Tissue engineering was defined by Vacanti and Langer as the field combining engineering and the sciences to “restore, maintain, and improve tissue function or whole organ” function [2, 3]. This is done by observation of the relationship between structure of the tissue and the function that it then performs. After, a construct can be created to mimic those functions.
1.1 Need for Tissue Engineering
The field of tissue engineering is one that is crucially in demand today. Whether the defect is caused through disease, trauma, or other causes, the need for the regeneration of tissues and organs continues to become clearer. Typically, these defects and traumas involve procedures and treatment using the gold standard donor graft such as autografts or allografts [4-6]. An autograft is a graft that is taken from another place in the same person’s body while allografts are where the graft is taken from another human (typically a donor or cadaver) .
While these grafts are useful in the performance of the intended function, there are disadvantages that can be associated. These include transmission of diseases, donor site morbidity, pain and a possible immune response. In addition to this, there is often a shortage in terms of donor organs and tissues. In terms of immune response, rejection is also probable because the body is not acclimated with the new tissue or organ [4-6]. Thus, tissue engineering aims to provide the same intended function with use of biological substitutes instead .
1.1 Hard versus Soft Tissue Engineering
When studying tissues and looking at regeneration, it is important to specify whether it is a hard or soft tissue. The hard tissues typically include bone and cartilage and are more commonly studied in tissue engineering. For bone, osteoprogenitor or stem cells will be used as a cell source and the scaffold on which they are implanted acts to facilitate key cellular activities such as migration, proliferation and differentiation [8-10]. The scaffold environment needs to be like that of the native bone . It is important for the scaffold to have an interconnected pore structure for vascularization purposes [8, 9, 11-13]. For cartilage tissue engineering, the main challenge is in regenerating the complex structure. Embryonic stem cells have been studied in this application because they are said to be “immortal” which means that they will continue to self-renew and proliferate . In terms of scaffold structure, hydrogels have been studied frequently and determined as a good material for this application. The scaffold needs to be able to provide an ideal environment for the cells .
Soft tissues on the other hand include nerves, muscles, fat, etc. and aim to support and surround organs and other types of tissues . Currently, injuries and diseases affecting these types of tissues are treated using autologous grafts or fillers. However, these treatments can cause a “volume loss over time” and can cause the expected outcome with these types of grafts, donor site morbility . In nerve tissue, researchers have considered the use of stem cells (ipsc and embryonic). The scaffold used needs to be carefully designed to include the necessary properties and the correct growth factors for intended cell function . In regenerating muscle tissue, the tissues need to be able to contract either voluntarily and involuntarily. In skeletal muscle, the typical treatment is to introduce a muscle flap that covers the area that is injured . However, research has begun to culture cells that can act in place of the damaged cells. The scaffolding applied in this application is typically a hydrogel, or something similar that can “stimulate vascularization and innervation, induce contractility and support cell alignment” .
2. The Tissue Engineering Triad
Tissue Engineering relies on three major components to produce a full construct that is capable of the desired function. These three components are cells, signaling molecules (including growth factors), and scaffolds [6, 17].
The first major component is the signaling molecules. These signaling molecules are incorporated within the designed construct. They help cells in terms of giving directions so that the cell will “express the desired tissue phenotype” . They also help cells in constructs to differentiate and perform their key functions .
The second major component is cells. Cells are found to be the one of the key building blocks that make up and develop the tissue. The cells rely on the extracellular matrix support structure in order to influence their behavior, and to help them in performing key functions such as migration, proliferation, and differentiation in the tissue [4, 19-21].
2.1.1 Sources for Cells
There are multiple different types of cells that have been explored in the implementation of a scaffold for tissue engineering. For these cells, it is crucial that they integrate into the desired tissue and further begin activities such as producing growth factors and other biomolecules . The typical cell sources include: autologous cells, allogeneic cells, stem cells and induced pluripotent stem cells among others. Autologous cells are from the same patient and are ideal because they allow for a low risk of rejection. Allogeneic cells are from the body of a donor. These can be useful but run the risk of pathogen transmission and rejection. Stem cells are undifferentiated cells that are also able to differentiate into multiple cell types. They are able to self-renew in culture conditions. Mesenchymal stem cells are often used because they have many ideal properties such as the fact that they proliferation well and are available [3, 23].
