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Tissue engineering is a
biomedical engineering Biomedical engineering (BME) or medical engineering is the application of engineering principles and design concepts to medicine and biology for healthcare purposes (e.g., diagnostic or therapeutic). BME is also traditionally logical sciences ...
discipline that uses a combination of cells,
engineering Engineering is the use of scientific principles to design and build machines, structures, and other items, including bridges, tunnels, roads, vehicles, and buildings. The discipline of engineering encompasses a broad range of more speciali ...
,
materials Material is a substance or mixture of substances that constitutes an object. Materials can be pure or impure, living or non-living matter. Materials can be classified on the basis of their physical and chemical properties, or on their geolog ...
methods, and suitable biochemical and physicochemical factors to restore, maintain, improve, or replace different types of biological tissues. Tissue engineering often involves the use of cells placed on tissue scaffolds in the formation of new viable tissue for a medical purpose but is not limited to applications involving cells and tissue scaffolds. While it was once categorized as a sub-field of
biomaterial A biomaterial is a substance that has been engineered to interact with biological systems for a medical purpose, either a therapeutic (treat, augment, repair, or replace a tissue function of the body) or a diagnostic one. As a science, biomateria ...
s, having grown in scope and importance it can be considered as a field of its own. While most definitions of tissue engineering cover a broad range of applications, in practice the term is closely associated with applications that repair or replace portions of or whole tissues (i.e.
bone A bone is a rigid organ that constitutes part of the skeleton in most vertebrate animals. Bones protect the various other organs of the body, produce red and white blood cells, store minerals, provide structure and support for the body, ...
, cartilage,
blood vessel The blood vessels are the components of the circulatory system that transport blood throughout the human body. These vessels transport blood cells, nutrients, and oxygen to the tissues of the body. They also take waste and carbon dioxide away ...
s,
bladder The urinary bladder, or simply bladder, is a hollow organ in humans and other vertebrates that stores urine from the kidneys before disposal by urination. In humans the bladder is a distensible organ that sits on the pelvic floor. Urine enters ...
,
skin Skin is the layer of usually soft, flexible outer tissue covering the body of a vertebrate animal, with three main functions: protection, regulation, and sensation. Other animal coverings, such as the arthropod exoskeleton, have different de ...
, muscle etc.). Often, the tissues involved require certain mechanical and structural properties for proper functioning. The term has also been applied to efforts to perform specific biochemical functions using cells within an artificially-created support system (e.g. an
artificial pancreas Automated insulin delivery systems are automated (or semi-automated) systems designed to assist people with diabetes, primarily type 1, by automatically adjusting insulin delivery to help them control their blood glucose levels. Currently availabl ...
, or a bio artificial liver). The term ''
regenerative medicine Regenerative medicine deals with the "process of replacing, engineering or regenerating human or animal cells, tissues or organs to restore or establish normal function". This field holds the promise of engineering damaged tissues and organs by st ...
'' is often used synonymously with tissue engineering, although those involved in
regenerative medicine Regenerative medicine deals with the "process of replacing, engineering or regenerating human or animal cells, tissues or organs to restore or establish normal function". This field holds the promise of engineering damaged tissues and organs by st ...
place more emphasis on the use of stem cells or
progenitor cells In genealogy, the progenitor (rarer: primogenitor; german: Stammvater or ''Ahnherr'') is the – sometimes legendary – founder of a family, line of descent, clan or tribe, noble house, or ethnic group.. Ebenda''Ahnherr:''"Stammvater eines G ...
to produce tissues.


