogels to the clinical practice as it was limited due to their poor mechanical characteristics, low adhesive strength, and their weakness to inhibit bacterial colonization.

Reinforcement of hydrogels can be achieved through interpenetrating secondary networks.⁷⁵ The addition of a second network enables conventional hydrogels to be used in many emerging biofabrication techniques toward achieving hierarchical architectures and developing personalized medicine. These interpenetrating hydrogels can find applications in tissue engineering and drug delivery systems as well as in developing in vitro disease models for drug discovery and screening. Hybrid hydrogels were found to have greater adhesive strength to the tissue being engineered, as compared with commercially available tissue adhesives. A great potential of hydrogels is their use as injectable materials to deliver cells, drugs, and biomolecules¹⁶ for regenerative purposes that can often be achieved by using minimally invasive techniques.^76,77

A recent study⁷⁸ looking into cartilage repair found that HA hydrogels could be used to encapsulate chondrocytes and support cell survival and the regeneration of cartilaginous tissue. Aside from HA, alginate, and collagen, ECM hydrogels have been used in tissue engineering, and also for cell encapsulation. In addition, microencapsulation of cells to produce microgels was also explored.^79–81 Hydrogels such as these can be blended and processed through 3D bioprinting, where cells are encapsulated and printed into designed structures and then crosslinked to provide appropriate mechanical properties. For example, 3D-printed scaffolds of collagen/alginate hydrogel have been used for cartilage tissue engineering⁸² and ECM hydrogel for cardiac patches.⁸³

The regeneration of damaged tissue can be achieved either via ex vivo or in situ methods. In ex vivo tissue engineering, scaffolds are combined with cells and biomolecules outside the body to obtain cell-laden tissue constructs for implantation (Fig. 2A).¹⁶ However, the ex vivo tissue regeneration has limitations, such as tissue morbidity and the lack of reliable cell sources. On the other hand, in situ tissue engineering requires precise control of the biochemical and biophysical cues to stimulate resident host cells and attract cells to the site of injury requiring regeneration (Fig. 2B). On the other hand, in situ tissue regeneration can be achieved by stimulating endogenous cells using either extracellular signals or cell reprogramming. In the first approach, cells are primed via extracellular factors, such as through modulating the biophysical and biochemical characteristics of the biomaterial (Fig. 2B).¹⁶ In the second approach, direct manipulation of the cellular gene- expression program is accomplished through cellular reprogramming (Fig. 2B).¹⁶ Because of its relation to biomaterials, we review the first approach in this section.

FIG. 2.

Schematic illustration showing (A) Use of traditional ex vivo tissue engineering approach, which is based on the use of cells (1) cultured (2) with growth factors (3) and scaffolds (4) to develop pre-seeded constructs (5) outside the body before their implantation (6). (B) Use of in situ tissue regeneration to harness the innate regenerative capacity in the body either through extracellular signal manipulation by using bioactive (1) or immunomodulatory (2) biomaterials or bioactive molecules (3), or through an intracellular reprograming approach, which employs epigenetic transformation (4), transcription factors (5), gene editing (6), or an RNA-based approach (7). Created with Biorender.com. Color images are available online.

Smart and functional polymers

Smart polymers used in tissue engineering include those with self-healing,^84–86 shape memory,^76,87–89 or stimuli-responsiveness^18,90–92 properties. The ability to change the shape of 3D-printed objects via environmental stimuli, such as heat, moisture, water, pH, or light as a function of time, is known as four-dimensional (4D) printing, and it also has recently gained considerable interest.⁹³ For example, Invernizzi et al. have developed a novel 4D-printable smart material, using PCL and 2-ureido-4[1H]-pyrimidinone (UPy), which is a thermally activated shape memory polymer with self-repairing abilities.⁹⁴ The incorporation of methacrylates bearing UPy (UPyMA) monomers had provided self-healing properties to the 4D-printed structures, and the possibility to print actuators for soft robotics had been shown for the first time in this work. Synthesizing smart hydrogels that provide both self-healing and shape memory properties at the same time is expected to be further investigated in the following years.^95–97

Although there are many clear reasons why smart and functional polymers have gathered attention, they do have drawbacks that should be considered. For example, in the body environment, triggering thresholds of changes, for example, temperature or pH, may affect the responsiveness of these polymers, which, in turn, will affect controllability of the construct and the included cells.⁶⁹ A shape memory material implanted in the body may lead to injury of the neighboring tissues or loss of function when it returns to its original shape.⁹⁸ These challenges have to be mitigated before full benefits can be gained from utilizing smart polymers for tissue engineering.

Stimuli-responsive and self-healing hydrogels have also emerged as pharmaceutical carriers for tissue engineering.^84,94–105 One such example application of these self-healing hydrogels that is being explored is bone regeneration that can be achieved by providing an optimal microenvironment for new bone formation and for therapeutic drug delivery. Unlike conventional hydrogels, these constructs can resist mechanical stress, while protecting their therapeutic cargos from degradation and maintaining their sustained release for the long-term performance required for bone tissue healing.^84,99 To this end, a hydrogel made of chondroitin sulfate (ChS), known for its regenerative capacities, was developed for bone tissue repair with the material being cross-linked to mimic cranial bone characteristics.¹⁰⁰

With excellent self-healing, injectability, and in vivo tissue adhesion abilities, ChS-based hydrogel exhibited good cytocompatibility when it was used to encapsulate rat-derived mesenchymal stem cells (MSCs). Most importantly and compared with phosphate-buffered saline-loaded hydrogel, the injection of bone morphogenetic protein (BMP)-4 loaded hydrogel into a murine bone defect model led to defect repair through the formation of new cranial bone tissue, with a significant decrease in the defect size after 12 weeks.

Over the past few years, significant progress in the development of advanced functional polymers with tunable chemical, physical, and biological properties has been achieved.^101,102 This resulted in novel applications in 3D and 4D bioprinting¹⁰³ and drug delivery.^104–106 For example, Zhang et al. recently developed a biocompatible hydrogel ink, which contains self-healing precrosslinked hydrogel microparticles of chitosan methacrylate and polyvinyl alcohol hybrid hydrogels. Their results showed advanced structures with a high aspect ratio, and excellent shape accuracy at organ-proper scales could be quickly produced.¹⁰⁷

Several drug delivery systems have been based on the use of advanced polymers. Consequently, functional hydrogels that can provide the required dosage in both proper chronicity and location can mitigate complications and enhance success with clinical application.^108–110

Advances in bioceramic biomaterials

Ceramics are attractive materials for tissue engineering, because of their highly bioactive and biocompatible characteristics.¹¹¹ Ceramics, including bioactive glass ceramics, have been used in bone tissue engineering applications for many years due to their well-matched mineral characteristics.^112–114 They are strong and osteoconductive, which makes them ideal for application in hard tissue engineering.^66–69 They are strong in compression, however, weak in tension, and very brittle. Bioactive ceramic and glass-ceramic scaffolds were also produced by 3D printing processes such as “robocasting”¹¹⁵ and the “freeze extrusion fabrication” that combines extrusion printing with freeze-drying.¹¹⁶ The high strength values of scaffolds fabricated by additive manufacturing result from their ability to maintain highly interconnected channels with high alignment, at a porosity of 50–60%. These scaffolds presented an elastic response under compression, with an average compressive strength of 140 MPa and an elastic modulus of 5–6 GPa, which are comparable to those of the human cortical bone.

Although bioactive ceramic and glass-ceramic scaffolds can effectively mimic porous bone, provide required compressive strength,¹¹⁷ and contain channels in their 3D structure for tissue ingrowth,¹¹⁵ they are brittle and not suitable for applications in locations exposed to cyclic or high loads. Thus, scaffolds made of pure ceramics were not very successful when they were used in load-bearing regions of the body.¹¹⁸ It is also imperative to note that these scaffolds, in particular once seeded with cells and remodeled by biologic ingrowth and calcified ECM production, will change their structural capacity and thus often can be used as a bridge technology. Therefore, the development of advanced scaffolds that can maintain bioactivity properties is required. To achieve this, the obvious engineering solution that has been implemented was the development of composite materials.