For tissue engineering purposes, the stem cells used are usually from one of three main sources: embryonic, bone marrow, and cord derived. Embryonic stem cells are harvested from the epiblast tissue of the blastocyst . Use of these cells requires an implemented method for initiating differentiation and it needs to be controlled . Bone marrow derived stem cells are used in bone and cartilage tissue engineering where the cells need to differentiate to an osteogenic lineage . Cord derived stem cells have the ability to differentiate into multiple lineages based on necessity and provided growth factors . One new cell type used is induced pluripotent stem cells that are differentiated and been reprogrammed to be pluripotent .
2.1.2 Co-culturing Cells
In many cases, the tissues and organs that are being regenerated are more complex than initially thought and thus require a method of regeneration that is also complex. This has been one of the major challenges in the field thus far. Often, tissues will contain more than one dominant cell type and these cells crosstalk with each other to achieve homeostasis within . One example of this is in bone, where often osteoblasts are present but endothelial cells are also necessary for the vascularization which provides nutrients and oxygen to the cells throughout the bone matrix [11-13, 25]. In this example, both cell types present allow for proangiogenic factors (such as VEGF) to be produced [13, 24, 26-28]. To replicate this crosstalk in vitro, multiple cell types are typically co-cultured and seeded on the scaffold in a manner to promote the complex tissue regeneration . One example of this co-culture work is shown in the attempt to regenerate a human liver with full vascularization. In this study done by takebe et al, induced pluripotent stem cells were co-cultured with hepatic cells and mesenchymal stem cells . Through the co-culture method, researchers are better able to mimic the native complex tissue or organ. This co-culture technique is something that needs to be further explored but has been done in bone and cartilage among other tissues and organs [24, 30].
The third major component of the biomaterial construct is the scaffold. The scaffold is a template made from different materials that hold the cells and allow them to secrete ECM. They are also key regulators in cell activity [4, 19-21, 31]. These scaffolds are designed to mimic the native ECM by mimicking the mechanical properties and providing adequate support [4, 19-21, 31].
In terms of fabrication, it is first important to consider the material. In scaffolds, these materials can be natural or synthetic polymers, ceramics or composites.
Natural polymers tend to include materials such as alginate, collagen, chitosan, etc . This type of material tend to be biologically active, biocompatible and biodegradable which can be extremely useful . The material is thus able closely mimic the native environment. Natural materials are usually commercially available and easy to process [4, 17]. However, some disadvantages to these materials include non-homogeneous structure and possible batch to batch variation along with the possibility of poor mechanical properties. These materials are in short supply and can be expensive [4, 17].
Synthetic polymers includes materials such as polyglycolide, polylactide, etc.  These materials allow for biodegradability and controlled degradation rate along with a tailored architecture for the desired application . However, there is a possibility of inflammatory response and potential risk of rejection because this is not a natural material . Also degradation is a concern due to the degradation process of hydrolysis (CO2 production) [4, 17].
Bio-ceramics are split into three classifications: non-resorbable, bioactive, and resorbable. Non-resorbable materials are inert and include materials such as alumina, and zirconia . Bioactive materials are semi inert . Resorbable materials are not inert and include materials such as calcium phosphate, coralline, etc. . The main advantages include high mechanical stiffness and brittle surfaces . In addition, the materials have good biocompatibility and enhance osteoblast differentiation and proliferation. However, the disadvantages include difficulty in producing and the sub-par mechanical loading capabilities [4, 17].
Composite materials are comprised of combinations of multiple materials. This allows for researchers to control the degradation and mechanical properties of the entire scaffold and tailor the properties in order to best fit the desired application [4, 17].
2.3 Factors for Consideration
In order for tissue engineering constructs to mimic the natural ECM, there are a variety of factors that need to be considered. The mechanical properties are typically designed to match the native or host tissue so it can effectively serve as a substitute. The scaffold/construct needs to be strong enough to handle implantation [4, 17]. It also needs to protect the cells from the different forces that are acting. The scaffold needs to be load bearing in bone and cartilage engineering and have easy processibility [4, 17]. The bioactivity needs to be considered as the scaffold needs to be biocompatible and have a negligible immune reaction . The scaffold should be non-toxic and degradable to allow for tissue growth (with non-toxic byproducts) or non-degradable but able to fully integrate into the body [4, 17].