Overview

A commonly applied definition of tissue engineering, as stated by Langer and Vacanti, is "an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve iological tissuefunction or a whole organ". In addition, Langer and Vacanti also state that there are three main types of tissue engineering: cells, tissue-inducing substances, and a cells + matrix approach (often referred to as a scaffold). Tissue engineering has also been defined as "understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use". A further description goes on to say that an "underlying supposition of tissue engineering is that the employment of natural biology of the system will allow for greater success in developing therapeutic strategies aimed at the replacement, repair, maintenance, or enhancement of tissue function". Developments in the multidisciplinary field of tissue engineering have yielded a novel set of tissue replacement parts and implementation strategies. Scientific advances in
biomaterials A biomaterial is a substance that has been engineered to interact with biological systems for a medical purpose, either a therapeutic (treat, augment, repair, or replace a tissue function of the body) or a diagnostic one. As a science, biomateria ...
, stem cells, growth and differentiation factors, and
biomimetic Biomimetics or biomimicry is the emulation of the models, systems, and elements of nature for the purpose of solving complex human problems. The terms "biomimetics" and "biomimicry" are derived from grc, βίος (''bios''), life, and μίμησ ...
environments have created unique opportunities to fabricate or improve existing tissues in the laboratory from combinations of engineered extracellular matrices ("scaffolds"), cells, and biologically active molecules. Among the major challenges now facing tissue engineering is the need for more complex functionality, biomechanical stability, and vascularization in laboratory-grown tissues destined for transplantation.


Etymology

The historic origin of the term is unclear as the definition of the word has changed throughout the past few decades. The term first appeared in a 1984 publication that described the organization of an endothelium-like membrane on the surface of a long-implanted, synthetic ophthalmic prosthesis. The first modern use of the term as recognized today was in 1985 by the researcher, physiologist and bioengineer
Yuan-Cheng Fung Yuan-Cheng "Bert" Fung (September 15, 1919 – December 15, 2019) was a Chinese-American bioengineer and writer. He is regarded as a founding figure of bioengineering, tissue engineering, and the "Founder of Modern Biomechanics". Biography Fung ...
of the Engineering Research Center. He proposed the joining of the terms tissue (in reference to the fundamental relationship between cells and organs) and
engineering Engineering is the use of scientific principles to design and build machines, structures, and other items, including bridges, tunnels, roads, vehicles, and buildings. The discipline of engineering encompasses a broad range of more speciali ...
(in reference to the field of modification of said tissues). The term was officially adopted in 1987.


History


Ancient era (pre-17th century)

A rudimentary understanding of the inner workings of human tissues may date back further than most would expect. As early as the Neolithic period, sutures were being used to close wounds and aid in healing. Later on, societies such as ancient Egypt developed better materials for sewing up wounds such as linen sutures. Around 2500 BC in ancient India, skin grafts were developed by cutting skin from the buttock and suturing it to wound sites in the ear, nose, or lips. Ancient Egyptians often would graft skin from corpses onto living humans and even attempted to use honey as a type of antibiotic and grease as a protective barrier to prevent infection. In the 1st and 2nd centuries AD, Gallo-Romans developed wrought iron implants and dental implants could be found in ancient Mayans.


Enlightenment (17th century–19th century)

While these ancient societies had developed techniques that were way ahead of their time, they still lacked a mechanistic understanding of how the body was reacting to these procedures. This mechanistic approach came along in tandem with the development of the empirical method of science pioneered by René Descartes. Sir Isaac Newton began to describe the body as a "physiochemical machine" and postured that disease was a breakdown in the machine. In the 17th century, Robert Hooke discovered the cell and a letter from Benedict de Spinoza brought forward the idea of the homeostasis between the dynamic processes in the body. Hydra experiments performed by Abraham Trembley in the 18th century began to delve into the regenerative capabilities of cells. During the 19th century, a better understanding of how different metals reacted with the body led to the development of better sutures and a shift towards screw and plate implants in bone fixation. Further, it was first hypothesized in the mid-1800s that cell-environment interactions and cell proliferation were vital for tissue regeneration.


Modern era (20th and 21st centuries)