Advances in ceramic polymer composite biomaterials

Bioactive glass was used in the form of particles,¹¹⁹ fibers,¹²⁰ or scaffolds,¹²¹ and it was combined with polymers to develop various composites for tissue engineering. For example, bioactive glass nanoparticles have been incorporated into freeze-cast gelatin-chitosan foams with a pore size range between 150 and 300 μm.¹²² The low strength of the composite was improved by a decrease in its porosity. Bioactive glass–collagen–phosphatidylserine scaffolds (65 wt.% 58S sol–gel bioactive glass) were developed with 75% porosity, a pore size of 300 μm, and a compressive strength of 1.5 MPa.¹²³ However, connectivity between pores was poor, limiting scaffold application in tissue engineering.

In addition, Nikpour et al.¹²⁴ developed a composite with bioactive glass-ceramic and dextran hydrogel because of its biocompatibility and hydrophilicity, which enable the incorporation of cells and nanoparticles in the structure. Chatzistavrou et al. also looked at the combination of bioactive glass-ceramic particles with appropriate matrixes (e.g., ECM, collagen–fibrin microspheres) and stem cells to enhance odontogenic differentiation and trigger new dentine formation in dental tissue engineering approaches.^125,126 Another study also looked at producing nano-bioactive glass-ceramic particles that were incorporated with Calcarea phosphorica aiming at assessing the effect of these nanoparticles on osteoblast differentiation.¹²⁷ It was found that these particles had osteogenic potential, as they promoted mouse mesenchymal cell proliferation.

Ceramics are also being coated with polymers, which can help in achieving surface functionalization, controlled delivery of growth factors and drugs, and enhanced bioactivity.¹²⁸ In one example, Luginina et al.⁶⁴ combined bioactive glass particles with electrospun PGS/PCL for the engineering of cartilage. This combination helped to maintain smaller projected cell areas as well as rounded cell phenotype. Scaffolds made of 13–93 bioactive glass were seeded with rat-bone-marrow-derived MSCs and implanted in the subcutis of rats for 4 weeks, which resulted in tissue infiltration of the scaffolds.¹²¹ Moreover, vessels can form inside scaffolds in in vitro cultures and when the construct is implanted in vivo newly formed vessels may connect to the host blood vessels.¹²⁹ Further, scaffolds made from bioactive glass fibers and PLGA mesh were also developed and investigated for bone tissue engineering using osteoprogenitor cells representing craniosynostotic osteoprogenitors, with the view of using this approach for the reconstruction of the crania of these patients using autologous cells derived from removed tissues.¹³⁰

Advances in metal biomaterials

Metals are a group of interesting materials that can also be used for developing scaffolds for tissue engineering. At a historic level, gold and other materials with malleable properties have been used before the time of Hippocrates. Because most of metals are not biodegradable, they cannot be replaced by tissues. Therefore, the use of this group of materials for tissue engineering has been very limited except for the recent activity in biodegradable metal alloys, which represents an expanding research frontier. These materials combine both the properties of metals and biodegradability sought in polymers. In addition, their use helps to avoid many problems associated with the use of biodegradable polymers, such as inflammation^131,132 and osteolysis.¹³³ In this group of biodegradable metals, magnesium-based alloys have been explored and various implants have been developed especially for application in the treatment of bone tissue.¹³⁴ A combination of both biodegradable polymers and metals has also been investigated,¹³⁵ for example, a biodegradable magnesium-reinforced biodegradable PLA membrane was developed for application in guided bone regeneration.¹³⁶ In future, it is expected to see more studies on the combination of metals with polymers.

Metals, in general, exhibit improved mechanical properties (i.e., yield strength, ultimate tensile strength, hardness, etc.), and they are considered the best alternative for structural support. In addition to mechanical performance, absorbable metals should be compatible and nontoxic with controlled corrosion behavior. A metal that can be considered absorbable should corrode in the body's environment without generating toxic corrosion products. Thus, they should meet an appropriate balance between maintaining the required mechanical performance and corroding within a required period while the native tissue is regenerating.

Iron (Fe), magnesium (Mg), and their alloys have been investigated as absorbable metals, for biomedical applications in cardiovascular and orthopedic surgery. Mg is biocompatible, reduces thrombogenicity, and is critical for several cellular functions, such as intracellular transport, signal transduction, and energy metabolism.¹³⁷ Absorbable stents¹³⁸ and bone screws¹³⁹ made of Mg-based alloys are already commercially available.^140,141

However, the uncontrolled and fast corrosion of Mg in biological environments remains a challenge.¹⁴² Mg-based alloys are still being optimized toward meeting the expectations of absorbable metallic implants.¹³² Zinc (Zn) was incorporated as an alternative to Mg, because of its moderate corrosion rate in simulated body fluid.¹⁴³ In one study, Bowen et al.¹⁴⁴ presented an outstanding corrosion behavior and biocompatibility of Zn vascular stents in rat aorta. Current research proposes that Zn alloys could potentially overcome the challenges of Mg alloys used as absorbable implants. Current research work also includes advances in other biomedical applications such as 3D porous Zn scaffolds.¹⁴⁵

Through previous research it became clear that each biomaterial brings with it certain advantages and disadvantages, and to create specialized scaffolds and other tissue engineering constructs we must be able to utilize multiple materials in combination, so that their varied advantages can be exploited. In some sense, this requires that the engineering process begins with a clinical problem that dictates the design and needs of the bioresorbable material properties. Early tissue engineering began with a polymer or a construct and looked to find an application. Therefore, as the field has advanced, our approach to design and fabrication should also evolve.

Advances in stem cells and their application in tissue engineering

Although primary cells can be used for tissue engineering,¹⁴⁶ the use of stem cells offers the advantage of access to cells that can be directed to differentiate to the desired cell type.¹⁴⁷ The use of autologous cells, in particular, can help to avoid the problems associated with allo-transplantation. Therefore, stem cells represent a very important and almost inexhaustible source for tissue engineering^148,149 and regenerative medicine.^150,151 Stem cells can also be used for engineering tissues either with or without a biomaterial as a matrix¹⁵² (Fig. 3). In addition to engineering tissues for regenerative purposes, stem cells were recently used for the engineering of cancer spheroids to develop models for cancer studies.¹⁵³

FIG. 3.

Engineering of cell sheets composed of cells only using a modified poly(N-isopropylacrylamide) (PNIPAAm) surface.⁴³⁰ Color images are available online.

Advances in stem cell sources

Stem cells are divided according to their differentiation potency into various lineages as totipotent, pluripotent, and multipotent. Totipotent stem cells can give rise to the three primary germ cell layers of the embryo and also give rise to extra-embryonic tissues.¹⁵⁴ Pluripotent stem cells can give rise to all tissues in the body, except the placenta and umbilical cord. Embryonic stem cells (ESCs) represent an important type of pluripotent stem cells that were explored for cell therapy and tissue regeneration.^155,156 Because of the associated ethical issues and regulatory restrictions, research continued to explore other possibilities.¹⁵⁷

In 2006, scientists succeeded in developing pluripotent stem cells from adult somatic cells,¹⁹ that is, iPSCs, by using clustered regularly interspaced short palindromic repeats (CRISPR) technology, which can be used for either the activation (CRISPRa) or interference (CRISPRi) with the expression of certain genes.¹⁵⁸ The iPSCs are currently being intensively studied because of their pluripotency but without having many of the issues associated with ESCs.¹⁵⁹ Today, they represent an advancing frontier in tissue engineering, because of their potential to differentiate into many cell types.¹⁵⁸ For example, iPSCs that were generated from human anterior cruciate ligament were used in the repair of ligaments and tendons. Further, iPSCs will be an invaluable tool for the development of personalized therapeutics.^160,161

Compared with pluripotent cells, multipotent stem cells such as MSCs can produce only certain cell types.¹⁶² MSCs are derived from mesodermal embryonic tissues, have high regenerative ability, and are precursors of different mesenchymal tissues such as bone and cartilage.¹⁶³ MSCs can be isolated from different tissues such as bone marrow,¹⁶⁴ adipose tissue,^165–167 amniotic membrane,¹⁶⁸ umbilical cord,^168–170 placenta,¹⁷¹ dental pulp,¹⁷² and other sources that are being continuously explored.¹⁷³