In terms of structure, the architecture has been shown to significantly influence the functions, conditions, and organization of the new tissue formation. The ideal scaffold would have a porous, interconnected pore structure. By allowing a large surface area, cells have a greater ability to attach and grow [4, 32] The interconnected pore structure allows for increased cell migration and transport throughout the scaffold along with vascularization, nutrient diffusion and waste removal from the scaffold .
3. Method #1 – Pre-Made Porous Scaffolds for Cell Seeding
The first method of scaffold fabrication is to create the porous scaffold (including conventional fabrication, rapid prototyping or woven/non-woven fiber) and then seeding cells.
3.1 Conventional Fabrication
Conventional fabrication is the category that contains many classic fabrication methods such as: particulate leaching, freeze drying, phase separation, and gas foaming. In particulate leaching, porogens are incorporated into the polymer solution. The polymer hardens, the solvent evaporates and the salts left are leached out by an immersion in water [33-35]. The pore size is controlled by porogen size included . The main disadvantage of this method is that the pore shape and connectivity are not able to be controlled . Freeze drying occurs when the polymer is frozen so that the solvent will crystallize [33-35]. The solvent is removed through sublimation which results in pore formation whose shape is dependent on the freezing temperature chosen . This method does not need cross-linking but needs to be tightly regulated so that the resulting scaffold will be homogeneous .For phase separation, the polymer solution is quenched and forms two phases. The polymer poor phase is removed after the polymer rich phase is solidified [33-35]. This technique can form nano-scale fibrous structures which are more like the native ECM that is desired in the tissue . The gas foaming technique occurs using different gases as the porogens . The polymer solution is formed into a solid disk which is then placed under applied pressure (with CO2 and nitrogen) where gas bubbles form [33, 34]. In this method, no chemical solvents are used. In addition, this method has a low fabrication time and the scaffolds are found to be highly porous [33, 34]. The main disadvantage is that it is hard to ensure that the pores are connected and there is no way to control the size of the pores that are formed .
3.2 Rapid Prototyping
Solid free-form or rapid prototyping is the category that incorporates computer aided design or manufacturing to influence and control the microstructure of the scaffold . These methods include 3D printing, selective laser sintering, stereolithography and fused deposition modeling. 3D printing occurs when the scaffold is built in a layer-by-layer fashion where the layer of binder solution is merged to the previous layer on a platform controlled by a computer [33, 34]. This method allows more complex scaffolds to be fabricated, and controlled pore size . However, only certain polymers can be used at the high temperatures, so it is not ideal .
Selective laser sintering occurs when the scaffold is fused layer by layer using a laser beam controlled by a computer . This method is fast and cost effective and has high mechanical strength [33, 34]. However, the laser beam can sometimes cause degradation. Stereolithography uses a UV laser to “polymerize liquid UV curable photopolymer resin” in a layer by layer fashion . This is then solidified, cured and the excess resin is then drained. This method helps to “achieve small features” and offers a wide variety of shapes [33, 34].
Fused deposition modeling is constructed layer by layer due to deposition of a thermo-plastic material from an extruder that is temperature controlled [33, 34]. This method is temperature controlled which limits the type of biomaterial able to be used .
3.3 Woven or non-woven fibers
Another method that has been studied to create porous scaffolds is electrospinning. In this method, the polymeric solution is ejected from the spinneret (needle) and a voltage is applied to create an electric field between the solution and the collector plate . The solution is twisted to form fibers as it draws closer to the collector plate and form a porous structure when collected. The fiber matrix created has a high surface area which makes it ideal for nutrient exchange and waste exchange along with cell attachment . However, this process can involve toxic solvents.
4. Method #2 – Decellularized Extracellular Matrix
The second method for scaffold assembly is decellularized extracellular matrix. This method produces one of the most similar scaffolds to the natural ECM. In this method, donor tissues or organs are taken when they are not suitable for transplantation and the cells are removed. With this, the assembly of the tissue or organ still remains intact along with the proteins. When implanted after recellularization, this allows for the scaffold to have the desired properties . The goal therefore is to remove the cells efficiently without influencing the composition or properties of the matrix left behind. When foreign cellular components remain in the scaffold when implanted, it can elicit response from the body which leads to rejection [34, 36]. The typical treatments for decellularization are often combined to ensure for complete decellularization of the organ or tissue.