As time progresses and technology advances, there is a constant need for change in the approach researchers take in their studies. Tissue engineering has continued to evolve over centuries. In the beginning people used to look at and use samples directly from human or animal cadavers. Now, tissue engineers have the ability to remake many of the tissues in the body through the use of modern techniques such as microfabrication and three-dimensional bioprinting in conjunction with native tissue cells/stem cells. These advances have allowed researchers to generate new tissues in a much more efficient manner. For example, these techniques allow for more personalization which allow for better biocompatibility, decreased immune response, cellular integration, and longevity. There is no doubt that these techniques will continue to evolve, as we have continued to see microfabrication and bioprinting evolve over the past decade. In 1960, Wichterle and Lim were the first to publish experiments on hydrogels for biomedical applications by using them in contact lens construction. Work on the field developed slowly over the next two decades, but later found traction when hydrogels were repurposed for drug delivery. In 1984, Charles Hull developed bioprinting by converting a Hewlett-Packard inkjet printer into a device capable of depositing cells in 2-D. Three dimensional (3-D) printing is a type of additive manufacturing which has since found various applications in medical engineering, due to its high precision and efficiency. With biologist James Thompson's development of first human stem cell lines in 1998 followed by transplantation of first laboratory-grown internal organs in 1999 and creation of the first bioprinter in 2003 by the University of Missouri when they printed spheroids without the need of scaffolds, 3-D bioprinting became more conventionally used in medical field than ever before. So far, scientists have been able to print mini organoids and organs-on-chips that have rendered practical insights into the functions of a human body. Pharmaceutical companies are using these models to test drugs before moving on to animal studies. However, a fully functional and structurally similar organ hasn't been printed yet. A team at University of Utah has reportedly printed ears and successfully transplanted those onto children born with defects that left their ears partially developed. Today hydrogels are considered the preferred choice of bio-inks for 3-D bioprinting since they mimic cells' natural ECM while also containing strong mechanical properties capable of sustaining 3-D structures. Furthermore, hydrogels in conjunction with 3-D bioprinting allow researchers to produce different scaffolds which can be used to form new tissues or organs. 3-D printed tissues still face many challenges such as adding vasculature. Meanwhile, 3-D printing parts of tissues definitely will improve our understanding of the human body, thus accelerating both basic and clinical research.