Recently, MSCs that were isolated from the synovial fluid and synovial membrane were investigated for cartilage tissue engineering.¹⁷⁴ Among these, adipose tissue represents an attractive source¹⁷⁵ because of its abundance, easiness of accessibility, and the possibility of retrieval of stem cells that were proved to differentiate to different lineages such as fat, bone, and cartilage.¹⁷⁶ Generally, MSCs have been the most widely investigated stem cells^177,178 for various tissue engineering applications.¹⁷⁹ Their versatile behavior in vivo and in vitro made stem cells favorable for research and clinical applications.^180,181 Stem cells can also be used for immunomodulation,^182,183 which can be explored for application in tissue engineering and regenerative therapy.¹⁸⁴

Recent advances in stem cell-derived extracellular vesicles

In addition to iPSCs, advancing frontiers in stem cell technology and its application in tissue engineering include stem cell-derived EVs. EVs are produced by cells in the form of exosomes, microvesicles, or apoptotic bodies; they carry peptides, lipids, or nucleotides such as RNA and DNA, and they have been increasingly recognized as an important means of molecular communication between cells and organs.¹⁸⁵ In particular, EVs secreted by MSCs have been investigated for tissue regeneration since they can produce important effects without the need to use cells. They have been investigated for skin, bone, cartilage, and neuronal regeneration.^178,186

To increase the efficacy of EVs, MSCs were preconditioned by using hypoxia to produce primed MSCs.¹⁸⁷ This paves a new way of devising regenerative strategies based on the use of stem cell-derived EVs, which will help to eliminate many of the problems associated with the use of cell-based products. It is expected that research in this area will expand and extend to clinical translation in the future. It also underscores the importance of closing the knowledge and understanding gap that we still have in relation to the stem cell microenvironment.

Advances in stem cell differentiation

The most important challenge in the development of stem cell-based treatments in tissue engineering applications is the identification of biophysically and biochemically different tissue-specific environments.¹⁸⁸ In this process, besides defining differentiation and growth factors that mimic the stem cell environment, it has been reported that determining the physical properties and mechanical forces of stem cell matrix such as morphology and stiffness are also important.¹⁸⁹ Studying the effects of ECM biological, physical, and chemical effects on stem cells will help to develop methods that can influence cell differentiation.¹⁹⁰ It was also found that making the surface architecture and the stiffness properties of the biomaterials similar to those of certain native tissues favors the differentiation of the stem cells to cells specific to these tissues.¹⁹¹

Stem cell fate control is a crucial issue for stem cell research and applications. In a recent study, magnetic nanoparticles were used to guide stem cell differentiation,¹⁹² with the help of an externally applied magnetic field that was used to pull iron oxide particle-laden ESCs together and form spheroids. Then, opposing magnetic fields were used to stretch them and lead to cardiac lineage differentiation. When both physical and chemical factors were combined and applied to ESC, significantly higher myogenic differentiation was observed.^193,194

Differentiation of stem cells into desired cell type is possible by identifying factors such as matrix microenvironment and epigenetic mechanisms¹⁹⁵ that regulate the fate of stem cells.^190,194,196 For instance, an injectable hydrogel was developed by using HA, horseradish peroxidase, galactose oxidase, and tyramine; it was used as a crosslinker. Experiments in mice demonstrated the biocompatibility of the material, which makes it a good candidate for use in biomedical applications such as tissue engineering applications.¹⁹⁷

In a recent study, the porosity of hydrogel biomaterial was shown to influence MSCs and their response to insulin-like growth factor-1 (IGF-1).¹⁹⁸ Unlike nanoporous alginate hydrogel, microporous ones could sensitize MSCs to the growth factor. Adding cell–cell adhesion mediating molecule (N-cadherin) mimicking peptide to nanoporous alginate added the effect that macroporous had in eliciting MSCs paracrine activity in response to IGF-1. This demonstrated the role of physical properties of the biomaterials further, and also the possibility to influence this by using chemical ways. Combined, these methods will help us to control the behavior of stem cells further in the future and tailoring it toward desired activity and fate, for regenerating desired tissues.

Advances in cell maturation strategies

There are several technologies that have been developed to increase cell maturation by using electrical and physical cues. For influencing cell maturation, physical cues such as surface patterning¹⁹⁹ or mechanical stretching²⁰⁰ have been investigated. For example, physical conditioning of cardiomyocytes (both primary myocytes and human pluripotent stem cell-derived cardiomyocytes) embedded in a collagen hydrogel was achieved by using an automated stretch device.²⁰⁰ More recently, the effect of electrical stimulation on cell maturation and the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) was investigated.²⁰¹ It was found that the application of electrical stimulation during cell differentiation makes hiPSC-CMs behave similar to cardiac cells. It was demonstrated that hiPSC-CMs derived by using electromechanical stimulation can be used to engineer heart tissue.²⁰² In fact, the maturation of early stage cardiomyocytes was achieved by using electrical stimulation for two weeks in the presence of various growth factors. This area represents an advancing frontier in tissue engineering, and it is worth investigating further to also look at the outcome of in vivo applications of cells matured using electrical stimulation.

In another recent study, electrical stimulation of neonatal rat cardiomyocyte-embedded in gelatin hydrogel led to their maturation.²⁰³ It was also shown that the organization of the cells within the gelatin hydrogels was improved by employing this strategy. For skeletal muscle tissue engineering, a gelatin-carboxymethyl cellulose biomaterial was combined with carbon nanotubes to increase electrical properties of the biomaterial.²⁰⁴ Electric pulse stimulation was applied and led to enhanced myogenic differentiation and maturation of C2C12 myoblasts to form a skeletal muscle tissue.

Advances in culture systems and their application in tissue engineering

Cell culture is an important and integral part of ex vivo tissue engineering. Because of the rarity of certain cell types in the human body and the potential donor site morbidity associated with the retrieval of cells in large numbers,^22,205 cells are propagated outside the body in an environment that can provide nutrients and possible stimulation of cells to proliferate, differentiate, and function.²² This is carried out using static or dynamic culture methods. In recent years, there have been advances in cell culture methods such as 3D, 4D, and microfluidic OoC culture systems. Significant progress has been made in these areas, and therefore, they will be highlighted in this section.

Advances in cell culture microenvironment

The most common drawback of any of cell culture techniques is the need to use media, which may contain animal serum. There is evidence that fetal serum could be the source of endotoxins, mycoplasma, or viral contaminants.²⁰⁶ Also, the serum itself has ECM components that may alter the cell expression of proteins. Human autologous serum can alternatively be used, and it has been shown to be equivalent to fetal animal sera. However, it is often difficult to isolate human autologous serum in sufficient quantities, especially for use in prolonged and large-scale applications.²⁰⁷ Serum-free media tend to substitute individual key components found in serum-containing media, which can be a safer approach.²⁰⁸

Among the key components, are the growth factors specific for the stem cell type and tissue culture application. For instance, Hasegawa and colleagues have created a medium for stem cell culture containing a replacement of wnt with a GSK3β and NFAT inhibitor.²⁰⁹ Despite significant progress made with serum-free media, the use of new approaches for the elimination of protein from the media will make this technology more cost-effective and possible to scale up.

Other parameters of cell culture such as temperature can also be used to influence cells, for example, improving adipogenic differentiation.²¹⁰ Another area of interest is the co-cultivation induction, where the concomitant culture of stem cells and committed cells is carried out. This technique has been shown to upregulate the properties of the stem cells and to induce a “physiologic” differentiation process without the need for the use of morphogens and other differentiation induction media. In a recent study, cardiomyocytes were cultured together with iPSCs, and it was found that older cardiomyocytes serve as an adequate inducer for stem cell differentiation, recapitulating the environment necessary for cardiac cell differentiation.²¹¹

Imprinted micropatterns on the surface of plates allow cell-to-cell adhesion and determine the formation and characteristics of the culture. This approach has seen several potential applications. For example, the use of micropatterned surfaces allowed homogenous stem cell differentiation to chondrocytes.²¹² In another study, a 3D micropatterned plate was used to culture hepatic endoderm iPSCs.²¹³ The cells quickly reaggregated and formed hundreds of round-shaped spheroids while they efficiently differentiated into hepatocyte-like cells expressing hepatic gene makers. In addition, growth factors can be printed in micropatterned surfaces. For example, a micropattern-immobilized nerve growth factor nanolayer was found to induce neurite growth and regulate neurite formation.²¹⁴

Advances in 3D culture

Conventional two-dimensional (2D) systems are classically used for stem cell culture. Such culture uses a feeder layer of cells complemented with tissue culture media supplemented with growth factors or cytokines containing cues that support cells and drive them to proliferate or differentiate.²¹⁵ Two-dimensional cultures have several limitations, including: (1) the deformation of the cells during culture (flattening and elongation), (2) poor differentiation and cell junction formation, (3) unnatural high proliferation rates, and (4) significant differences in gene expression and phenotypes.²¹⁶ Recent advances in 2D cultures have tried to overcome some of these drawbacks. Adaptations of the tissue culture biomaterial properties have been shown to modify cell fate.