4.1 Physical Treatments
The physical treatment of the tissue or organ is performed to lyse the membrane. By completing this, the cells contents are then able to be released and removed from the ECM. Typically, the physical treatment will include methods such as: agitation or sonication, massage or pressure, or freeze/thaw.
Agitation or sonication is the process that is used to allow the tissue or organ to gain exposure to the chemical reagent later introduced. It works to disrupt the membrane, so the cellular components can then be removed. Massage or pressure is another method that is used to lyse the cells by causing the cells to burst. Finally, the freeze/thaw method is done by rapidly freezing the tissue or organ which causes ice crystals to form around the cell membrane and through the alternation between the temperatures, the cell is lysed . Often times these methods do not completely remove all cellular components and therefore either chemical or enzymatic processes need to be performed in addition 
4.2 Enzymatic and Chemical Treatments
Both the enzymatic and chemical treatments are done to disrupt the membrane. The enzymatic treatment is used to detach the cellular components from the ECM. Typically, the chemical treatment will include methods such as: triton X-100, and sodium dodecyl sulfate (SDS). The enzymatic treatment will include methods such as trypsin and others.
Triton X-100 is a non-ionic detergent that works to disrupt the interaction of lipid-lipid and lipid-protein. SDS is an ionic detergent that works to solubilize the membrane but is found to also leave an impact on the collagen fibers of the matrix [36, 37]. Trypsin is an example of an enzymatic treatment that breaks down the cell-matrix adhesions.
The extent of decellularization that is achieved is extremely important. When overdone, the residue will harm the ECM and thus the properties will be altered. When underdone, there is residue of the cellular components which can cause a response in implantation .
Typically, the decellularization process will be followed be cell seeding where the cells are inserted and distributed throughout the matrix. This is followed by perfusion to allow adequate distribution. One major example of the use of this method is published in the paper Organ Reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix by Uygun et al. The objectives, methods and results are discussed further below.
In the above paper, the objective was to modify the previously discovered method for decellularization for use in the liver. The decellularization of livers (donor organs not in optimal shape for transplant) can provide a different alternative for liver disease ,which typically has been treated with a transplant, to account for the shortage of viable organs and tissues for transplant. A portal perfusion is first performed for 72 hours using SDS. In figure 1 below, the rat liver is shown at various time points throughout the course of 72 hours. The result is a translucent liver that is still similar in shape to that of the original liver . Immunostaining was also done to compare the translucent liver to that of the original in terms of proteins in the matrix. It was found that the components were similar to that of the native tissue with cells .
Figure 1. Rat liver decellularization via SDS perfusion at 0, 18, 48, 52, 72 hours respectfully. 
The remaining vasculature found within the decellularized matrix was tested to ensure functionality. The team used an allura red dye which showed a clear vascular tree in decellularization shown in figure 2 below . The dye flowed in typical patterns . Next, corrosion casting was performed with the portal vein in red and the venous vasculature in blue. Through this researchers wanted to show that most of the small branches of the liver matrix were conserved .
Figure 2. A) Allura Red Dye test to check for vascular network. b) corrosion casting 
The matrix was then recellularized in 4 steps, at 10-minute intervals with 12.5 million cells each. It was found that the engraftment efficiency was around 95.6 % . The team then perfused the organ and studied viability of the cells and found minimal cell death .
To test the recellularized matrix, the scaffold was inserted into a rat that had undergone a unilateral nephrectomy. It was found that efflux was established within 5 minutes of unclamping the artery . Images of this are found in figure 3 below. The laboratory then studied the hepatocytes to make sure they were still functional. This was done through the performance of immunohistochemical staining for albumin, G6PC, and UGT1A. Minimal indication of ischemic damage was found .
Figure 3. Showing the transplant site, graft at the transplant site, graft before perfusion, graft when declamped, 1, 2, 4 minutes after and a comparison to what the native liver would look like
This example used the previous decellularization protocol for the heart and applied it to the liver. They showed that with decellularization, the liver matrix and vascularization remain intact. The researchers recellularized the liver using a step by step model where cells were added in bunches and not all at once and the whole construct was implanted into a mouse model. This is the first step in creating a functional liver transplant using the decellularization method .