Examples

As defined by Langer and Vacanti, examples of tissue engineering fall into one or more of three categories: "just cells," "cells and scaffold," or "tissue-inducing factors." * In vitro meat: Edible artificial animal muscle tissue cultured ''in vitro''. * Bioartificial liver device, "Temporary Liver", Extracorporeal Liver Assist Device (ELAD): The human
hepatocyte A hepatocyte is a cell of the main parenchymal tissue of the liver. Hepatocytes make up 80% of the liver's mass. These cells are involved in: * Protein synthesis * Protein storage * Transformation of carbohydrates * Synthesis of cholesterol, ...
cell line (C3A line) in a hollow fiber
bioreactor A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances ...
can mimic the hepatic function of the liver for acute instances of liver failure. A fully capable ELAD would temporarily function as an individual's liver, thus avoiding transplantation and allowing regeneration of their own liver. *
Artificial pancreas Automated insulin delivery systems are automated (or semi-automated) systems designed to assist people with diabetes, primarily type 1, by automatically adjusting insulin delivery to help them control their blood glucose levels. Currently availabl ...
: Research involves using
islet cell The pancreatic islets or islets of Langerhans are the regions of the pancreas that contain its endocrine (hormone-producing) cells, discovered in 1869 by German pathological anatomist Paul Langerhans. The pancreatic islets constitute 1–2% of ...
s to regulate the body's blood sugar, particularly in cases of
diabetes Diabetes, also known as diabetes mellitus, is a group of metabolic disorders characterized by a high blood sugar level ( hyperglycemia) over a prolonged period of time. Symptoms often include frequent urination, increased thirst and increased ...
. Biochemical factors may be used to cause human pluripotent stem cells to differentiate (turn into) cells that function similarly to
beta cell Beta cells (β-cells) are a type of cell found in pancreatic islets that synthesize and secrete insulin and amylin. Beta cells make up 50–70% of the cells in human islets. In patients with Type 1 diabetes, beta-cell mass and function are di ...
s, which are in an
islet cell The pancreatic islets or islets of Langerhans are the regions of the pancreas that contain its endocrine (hormone-producing) cells, discovered in 1869 by German pathological anatomist Paul Langerhans. The pancreatic islets constitute 1–2% of ...
in charge of producing insulin. * Artificial bladders: Anthony Atala (
Wake Forest University Wake Forest University is a private research university in Winston-Salem, North Carolina. Founded in 1834, the university received its name from its original location in Wake Forest, north of Raleigh, North Carolina. The Reynolda Campus, the un ...
) has successfully implanted artificial bladders, constructed of cultured cells seeded onto a bladder-shaped scaffold, into seven out of approximately 20 human test subjects as part of a
long-term experiment A long-term experiment is an experimental procedure that runs through a long period of time, in order to test a hypothesis or observe a phenomenon that takes place at an extremely slow rate. What duration is considered "long" depends on the academ ...
. * Cartilage: lab-grown cartilage, cultured ''in vitro'' on a scaffold, was successfully used as an
autologous Autotransplantation is the transplantation of organs, tissues, or even particular proteins from one part of the body to another in the same person ('' auto-'' meaning "self" in Greek). The autologous tissue (also called autogenous, autogenei ...
transplant to repair patients' knees. * Scaffold-free cartilage: Cartilage generated without the use of exogenous scaffold material. In this methodology, all material in the construct is cellular produced directly by the cells. * Bioartificial heart: Doris Taylor's lab constructed a
biocompatible Biocompatibility is related to the behavior of biomaterials in various contexts. The term refers to the ability of a material to perform with an appropriate host response in a specific situation. The ambiguity of the term reflects the ongoing de ...
rat heart by re-cellularising a de-cellularised rat heart. This scaffold and cells were placed in a
bioreactor A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances ...
, where it matured to become a partially or fully transplantable organ. the work was called a "landmark". The lab first stripped the cells away from a rat heart (a process called "decellularization") and then injected rat stem cells into the decellularized rat heart. * Tissue-engineered blood vessels: Blood vessels that have been grown in a lab and can be used to repair damaged blood vessels without eliciting an
immune response An immune response is a reaction which occurs within an organism for the purpose of defending against foreign invaders. These invaders include a wide variety of different microorganisms including viruses, bacteria, parasites, and fungi which could ...
. *
Artificial skin Artificial skin is a collagen scaffold that induces regeneration of skin in mammals such as humans. The term was used in the late 1970s and early 1980s to describe a new treatment for massive burns. It was later discovered that treatment of deep ski ...
constructed from human skin cells embedded in a hydrogel, such as in the case of bio-printed constructs for battlefield burn repairs. * Artificial bone marrow: Bone marrow cultured ''in vitro'' to be transplanted serves as a "just cells" approach to tissue engineering. * Tissue engineered bone: A structural matrix can be composed of metals such as titanium, polymers of varying degradation rates, or certain types of ceramics. Materials are often chosen to recruit osteoblasts to aid in reforming the bone and returning biological function. Various types of cells can be added directly into the matrix to expediate the process. * Laboratory-grown penis: Decellularized scaffolds of rabbit penises were recellularised with smooth muscle and endothelial cells. The organ was then transplanted to live rabbits and functioned comparably to the native organ, suggesting potential as treatment for
genital trauma Genital trauma is trauma to the genitalia. History of studying genital trauma Doctors and nurses have been conducting sexual assault examinations and have been collecting evidence for victims of assault for 20 years. But the amount of scientific ...
. * Oral mucosa tissue engineering uses a cells and scaffold approach to replicate the 3 dimensional structure and function of
oral mucosa The oral mucosa is the mucous membrane lining the inside of the mouth. It comprises stratified squamous epithelium, termed "oral epithelium", and an underlying connective tissue termed '' lamina propria''. The oral cavity has sometimes been des ...
.


Cells as building blocks

Cells are one of the main components for the success of tissue engineering approaches. Tissue engineering uses cells as strategies for creation/replacement of new tissue. Examples include fibroblasts used for skin repair or renewal, chondrocytes used for cartilage repair (MACI–FDA approved product), and hepatocytes used in liver support systems Cells can be used alone or with support matrices for tissue engineering applications. An adequate environment for promoting cell growth, differentiation, and integration with the existing tissue is a critical factor for cell-based building blocks. Manipulation of any of these cell processes create alternative avenues for the development of new tissue (e.g., reprogramming of somatic cells, vascularization).


Isolation

Techniques for cell isolation depend on the cell source. Centrifugation and apheresis are techniques used for extracting cells from biofluids (e.g., blood). Whereas digestion processes, typically using enzymes to remove the extracellular matrix (ECM), are required prior to centrifugation or apheresis techniques to extract cells from tissues/organs. Trypsin and collagenase are the most common enzymes used for tissue digestion. While trypsin is temperature dependent, collagenase is less sensitive to changes in temperature.