On the other hand, 3D culture systems are better at recapitulating in vivo conditions. Several studies showed the effect of 3D culture systems on improving cell morphology, proliferation, differentiation, and response to stimuli.²¹⁷ Three-dimensional culture could be divided into either anchorage-dependent (scaffold-based) or anchorage-independent ones using specialized 3D platforms.²¹⁸ The former can benefit from recent advances in processing techniques mentioned earlier, such as 3D bioprinting^20,219,220 and electrospinning,^221,222 to create complex structures.²²³ Such cell culture models should mimic cells' natural environment, providing interactions between the cells and the microenvironment, nutrients, O₂, and waste product removal.

Despite several advantages of 3D over 2D culture, 3D culture still have some drawbacks such as uneven distribution of nutrients, growth factors, and O₂, which often results in making cells residing far away from the surface of the matrix inactive.²²⁴ In addition, many tissue-engineered constructs are looking for regenerative models of culture as opposed to mature quiescent ECM–cell relationships. Increased costs, differences in experiment replication, and data interpretation are additional drawbacks of this type of culture²²⁵ that remain to be addressed in future development activities.

Scaffold-based anchorage-dependent culture techniques utilize a scaffold of variable architecture ranging from a simple extracellular-like matrix to complex multilayer structures. Scaffold selection is largely dependent on the target tissue to be engineered, advantaging physical factors providing structural stability and the cellular composition of the target tissue. Three-dimensional bioprinting has revolutionized the construction of such complex structures. However, because the development of functional vasculature in transplantable devices has not been achieved, successful in vivo applications and clinical translation are largely affected.

Special 3D anchorage-independent techniques include the use of a low attachment vessel,²²⁶ magnetic levitation,²²⁷ or hand-drop technique,²²⁸ including the use of magnetic forces.¹⁹² The low attachment plate technique uses a culture vessel with an ultra-low attachment coating. Anchorage-independent techniques force cells to aggregate, form spheres, and subsequently create their own ECM. The most common form of these techniques is the spheroid culture, which is used in the engineering of cartilage.²²⁹

Magnetic levitation utilizes a magnetic force to levitate cultured cells mixed with magnetic nanoparticles. This technique is shown to have reproducible results and to reduce necrosis in the spheroid core. Stem cells cultured in these conditions maintain their properties and remain quiescent for subsequent clinical use.²³⁰ One area of interest in anchorage-independent culture is the development of organoids. Organoid formation involves the utilization of a tissue culture technique that allows self-organizing and self-renewing of 3D cultures. Organoid cultures have been described for several organs, including the kidney, eye, brain, gut, and lungs.²³¹

More recently, Tseng et al. demonstrated the capacity of assembling adipospheres from multiple cell types, including adipose tissue-derived stem cells, endothelial cells, and leukocytes, that recreate tissue organization.²³² This technique enabled the formation of vessel-like endothelial structures with lumens and differentiation of unilocular adipocytes. The hand-drop technique utilizes the self-aggregation properties of cells when no attachment wall is found. The cells aggregate to form spheroids, and the control of the volume of the cell suspension enables the control of the spheroid size. The outcome of this type of cell culture is better as compared with that of static cultures.

Recently, investigators explored the conversion of adipocytes to cardiomyocytes for application in cardiovascular tissue engineering.²³³ In applications for retinal degeneration, the hand-drop technique was utilized to convert adipocytes to retinal precursors and showed improved differentiation yield, with these precursor-like cells responding to glutamate neurotransmitters.²³⁴ This technique has been used in many other preclinical studies, including cartilage repair, bone healing, and cardiac tissue regeneration.^235,236

Four-dimensional culture platforms utilize a complex 3D-bioprinted or imprinted structure with a predetermined time-dependent dynamic morphological change. This is achieved by the control and manipulation of the behaviors of stem cells responding to cues that aim at replicating the topographical and mechano-biological environment of the target tissue. These systems could find applications in studying tissue biology and pathophysiology, preclinical testing, and tissue biofabrication.^237–239 As far as tissue engineering is concerned, the use of 4D culture systems is in its infancy. However, some promising studies were published. For example, Miao et al. have utilized this technique to create neural tissue with a time-dependent self-morphing regulation of neural stem cells that enhances neural differentiation of cells along with significant axonal alignment.²³⁸ Further studies will be of interest in this area of research, as it is structurally most replicative of the regenerative process of healing.

Advances in microfluidic culture systems

Microfluidic systems are designed for cultures under perfusion, allowing a continuous supply of O₂ and nutrients (Fig. 4). This enables the long-term maintenance of constructs at physiologically relevant nutrient supply rates. The use of a microfluidic-based approach in cartilage regeneration allowed enhanced conjugation of the key growth factor, transforming growth factor-beta 3 and its sustained release.²⁴⁰ In another study, biomimetic neural tissue fibers having hierarchically ordered nerve fibers were created by using a microfluidic system, which contained a coaxial triple-channel chip and a stretching loading device.²⁴¹ Authors reported good performance of the resulting nerve fibers.

FIG. 4.

(A) Schematic illustration of the design of microfluidic chip that has three parallel gel regions, six gel filling ports, and two medium channels connected to four medium reservoirs. The device also contains a surrounding vacuum channel. Scale bar, 2 mm. (B) The device comprises a microfluidic layer on a polydimethylsiloxane membrane featuring two sets of two capped pillars (inset). The membrane is itself bonded to a coverslip. (C) Schematic illustration showing the final coculture arrangement: embedded in a hydrogel, muscle bundles that are wrapped around and exerted force to the pillars. They are innervated by neurospheres, which are placed in the opposite gel chamber separated by a 1-mm-wide gel region. (D) Schematic illustration showing the differentiation process of the ESCs into motor neurons (MNs). Row 2: Schematic illustration displaying the top and front views of the tissue in the microfluidic device. Row 3: Three-dimensional illustrations showing the version of the device used at the corresponding days. ChR2, and Channelrhodopsin-2; CNTF, ciliary neurotrophic factor; EBs, embryoid bodies; ESCs, embryonic stem cells; GDNF, glia-derived neurotrophic factors; HS, horse serum; RA, retinoic acid; SAG, smoothened agonist. Reproduced from Uzel et al.,⁴³¹ which is an open-access article distributed under the terms of the Creative Commons Attribution license. Color images are available online.

The microfluidic system was also used for the production of a gene delivery system composed of nanocomplexes of plasmids encoding for BMP-2 and chitosan.²⁴² The results demonstrated the potential of using this system for in situ bone tissue regeneration. Another application of microfluidic systems is the development of OoC platforms, which aim at reproducing the function of organs or tissues.^243,244 Applications of OoC are currently limited to the development of basic tissue functions and of certain disease models,^245–247 and it points to new avenues for the study of novel tissue engineering strategies.

Advances in processing techniques and their application in tissue engineering

There are several techniques that have been used to develop scaffolds, matrices, or tissue constructs, such as salt leaching, molding, spinning, freeze-drying,²⁴⁸ solvent casting and particulate leaching,²⁴⁹ electrospinning,^221,222 selective laser sintering and 3D printing,²⁵⁰ and 4D bioprinting.⁶⁹ However, we will highlight in this section only the recent developments in the most advancing frontiers of fabrication techniques²⁵¹ (Fig. 5).

FIG. 5.

Advances in fabrication techniques for tissue engineering. (A) Three-dimensional printing and electric-field-assisted techniques can be very useful for 3D construction of tissue defects (1) based on data-derived from imaging such as computed tomography (2), which is transferred to a design template for 3D bioprinting (4), to produce living constructs (5) that are transplanted to bridge defects and heal damaged tissue (6). (B) In 3D printing (1–3), layer-by-layer deposition of polymeric gel results in the formation of predesigned 3D constructs. In the electric-field-assisted technique (5–6), an electric field is used to control for directing and depositing polymeric fibers. In melt-electrospinning writing (4), both 3D printing and electric-field-assisted methods are combined. Created with Biorender.com. Color images are available online.