5. Method #3 – Cells Sheets with Self Secreted ECM
The third method of scaffold assembly is relatively new and involves a self-secreted ECM through the culturing of cells. In this method, the cells are cultured on a thermos-responsive polymer culture dish and as the cells become confluent, they can secrete their own matrix which is then detached without the use of any strong enzymes or chemicals. The sheets are detached by regulating the temperature of the culture surface. These layers can then be implanted [17, 39]. One of the main applications for this is in corneal repair.
6. Method #4 – Cell Encapsulation in Self-Assembled Hydrogel
A hydrogel is defined as a crosslinked polymer matrix that when formed can swell due to its intake of water. It is found to be one of the main scaffolds that is insoluble to water . The hydrogel is typically composed of either natural or synthetic materials. The natural materials include options such as collagen, fibrin, chitosan alginate etc. . Typically, the natural hydrogels are hard to control in terms of mechanical properties and the composition of the hydrogel can vary due to batch to batch variation. The synthetic hydrogels offer an alternative option that is more easily able to be reproduced. This means that there is not the batch to batch variation and it additionally offers more control in the properties both mechanical and degradation .
These scaffolds are typically injectable and the cells in these scaffolds are encapsulated within the liquid that is injected. By using an injection, the cells are able to be delivered directly to the desired site and it can then diffuse around to allow for enhanced adhesion. With this method, the liquid will be injected and crosslinked in vitro and will thus form a gel [42, 43]. The crosslinking mechanism is done either through physical, chemical or covalent crosslinking. The covalent crosslinking is done through radical chain polymerization. The chemical crosslinking is also used because it does not require the presence of an initiator. The gelation process is found to not be disruptive for the cells . Another advantage to this method is that in use for treatments like bone defects, the gel has the capability to form complex structures that are difficult when the scaffold is pre-made.
In the paper Human Embryonic Stem Cell Encapsulation in Alginate Microbeads in Macro Porous Calcium Phosphate Cement for Bone Tissue Engineering by Tang et al. the objective was to encapsulate human embryonic stem cells into alginate microbeads which were then incorporated into a calcium phosphate cement and study osteogenic activity. Calcium phosphate was chosen because its injectable, and it has properties similar to native bone mineralization. Human embryonic stem cells were chosen because they can self-renew, allow for rapid proliferation, and are able to differentiate into different cell types. The cells were first encapsulated within the alginate beads and then incorporated into the calcium phosphate cement. The viability of the cells was tested and confirmed using a live dead assay shown in figure 4. Figure 4 also shows that the percentage of live cells throughout 21 days stayed between 85 – 95 %. The density showed no major comparable differences. From this data, it was shown that the cells in the encapsulation remained viable throughout the 21 days .
Figure 4. Shows viability (live/dead) throughout. c and shows the % of live cells and density .
Figure 5. ALP and OC expression at day 1- 21 .
Figure 6. Xylenol Orange Staining results for mineral synthesis .
The osteogenic gene expression of the cells when placed inside the microbeads was tested by looking at alkaline phosphatase (ALP) and osteocalcin expression shown in figure 5. Low ALP expression at day 1 and 7 was shown but increased at both day 14 and day 21. The osteocalcin expression followed a similar pattern .. Both are markers for osteogenesis and differentiation. The laboratory then tested for mineral synthesis using the xylenol orange stain on the encapsulated cells found in figure 6. To use the xylenol orange stain which chelates with calcium typically, the calcium phosphate cement was removed so as not to interfere with the results. As can be seen, at day 1 the area that was mineralized was quite small. However, as the days increased up to 14 and 21 days, the mineralization staining significantly increased . Overall, this paper found that the encapsulated cells were still able to differentiate to an osteogenic lineage and were capable of mineralization even when encapsulated. This experiment was able to look at osteogenic activity and show that encapsulation does not prohibit differentiation and function of the stem cells.
Overall, it is found that tissue engineering is a field that has grown and seen many improvements over the past few decades. Tissue Engineering is a multidisciplinary field that uses the knowledge of engineering and the various sciences in order to develop constructs that will allow for the regeneration of tissues and organs in vivo. These constructs are typically made of cells incorporated within a scaffold biomaterial matrix with regulatory signals included. These constructs are fabricated using: a premade scaffold with incorporation of cells, decellularization of donor tissues and organs and then subsequent recellularization, cell sheets made on a culture dish through the secretion of ECM from the cells and cell encapsulation into hydrogels. These constructs and methods can in the future provide a supply of soft and hard tissue on demand.