Cell sources

''Primary cells'' are those directly isolated from host tissue. These cells provide an ex-vivo model of cell behavior without any genetic, epigenetic, or developmental changes; making them a closer replication of in-vivo conditions than cells derived from other methods. This constraint however, can also make studying them difficult. These are mature cells, often terminally differentiated, meaning that for many cell types proliferation is difficult or impossible. Additionally, the microenvironments these cells exist in are highly specialized, often making replication of these conditions difficult. ''Secondary cells'' A portion of cells from a primary culture is moved to a new repository/vessel to continue being cultured. Medium from the primary culture is removed, the cells that are desired to be transferred are obtained, and then cultured in a new vessel with fresh growth medium. A secondary cell culture is useful in order to ensure that cells have both the room and nutrients that they require to grow. Secondary cultures are most notably used in any scenario in which a larger quantity of cells than can be found in the primary culture is desired. Secondary cells share the constraints of primary cells (see above) but have an added risk of contamination when transferring to a new vessel.


Genetic classifications of cells

Autologous: The donor and the recipient of the cells are the same individual. Cells are harvested, cultured or stored, and then reintroduced to the host. As a result of the host's own cells being reintroduced, an antigenic response is not elicited. The body's immune system recognizes these re-implanted cells as its own, and does not target them for attack. Autologous cell dependence on host cell health and donor site morbidity may be deterrents to their use. Adipose-derived and bone marrow-derived mesenchymal stem cells are commonly autologous in nature, and can be used in a myriad of ways, from helping repair skeletal tissue to replenishing beta cells in diabetic patients. Allogenic: Cells are obtained from the body of a donor of the same species as the recipient. While there are some ethical constraints to the use of human cells for in vitro studies (i.e. human brain tissue chimera development), the employment of dermal fibroblasts from human foreskin demonstrates an immunologically safe and thus a viable choice for allogenic tissue engineering of the skin. Xenogenic: These cells are derived isolated cells from alternate species from the recipient. A notable example of xenogeneic tissue utilization is cardiovascular implant construction via animal cells. Chimeric human-animal farming raises ethical concerns around the potential for improved consciousness from implanting human organs in animals. Syngeneic or isogenic: These cells describe those borne from identical genetic code. This imparts an immunologic benefit similar to autologous cell lines (see above). Autologous cells can be considered syngenic, but the classification also extends to non-autologously derived cells such as those from an identical twin, from genetically identical (cloned) research models, or induced stem cells (iSC) as related to the donor.


Stem cells

'' Stem cells'' are undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells. Stem cells are divided into "adult" and "embryonic" stem cells according to their source. While there is still a large ethical debate related to the use of embryonic stem cells, it is thought that another alternative source –
induced pluripotent stem cells Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed in ...
may be useful for the repair of diseased or damaged tissues, or may be used to grow new organs. ''
Totipotent Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta. According to Latin terms, Pluripotentia means the ability for many thin ...
'' cells are stem cells which can divide into further stem cells or differentiate into any cell type in the body, including extra-embryonic tissue. ''
Pluripotent Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta. According to Latin terms, Pluripotentia means the ability for many thin ...
'' cells are stem cells which can differentiate into any cell type in the body except extra-embryonic tissue.
induced pluripotent stem cells Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed in ...
(iPSCs) are subclass of pluripotent stem cells resembling embryonic stem cells (ESCs) that have been derived from adult differentiated cells. iPSCs are created by altering the expression of transcriptional factors in adult cells until they become like embryonic stem cells. ''
Multipotent Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta. According to Latin terms, Pluripotentia means the ability for many thin ...
'' stem cells can be differentiated into any cell within the same class, such as
blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the cir ...
or
bone A bone is a rigid organ that constitutes part of the skeleton in most vertebrate animals. Bones protect the various other organs of the body, produce red and white blood cells, store minerals, provide structure and support for the body, ...
. A common example of multipotent cells is
Mesenchymal stem cell Mesenchymal stem cells (MSCs) also known as mesenchymal stromal cells or medicinal signaling cells are multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cel ...
s (MSCs).