Advances in 3D printing

Tissue engineering has adopted the 3D printing technique²⁵² for the fabrication of scaffolds and later to create cell-laden multi-cellular²⁵³ and complex²⁵⁴ tissue constructs, and the technique was termed “3D bioprinting.” Three-dimensional bioprinting is gaining increasing popularity, with more companies innovating to produce 3D bioprinters. The method employs most commonly extrusion, inkjet, laser, or stereolithography, with each of these methods having its own advantages and limitations.^25,255 Therefore, new approaches include combining 3D bioprinting with conventional manufacturing methods. Different combinations of various fabrication techniques can be used, for example, combining electrospinning with 3D bioprinting²⁵⁶ or 3D printing with 3D bioprinting^257,258 to produce advanced scaffolds.

Three-dimensional bioprinting has several advantages over other tissue engineering techniques.²⁵⁹ It allows the creation of well-defined, customized structures that mimic native tissues. These tissues have functional cellular components; therefore, cellular migration from the host is not essential. Further, cellular interaction and key signaling molecules can be incorporated into the design of the printed constructs. The overall cost of 3D bioprinting is lower when compared with currently used graft materials with no donor site morbidity.²⁶⁰ Host tissue regeneration that occurs in pace with the degradation of the implanted 3D-bioprinted construct can hopefully be achieved in future by controlling material properties of the construct bioink. The field is still in its infancy, and therefore we expect to still see shortcomings of current 3D bioprinted constructs.

One problem is the choice of the material that can address both the biology and the anatomy of the tissue to be treated. A lot of our current understanding of these issues is based on our experiments on animals, which may make the design of structures with appropriate properties that fit human tissue structure and function challenging. Further, several aspects of the 3D bioprinting process, such as the isolation of cells, culture conditions, and identification of the signals and growth factors, need to be considered. Our current inability to incorporate vasculature and the potential degradation of the structures limits the success of implanted constructs, and it requires the attention and development of innovative solutions.^259,261,262 There are two areas of 3D bioprinting that deserve special discussion, the 4D²⁶³ and the in situ^264,265 bioprinting.

Despite the numerous challenges, in the past decade, an increasing number of studies were published regarding the creation of biomimetic constructs for future clinical applications. The most important applications so far include skin,²⁶⁶ musculoskeletal²⁶⁷ cardiovascular,²⁶⁸ neural,²⁶⁹ and other tissues.²⁷⁰ The studies present developments made in bioinks that are composed of different biomaterials, cell types, and additives such as growth factors, drugs, or osteoconductive elements, which were tested either in vitro or in vivo.²⁷¹ In addition, 3D-bioprinted products often have significant contributions from established FDA-approved component parts, including cells, signals, and scaffolds, and thus the fabrication technique can often produce and amalgamate products that incorporate existing technology with a novel effect. Available evidence is encouraging; however, we are far from achieving the full complex organ engineering that tissue engineering has promised.

Advances in 4D, in situ, and spheroid printing

Four-dimensional bioprinting uses smart stimuli-responsive materials,^18,90 which are programmed to change their properties and bioactivity over time in response to local or external stimuli.²⁷² An example of the application of 4D bioprinting can be in guided nerve generation, using materials such as graphene hybrid in a 4D construct, which can provide physical guidance, chemical cues, dynamic self-entubulation, and seamless integration.²⁷³

In addition, in situ bioprinting²⁶⁵ is of great interest. It employs special hardware and it completely eliminates the need for ex vivo manipulation of the grafts.²⁶⁴ This approach can find applications in cases where the exact dimensions of the tissue are not known preoperatively, such as, for example, after debridement of tissues following trauma, infection, or cancer resection. Several hand-held²⁶⁵ or scanner-controlled 3D printing devices have been developed and the available studies show that such structures retain the high resolution of the 3D bioprinting technique and can match the exact needs of tissues to be constructed.²⁷⁴

Spheroids were recently used as building blocks of constructs that were produced by 3D bioprinting. In this method, spheroids are sucked in, and then released in a controlled fashion. This approach tries to lend developmental biology approaches. This technique will allow the development of 3D constructs using biomaterial-free bioinks and precise deposition of spheroids into the resulting construct.²⁷⁵ It was also possible to use spheroid-based 3D bioprinting in combination with the freeform 3D bioprinting method that enabled patterning of the printed spheroids into the desired shape of constructs (Fig. 6A).²⁷⁶

FIG. 6.

Various fabrication techniques for tissue engineering constructs. (A) Use of mesenchymal stem cell spheroids to 3D bioprint a helix shape (i), Penn State University initials (ii), 5-layer tubular structures (iii), and double helix-shape constructs (iv). One hundred fifty micrometers (F-actin) and 450 μm (Hoechst) in radius in 1.2% Carbopol yield-stress gel. Magnified zone is indicated by dashed red line. Reproduced from Ayan et al.,²⁷⁶ which is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). (B) Cell-electrospinning process with the processing parameters. Reproduced from Hong et al.,⁴³² which is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). (C) Combined electrospinning and 3D printing. (i) Schematic illustration of the composite scaffold with electrospinning and 3D printing. Step (1) polymer polycaprolactone was used to 3D print the constructs and electrospinning to produce nanofibers, resulting in the formation of dual-scale scaffolds. Created with Biorender.com. Scanning electron microscope images of the scaffolds that were produced by using 3D printing (ii) and dual-scale scaffolds that were produced by using electrospinning and 3D printing (iii) (scale bar = 300 μm). Confocal laser scanning microscopy images of the scaffolds that were produced by using 3D printing (iv) and dual-scale scaffolds that were produced by using electrospinning and 3D printing (v) (scale bar = 300 μm). Reproduced from Vyas et al.,⁴³³ which is an open access article distributed under the terms of the Creative Commons CC BY license (ii–v). (D) Intravital 3D bioprinting, which is carried out by injecting a solution of the polymer into a certain tissue site to be treated in a living body. In this example, a two-photon excitation is used for the construction of a 3D object by gelating the polymer solution, and object intravital imaging is used for identification. Created with Biorender.com. Color images are available online.

Advances in other processing techniques

Electrospinning is a versatile technique relying on the use of an electric field to produce thin micro- and nanofibers²⁷⁷ that can also be combined with drugs.^222,278,279 This technique has also been used for some time to produce nanofiber-based scaffolds that mimic ECM in several aspects and explored for the engineering of various tissue constructs^277,280 such as bone,²⁸¹ cartilage,²⁸² nerve conduits,²⁸³ blood vessels,²⁸⁴ skin,²⁴ and other tissues. In addition to its use for ex vivo tissue engineering, electrospinning was also experimented for in situ applications, for example, for the treatment of skin wounds²⁴ and for ocular drug delivery.²⁸⁵

Different materials have been utilized for electrospinning, and various modifications of the technique of the spinning process have been developed to allow for combining the benefits and properties of more than one material.²⁸⁶ For example, in coaxial electrospinning, it is possible to use a material in the core and a different one in the shell that can have different degradation profiles and can be loaded with different molecules or drugs.²⁸⁷ Examples of the successful use of co-axial²⁸⁷ and triaxial²⁸⁸ electrospinning techniques include the engineering of osteochondral tissues.²⁸⁹ Among the interesting recent advances in electrospinning is cell-laden electrospinning (Fig. 6B), in which muscle cells were encapsulated in fibrin.²⁹⁰ It has been found that incorporating these cells and modifying electrospinning conditions significantly enhanced cell viability under a 4.5 kV electric field.

Another advancing frontier involves the use of electrospinning in combination with 3D printing and bioprinting to bring in various properties, such as reinforcement, to improve the mechanical properties of the resulting 3D-bioprinted constructs.²⁹¹ In addition, combining electrospinning with 3D printing provides a microporous structure that can enhance cell proliferation and infiltration of the structure²⁹² (Fig. 6C). Further, more control over the process of fiber laying of electrospinning, which classically randomly falls on the collecting surface, enabled the use of the techniques in a similar way as 3D printing.²⁹³ Once this is well controlled, it can be one form of 3D printing and bioprinting in future, used on its own.