Scaffolds

Scaffolds are materials that have been engineered to cause desirable cellular interactions to contribute to the formation of new functional tissues for medical purposes. Cells are often 'seeded' into these structures capable of supporting
three-dimensional Three-dimensional space (also: 3D space, 3-space or, rarely, tri-dimensional space) is a geometric setting in which three values (called ''parameters'') are required to determine the position of an element (i.e., point). This is the informa ...
tissue formation. Scaffolds mimic the extracellular matrix of the native tissue, recapitulating the ''in vivo'' milieu and allowing cells to influence their own microenvironments. They usually serve at least one of the following purposes: allow cell attachment and migration, deliver and retain cells and biochemical factors, enable diffusion of vital cell nutrients and expressed products, exert certain mechanical and biological influences to modify the behaviour of the cell phase. In 2009, an interdisciplinary team led by the thoracic surgeon Thorsten Walles implanted the first bioartificial transplant that provides an innate vascular network for post-transplant graft supply successfully into a patient awaiting tracheal reconstruction. To achieve the goal of tissue reconstruction, scaffolds must meet some specific requirements. High porosity and adequate pore size are necessary to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients.
Biodegradability Biodegradation is the breakdown of organic matter by microorganisms, such as bacteria and fungi. It is generally assumed to be a natural process, which differentiates it from composting. Composting is a human-driven process in which biodegradati ...
is often an essential factor since scaffolds should preferably be absorbed by the surrounding tissues without the necessity of surgical removal. The rate at which degradation occurs has to coincide as much as possible with the rate of tissue formation: this means that while cells are fabricating their own natural matrix structure around themselves, the scaffold is able to provide structural integrity within the body and eventually it will break down leaving the newly formed tissue which will take over the mechanical load. Injectability is also important for clinical uses. Recent research on organ printing is showing how crucial a good control of the 3D environment is to ensure reproducibility of experiments and offer better results.


Materials

Material selection is an essential aspect of producing a scaffold.  The materials utilized can be natural or synthetic and can be biodegradable or non-biodegradable. Additionally, they must be biocompatible, meaning that they don't cause any adverse effects to cells. Silicone, for example, is a synthetic, non-biodegradable material commonly used as a drug delivery material, while gelatin is a biodegradable, natural material commonly used in cell-culture scaffolds The material needed for each application is different, and dependent on the desired mechanical properties of the material. Tissue engineering of long bone defects for example, will require a rigid scaffold with a compressive strength similar to that of cortical bone (100-150 MPa), which is much higher compared to a scaffold for skin regeneration. There are a few versatile synthetic materials used for many different scaffold applications. One of these commonly used materials is polylactic acid (PLA), a synthetic polymer. PLA – polylactic acid. This is a polyester which degrades within the human body to form
lactic acid Lactic acid is an organic acid. It has a molecular formula . It is white in the solid state and it is miscible with water. When in the dissolved state, it forms a colorless solution. Production includes both artificial synthesis as well as nat ...
, a naturally occurring chemical which is easily removed from the body. Similar materials are
polyglycolic acid Polyglycolide or poly(glycolic acid) (PGA), also spelled as polyglycolic acid, is a biodegradable, thermoplastic polymer and the simplest linear, aliphatic polyester. It can be prepared starting from glycolic acid by means of polycondensation or r ...
(PGA) and
polycaprolactone Polycaprolactone (PCL) is a biodegradable polyester with a low melting point of around 60 °C and a glass transition temperature of about −60 °C. The most common use of polycaprolactone is in the production of speciality polyureth ...
(PCL): their degradation mechanism is similar to that of PLA,  but PCL degrades slower and PGA degrades faster. PLA is commonly combined with PGA to create poly-lactic-co-glycolic acid (PLGA). This is especially useful because the degradation of PLGA can be tailored by altering the weight percentages of PLA and PGA: More PLA – slower degradation, more PGA – faster degradation. This tunability, along with its biocompatibility, makes it an extremely useful material for scaffold creation. Scaffolds may also be constructed from natural materials: in particular different derivatives of the
extracellular matrix In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide s ...
have been studied to evaluate their ability to support cell growth. Protein based materials – such as collagen, or fibrin, and polysaccharidic materials- like
chitosan Chitosan is a linear polysaccharide composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated unit) and ''N''-acetyl-D-glucosamine (acetylated unit). It is made by treating the chitin shells of shrimp and other crustacean ...
or
glycosaminoglycan Glycosaminoglycans (GAGs) or mucopolysaccharides are long, linear polysaccharides consisting of repeating disaccharide units (i.e. two-sugar units). The repeating two-sugar unit consists of a uronic sugar and an amino sugar, except in the case ...
s (GAGs), have all proved suitable in terms of cell compatibility. Among GAGs, hyaluronic acid, possibly in combination with cross linking agents (e.g.
glutaraldehyde Glutaraldehyde is an organic compound with the formula . The molecule consists of a five carbon chain doubly terminated with formyl (CHO) groups. It is usually used as a solution in water, and such solutions exists as a collection of hydrates, c ...
, water-soluble carbodiimide, etc.), is one of the possible choices as scaffold material. Additionally, a fragment of an extracellular matrix protein, such as the RGD peptide, can be coupled to a non-bioactive material to promote cell attachment. Another form of scaffold is decellularized tissue. This is a process where chemicals are used to extracts cells from tissues, leaving just the extracellular matrix. This has the benefit of a fully formed matrix specific to the desired tissue type. However, the decellurised scaffold may present immune problems with future introduced cells.