In addition, melt-spinning has been used for tissue engineering.²⁹⁴ Melt electrospinning-based printing is an emerging printing technique that can print fibers with diameter in the range of nanometers, providing a high degree of resolution, porosity, and pore interconnectivity.^295–297 For example, Brown et al. combined melt electrospinning with a digitally controlled collector and developed a new class of 3D printer called melt-electrospinning writing (MEW), which enabled the deposition of well-defined filaments.²⁹⁸ The MEW has the advantage of avoiding problems related to solvents that are used in conventional electrospinning.²⁹⁹

Recently, and for the first time it was possible to have automated coupled melt-electrospinning and melt-electrowriting, by using a modified elongated nozzle to direct-write melt-electrospun polymeric thin fibers onto a collection surface.³⁰⁰ In one interesting development, multilayers were developed by electrospinning, and layers have complementary moieties that lead to the formation of covalent bonds (such as hydrazide and aldehyde groups) between electrospun fibers when they are brought together under mechanical loading.³⁰¹ The technique can be useful in tissue engineering of advanced structures in future, which can become stronger on exposure to stress, for example, blood vessel engineering,²⁸⁴ guided nerve regeneration,²⁸³ or tendon²⁸² and ligament engineering.³⁰²

All these advances open new avenues and application territories of the techniques and provide us with more options and versatility toward mimicking the complexity and heterogeneity of the native tissues to be engineered, by combining various processing techniques. With these novel approaches, we come one step closer toward developing successful engineered constructs, ex vivo or in situ.

Translational advances in tissue engineering

Successful transfer of technology from bench to industrial production of engineered tissue products has been progressive, but slow, in part because the clinicians often do not have embedded design and architecture input to the early stage tissue engineered constructs. Although there was a bolus of products, primarily focused on engineered skin tissue, that was approved in the late 1990s and early 2000s,³⁰³ only a few products have subsequently emerged.³⁰³ Although a few individuals have gained expertise in translating basic tissue engineering research to commercial products, researchers in the field of tissue engineering and regenerative medicine (TERM), overall, lack experience in translational sciences. One factor that may highlight the impact and enable faster commercial translation is academic–industry partnerships among tissue engineers, clinical investigators, clinicians, and industry partners.

Advances in clinical translation

Influencing factors

Clinical translation is affected by several factors that are related to the technology, approval, and acceptance by doctors^304,305 and patients.³⁰⁵ Although there have been advances in the field, clinical translation has been limited, not because of science or technology, but largely due to factors including scalability, cost, regulatory issues, and uptake.³⁰⁵ There are engineered tissue products that are in clinical use or are moving toward clinical translation such as skin, cartilage, bone, vascular grafts, cardiac tissues, and bladder.^305,306 More complex structures such as heart, lung, liver, and kidney have been recreated and are still in preclinical animal studies. Clinical translation of complex structures and whole organs face a completely different set of challenges.³⁰⁵

Acellular products

Successful clinical applications of engineered tissue products have flourished in the past couple of decades,³⁰⁴ and they were approved by the United States FDA, for example, Integra for skin and INFUSE for bone regeneration.^76,307,308 The former is composed of collagen, glycosaminoglycans, and polysiloxane, and it was approved by FDA in 2002 for use in the treatment of burns³⁰⁷ and then for the treatment of diabetic foot ulcers in 2016.³⁰⁹ INFUSE is a BMP2-containing collagen sponge that was also approved by the FDA in 2002 for use in lumbar fusion.³¹⁰ In addition to these biomaterials, the FDA has also approved native-tissue derived ECM for application in the treatment of complex wounds³¹¹ and nerve regeneration.³¹²

Currently, there are several clinical trials exploring the use of materials in achieving in situ regeneration of nervous,^10,313 cardiac,³¹⁴ and musculoskeletal³¹⁵ tissues. The Humacyte acellular vascular graft, which is made by laying smooth ECM on PGA with cultured smooth muscle cells that are subsequently removed from the graft,³¹⁶ is now in an open-label, nonrandomized, phase II clinical trial³¹⁷ for patients with life-threatening limb or torso vascular trauma. The primary outcome measures include primary graft patency along with frequency and severity of adverse outcomes.

For cartilage and osteochondral repair, TruFit and MaioRegen acellular devices have been developed. TruFit is composed of a PLGA, 10% calcium sulfate, PGA fibers, and surfactant and is used for cartilage repair.³¹⁸ Unfortunately, a 2-year clinical study showed no significant improvement in knee scores.³¹⁹ Compared with autologous osteochondral transplantation, knee scores were worse in the group that received TruFit.³²⁰ Other clinical studies with TruFit showed improvement in symptoms and radiologic outcomes but lack direct comparison with conventional surgical cartilage repair techniques.^321,322 Although there was excitement with MaioRegen (a three-layer scaffold composed of collagen I and hydroxyapatite) for the treatment of osteochondral lesions when medium-term results showed significant improvement of knee scores,³²³ the 5-year results showed failure of repair.³²⁴

Cellular products

Several notable cellular products have been approved by the FDA. In 1997, TransCyte, which is composed of fibroblasts and nylon mesh,³²⁵ was approved for the treatment of burns.³²⁶ In 1998, Apligraf, which is composed of fibroblasts, keratinocytes, and collagen matrix,³²⁷ and another product for the treatment of skin venous ulcers³²⁸ were approved. For the treatment of nonhealing diabetic foot ulcers,³²⁹ in 2001, the FDA approved Dermagraft, a construct made from a synthetic polymer, PGA with fibroblasts. OrCel is a collagen sponge-based scaffold seeded with keratinocytes and fibroblasts, which was used in the treatment of burns.³³⁰ Laserskin and Hyalograft are hyaluronan-based matrices seeded with fibroblasts and keratinocytes, which were used in the treatment of diabetic foot ulcers³³¹ and chronic wounds of the lower extremity.³³² Matriderm^® is an acellular matrix composed of coupled collagen and elastin,³³³ which can be seeded with fibroblasts and keratinocytes and used for the treatment of full-thickness skin loss.³³³

Most of the identified cellularized products are for skin regeneration.³³⁴ Although these products are very valuable, it is also important to develop and test cellular products for the treatment of tissues with much less intrinsic regenerative capacity such as the heart.³³⁵ Even though many of these products have not yet been approved or are not widely utilized in the field, it is important to mention that an iPSC-derived cell sheet based product (TERUMO BCT) for the treatment of heart failure was developed, but it has not yet been approved by the FDA.³³⁶ This cellular therapy may have a major impact on the treatment of heart failure, which constitutes one of the major causes of mortality in the world.³³⁷

Autologous cellular products

The FDA-approved autologous cell-based products include matrix-assisted autologous chondrocyte implantation (MACI), which is composed of matrix and chondrocytes and used for the treatment of full-thickness cartilage defects, and they were approved in 2001.^76,307 Since 2001, several MACI products have been commercialized,³¹⁸ including BioSeed^®-C³³⁸ and Hyalograft^® C.³³⁹ Fibrin glue is used in BioSeed as a cell carrier, and polyglactin 910/poly-p-dioxanone fleece is used as a scaffold. Significant benefits were demonstrated in clinical studies.^338,340 Hyalograft C, which uses HA as a matrix, has been investigated in 28 trials. Relative to microfracture therapy, Hyalograft C showed improved patient scores.³¹⁸ However, Hyalograft C did not undergo a phase III clinical trial and was withdrawn from the market³¹⁸ due to problems with manufacturing practices and comparative studies.³⁴¹

Other MACI products available outside of the United States include CaReS^®, which uses collagen type I hydrogel seeded with autologous chondrocytes. The results of a prospective multicenter clinical trial in 116 patients (49 women and 67 men; mean age, 32.5 ± 8.9 years) demonstrated significant improvement in the knee scores at 12–60 months after treatment with CaReS; there was a significant reduction in global pain scores and an improvement in the health-related quality of life (SF-36) scores.³⁴²

In 2017, phase III clinical trial of another MACI product, NeoCart^®, was completed. NeoCart relies on the use of type I collagen scaffold.³⁴³ At the 5-year follow-up, although magnetic resonance imaging showed significant improvements from earlier follow-up time points, subchondral bone lesions were seen in 80% of patients. NOVOCART^® 3D, a third-generation ACI, employs type I/III collagen biphasic scaffolds³⁴⁴; clinical trials have been performed.³⁴⁵ and phase III clinical trials are in progress.^346–349 One report showed significantly improved knee scores from the preoperative state.³⁵⁰ NOVOCART 3D may serve as the treatment of choice for children and adolescents.³⁵¹

Several other engineered tissues reached the clinical testing stage but faced several challenges. In 2010, tissue-engineered trachea that employed a decellularized allograft³⁵² along with autologous MSCs was implanted into a 12-year-old boy.³⁵³ Though successful, full restoration of the biomechanical properties of the trachea took a long time (18 months). Bladder tissue reconstruction was another success.

In 2005, Atala et al. reported implantation of engineered bladder in patients needing cystoplasty for end-stage bladder disease.³⁵⁴ Collagen or PGA-collagen scaffolds were seeded with the patient's own bladder cells. Unfortunately, a phase II study consisting of children with neurogenic bladder resulting from spina bifida showed no functional improvement in bladder compliance or capacity and the prevalence of serious adverse events prevented further development.³⁵⁵ The performance of the engineered bladder is still far from replacing that conventional use of gastrointestinal tissue for augmentation cystoplasty.³⁰⁶ As a result, a combination of techniques with neurovascular muscle transfer and tissue engineering was proposed.³⁵⁶ The common thread is that neovascularization had to occur in vivo on the construct, because the vascular component of tissue engineering is not optimized.

However, some clinical success was seen in large vessel tissue engineering related to congenital heart disease. In one patient, tissue-engineered pulmonary artery using PCL–PLA copolymer (weight ratio, 1:1) reinforced with woven PGA tubular scaffolds and seeded with autologous peripheral vein-derived cells was found to be successful, and follow-up at seven months showed no evidence of graft occlusion or aneurysms.³⁵⁷

Later, the same group showed both safety and absence of adverse events at four years in a cohort of 25 patients with congenital heart disease who had extracardiac total cavopulmonary surgeries with tissue-engineered vascular grafts serving as conduits.³⁵⁸ One major challenge hindering clinical application of engineered vessel grafts is related to standardizing the engineered parameters such as scaffold structure and materials, for which 3D printing may offer solutions in terms of reproducibility.³⁰⁶ Another interesting product is Holoclar^®, which employs autologous stem cells that are cultured on a fibrin matrix and transplanted to treat damaged outer layer of the cornea; Holoclar was approved by the European Commission in 2015.^306,359,360

Biomaterial-free cellular products

Biomaterial-free cellular grafts have also been tested in patients. In one case report, iPSC-derived retinal pigment epithelial (RPE) sheets were used in the treatment of age-related macular degeneration (AMD) of a 70-year-old woman.³⁶¹ Two phase I/II clinical studies have been conducted to assess the safety and tolerability of ESC-derived RPEs in the treatment of Stargardt's disease (n = 9 patients) and patients with AMD (n = 9) and provided first evidence of medium- to long-term safety, graft survival, and possible biological activity of these cells. No evidence of adverse proliferation or rejection was seen in these patients who were followed up for a median duration of 22 months.³⁶²

Three-dimensional bioprinted products

There are opportunities for translating 3D bioprinting technologies into the clinic. However, there remain significant challenges and limitations that need to be addressed. Some challenges include the production of tissue constructs that have a clinically relevant size, function, and vascularization.³⁶³ Tissues such as cartilage, bone, and skin are more feasible than complex tissues such as the myocardium. The physiologic components and functional requirements necessary for mimicking native tissues are significantly more challenging to engineer. Successful engineering of complex tissues requires time, development of multicomponent bioinks,²⁷¹ and improvements in materials, cell sourcing, and fabrication techniques.³⁶³ Scalability and costs are added barriers. To the best of our knowledge, there are no 3D-bioprinted products currently undergoing clinical testing. To advance translational aspects of 3D printing and bioprinting, we have organized sessions in the Annual meeting of the Society for Biomaterials, 2019 and World Biomaterials Congress 2020.

Industrial translational advances

Product concepts in the TERM field are challenging. Despite promising clinical outcomes,³⁶⁴ many therapeutics have limited insight into the target mechanism of action. This, in turn, leads to a poor understanding of the critical quality attributes that function, in part, to gauge acceptable levels of variability, either inherent to the biology or due to the process.³⁶⁵ Further, a critical eye needs to be kept on the efficacy and approach to developing therapies given the cost of resources for development and translation to the clinic.³⁶⁴

Many initial product concepts emerge from basic science research, largely supported by federal grants from the National Institutes of Health (NIH) and National Science Foundation (NSF). These funding mechanisms explicitly favor innovation, which may come at the cost of advancing simpler effective approaches. In addition, for some areas, the patent landscape is crowded and complicated requiring some product concepts to needlessly contort to remain unconflicted.³⁶⁶ The ultimate goal, however, is to develop a therapeutic that has a clear increase in efficacy over the standard of care,³⁶⁷ but if not careful the long path of translation may induce drift away from that goal.

As these products move from concept to realization, there are a host of business-related challenges that emerge.³⁶⁸ Even an efficacious approach needs to have a tenable business model to be ubiquitously and consistently available to patients. If the process to manufacture the product is not scalable, only a limited number of patients can benefit from the product. Further, if the financial models for generating the product, looking at both cost of goods and reimbursement levels, are not favorable, then eventually no corporate entity can support production. These considerations are often taken too late, leading to false starts as we try to develop therapies for aiding patients.

Limitations in supporting production infrastructure also comprise a concern for the nascent TERM industry. Although some larger corporations will develop full manufacturing and testing facilities internally, widespread translation will require contract organizations to support smaller businesses. However, there is a paucity of Contract Manufacturing Organizations (CMOs) and Contract Testing Organizations (CTOs) that are experienced in the technical aspects unique to the TERM field. In particular, the robust workforce for manufacturing and testing for this field is lacking. Fortunately, efforts by academic programs have recently emerged to address the specialized workforce required production.³⁶⁹

Another production concern is the availability of proper raw materials. For example, the lack of specialized cytokines and biomaterials that are cGMP grade will remain an issue until the demand for enough is established.³⁷⁰ Even for raw materials shared with other more established industries, such as Pharma, the TERM industry does not currently require the scale of material to provide the leverage needed to implement common supply chain strategies, in turn affecting the cost of goods. Unfortunately, only as more TERM products are translated toward the commercial scale will these resource issues be more fully addressed.

The challenges for the translation of TERM products include issues from tenable product concepts to manufacturing issues to regulatory hurdles.³⁷¹ One of the major hurdles in getting TERM products to the clinic has been the time for regulatory review and approval in the United States.³⁰⁴ The FDA has developed programs to accelerate the process, including the Regenerative Medicine Advanced Therapy (RMAT) designation, which was enacted in the 21st Century Cures Act in December 2016.³⁷² The RMAT designation applies to those regenerative medicine therapies that target serious or life-threatening conditions and has the potential to address unmet medical needs. Although this relatively new regulatory pathway will help ease one obstacle, many practical challenges still exist in commercializing TERM products. Only as the field continues to forge forward will some of these issues be overcome. Continued discussions among the community, such as ones at the TERMIS meetings, are critical for identifying the problems and sharing the solutions.

Challenges and Future Directions

There are already more than 100,000 publications and 9000 patents in the field of tissue engineering,³⁷¹ but many bottlenecks still exist at the translational interface. At this time, the pipeline for academic–industry collaborations with active participation by clinical investigators and clinicians is underdeveloped. Additional efforts in tissue engineering need to address both the scientific challenges and translational potential to achieve synergistic success that will have an impactful benefit on patients in the clinic.

One of the most important challenges in tissue engineering has been the death of cells in the scaffolds after their implantation in the body.³⁶⁵ Cells can survive on diffusion only at a distance of ∼100–200 μm away from the source of nutrient supply.³⁷³ Because angiogenesis takes time,³⁷⁴ various strategies have been explored to provide cells in the engineered tissue with essential nutrients and O₂ while awaiting new vessel formation.^375–377 One strategy that has been developed recently is to deliver O₂ into the engineered tissues by using O₂-generating biomaterials,³⁷⁸ which have been shown to be also effective when they are used as a part of 3D-bioprinted tissue constructs.³⁷⁹

To enhance angiogenesis, accurate cell positioning in printed constructs³⁸⁰ and angiogenic growth factors³⁸¹ can play important roles to avoid failure of engineered tissues³⁸² or implanted constructs.^383,384 To ensure continued blood supply to engineered constructs, strategies for vascularization,^375,385 or prevascularization of scaffolds through the use of microsurgery were investigated.³⁷⁷ The need for a functional vascular network increases with the complexity and size of the target tissue or organ. These vascular networks could be used to support the grafts during the immediate postfabrication period.

Although many of the early tissue engineering experiments were proof-of-concept and demonstrated function,³⁸⁶ they were mostly carried out in immune-deficient animals³⁸⁷ where normal immune reactions are not functional. After implantation in immunocompetent animals, immune response to the construct is a challenging problem.³⁸⁸ This response includes nonspecific inflammatory reactions to matrix materials and possible reactions to allogeneic cells.³⁸⁹

Various strategies to address these issues have been developed, such as the use of autologous cells and the use of biocompatible materials. In addition, the exploration of autologous native ECM derived materials has been pursued.³⁹⁰ The use of immune reaction modulating agents such as anti-inflammatory agents^391–393 embedded or integrated with the biomaterial has been explored for optimizing reactions toward implanted biodegradable materials. Further, cellular constructs that have no foreign materials added represent an interesting approach.³⁹⁴ Recent developments in the use of in situ tissue regeneration can be used as an alternative to ex vivo engineering, and it will help to avoid many of the current problems associated with the ex vivo engineering approach.³⁹⁵

Successful results of tissue engineering were demonstrated in early short- to medium-term animal experiments. Later, it was shown that function cannot be always sustained, such as it was seen in experiments with pancreatic endocrine³⁹⁶ and liver tissue engineering.³⁹⁷ Therefore, strategies to enhance the survival and function of implanted engineered tissue constructs were investigated.³⁹⁸ Durability of the implanted engineered tissue constructs is an important aspect, and long-term studies are required to demonstrate this. In vivo imaging³⁹⁹ and cell tracking⁴⁰⁰ have recently evolved and they enable better evaluation and monitoring during the postimplantation period. Sensor technology is an emerging area that can be taken as an enabling tool to advance our capabilities in monitoring our implants further and pursue timely intervention if needed.^401,402

Many of the engineered tissues are small in size because of limitation, partly imposed by difficulty in providing nourishment to deeper parts of the engineered tissue constructs and required vascularization for larger-sized constructs. Even with the most recent developments in the use of 3D bioprinting for engineering tissues, the production of clinically relevant sizes of constructs remains a challenge.²⁵⁸ Some strategies have been developed to address this, such as the printing of supportive structures²⁵⁸ or printing into a supportive sacrificial material.⁴⁰³ The latter is hoped to help produce larger constructs. It remains, however, to get mechanically stable constructs that can preserve their physical characteristics and mechanical properties for a time enough to support tissues while they are in the healing stage. Most challenges in this sense are related to constructs intended for use in hard tissue such as the bone. Important strategies can be sought by combining acellular frames and scaffolds with cellular constructs, as has been suggested earlier.²⁰

The use of engineered tissue products may be associated with safety issues related to cell, material, and molecule sources, during retrieval, processing, storage, transport, and application phases.^255,404 Ethical issues are especially related to the source of cells, for example, xenogeneic grafts, chimeric constructs, or ESCs.⁴⁰⁵ Also, aspects related to the use of stem cell therapies that are risky, untested, and unproven scientifically by unregulated clinics need to be addressed.⁴⁰⁶ Further, ethical aspects related to applications and making the therapy available when needed and for patients who need it are important, given the lack of availability of sufficient organs and tissues needed to provide vital functions and reduce the death of patients on the waiting list.⁴⁰⁷ Ethical aspects related to clinical trials should be properly analyzed and addressed. In clinical trials, it is sometimes difficult to design appropriate control groups because of ethical reasons, and therefore, results should be evaluated accordingly, and so also when applications are submitted for approval by regulatory bodies.

Because of financial reasons or the availability of other therapeutic alternatives, health service providers and insurance companies may not provide or approve engineered tissue products, which imposes another challenge facing wider application of engineered tissues. In addition, acceptance by doctors⁴⁰⁸ and patients is also an important factor that will influence the future of the use of engineered tissue therapeutics. Wider clinical application will, thus, be influenced by patient education, marketing, safety, and efficacy proof. Influencing factors will also include safety, efficacy, and price as well as coverage of the products by insurance companies for defined indications, where alternatives are not available, inefficient, or more expensive. It is clear that there is a niche for engineered tissue products in certain clinical indications, for example, skin for the treatment of face lesions, younger patients, and large burn wounds.⁴⁰⁹

In the future, we expect to see more of in situ tissue engineering that can be accomplished by creating an environment in vivo that stimulates an individual's own resident cells to achieve regeneration. This can be achieved via various approaches that may employ bioresponsive materials to influence immune cells, progenitors, or stem cells, or utilize transcription factors and RNA-based strategies to reprogram cells.¹⁶ Further, the use of in situ^264,265 and intravital⁴¹⁰ (Fig. 6D) 3D bioprinting will advance our capabilities further toward achieving less or minimally invasive delivery of regenerative therapeutics.⁷⁶

Using iPSCs⁴¹¹ and stem cell-derived EVs,⁴¹² as well as customized implants using 3D printing^413,414 allows for the development of more customized and personalized treatment modalities in the future. There is a significant need for the development of autologous endocrine tissues, for example, for the treatment of diabetes,³⁹⁶ and for musculoskeletal tissues that are important for craniomaxillofacial reconstruction,³⁹⁷ which can be addressed by using these new tissue and cellular engineering approaches.

One advancing frontier is related to the use of electroconductive materials for tissue engineering, which can be useful in many applications such as neural tissue engineering.^415,416 Care of the patients will be advanced by integrating capabilities of sensors and actuators; communication and remote control, which will enable real-time monitoring of implanted constructs and timely intervention by providing the right treatment at the right time.⁴¹⁷ Diagnosis, design of treatment, installation of implants, follow-up, and optimization will benefit from advances made through the Internet of Things.⁴¹⁸

Advances in microfluidic OoC systems²⁴⁵ have led to the publication of several experiments on studying tissues, developing disease models, and testing drugs.⁴¹⁹ However, their use for advancing tissue engineering for the purpose of regenerative medicine needs still to be harnessed and there are many untapped opportunities to be explored. In addition, OoC systems will enhance our ability to perform cell culture studies in a 3D dynamic environment, which makes it possible to mimic the in vivo microenvironment.²⁴³ Growth and spread of cells can be monitored in a controlled manner on microstructures created with 3D printers, and it can be possible to mimic the real-time events.⁴²⁰ Further, the use of such advanced in vitro systems will allow us to also test and decide on the ideal cell type for use in the engineering of certain tissues and defined clinical applications.

Next-generation studies in tissue engineering applications will be especially focused on the use of smart biomaterials,^421–423 stem cell studies,⁴²⁴ development of nanotechnology, new biofabrication techniques, and the integration of advances made in synthetic biology.^425,426 Especially with increasing studies and published results on stem cells, it will become easier to imitate target tissues and organs.⁴²⁷ Further studies in the area would allow overcoming the safety and efficacy concerns encountered with many stem cell types, such as the iPSCs and MSCs. Methodologies to isolate, ex vivo manipulate, and culture these cells need further evaluation.⁴²⁸

In tissue engineering applications, the selection and design of biomaterials that are suitable for target tissue and organ is one of the most important issues. In addition, the harmony and integrity of the cells with biomaterials have made the use of biological materials as scaffolds useful for the integration of organs with 3D systems.⁴²⁹ In this context, the most important issue will be to increase the use of technological applications such as computer modeling, artificial intelligence, OoC platforms, and 3D printing for understanding the interactions of cells and tissues with biomaterials in vivo. Thus, it will be possible to use the new-generation biocompatible smart materials in tissue engineering applications and to meet patient requirements in real time.³⁰⁵

Conclusions

Overall, it can be concluded that our current armamentarium in tissue engineering has made different advances at different levels, including biomaterials, stem cell technologies, fabrication techniques, industrial production innovation, and clinical applications. Through the integration of these facets by multidisciplinary teams with sustained funding, future developments should lead to optimized tissue constructs, successful products, and wider adoption for clinical application.

Disclosure Statement

No competing financial interests exist.

Funding Information

The authors acknowledge funding from the National Institutes of Health (1UG3TR003148-01) and the American Heart Association (18TPA34230036 and 442611-NU-80922). Dr. Nguyen receives grant funding from the American Heart Association (18TPA34170049), the National Institutes of Health (R01HL148182), and the Veterans Health Administration (I01-CX001901).