Synthesis

A number of different methods have been described in the literature for preparing porous structures to be employed as tissue engineering scaffolds. Each of these techniques presents its own advantages, but none are free of drawbacks.


Nanofiber self-assembly

Molecular self-assembly is one of the few methods for creating biomaterials with properties similar in scale and chemistry to that of the natural ''in vivo'' extracellular matrix (ECM), a crucial step toward tissue engineering of complex tissues. Moreover, these hydrogel scaffolds have shown superiority in in vivo toxicology and biocompatibility compared to traditional macro-scaffolds and animal-derived materials.


Textile technologies

These techniques include all the approaches that have been successfully employed for the preparation of non-woven meshes of different
polymer A polymer (; Greek '' poly-'', "many" + ''-mer'', "part") is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...
s. In particular, non-woven
polyglycolide Polyglycolide or poly(glycolic acid) (PGA), also spelled as polyglycolic acid, is a biodegradable, thermoplastic polymer and the simplest linear, aliphatic polyester. It can be prepared starting from glycolic acid by means of polycondensation or r ...
structures have been tested for tissue engineering applications: such fibrous structures have been found useful to grow different types of cells. The principal drawbacks are related to the difficulties in obtaining high
porosity Porosity or void fraction is a measure of the void (i.e. "empty") spaces in a material, and is a fraction of the volume of voids over the total volume, between 0 and 1, or as a percentage between 0% and 100%. Strictly speaking, some tests measur ...
and regular pore size.


Solvent casting and particulate leaching

Solvent casting and particulate leaching (SCPL) allows for the preparation of structures with regular porosity, but with limited thickness. First, the polymer is dissolved into a suitable organic solvent (e.g. polylactic acid could be dissolved into dichloromethane), then the solution is cast into a mold filled with porogen particles. Such porogen can be an inorganic salt like sodium chloride, crystals of saccharose, gelatin spheres or paraffin spheres. The size of the porogen particles will affect the size of the scaffold pores, while the polymer to porogen ratio is directly correlated to the amount of porosity of the final structure. After the polymer solution has been cast the solvent is allowed to fully evaporate, then the composite structure in the mold is immersed in a bath of a liquid suitable for dissolving the porogen: water in the case of sodium chloride, saccharose and gelatin or an
aliphatic In organic chemistry, hydrocarbons ( compounds composed solely of carbon and hydrogen) are divided into two classes: aromatic compounds and aliphatic compounds (; G. ''aleiphar'', fat, oil). Aliphatic compounds can be saturated, like hexane, ...
solvent like
hexane Hexane () is an organic compound, a straight-chain alkane with six carbon atoms and has the molecular formula C6H14. It is a colorless liquid, odorless when pure, and with boiling points approximately . It is widely used as a cheap, relatively ...
for use with paraffin. Once the porogen has been fully dissolved, a porous structure is obtained. Other than the small thickness range that can be obtained, another drawback of SCPL lies in its use of organic solvents which must be fully removed to avoid any possible damage to the cells seeded on the scaffold.


Gas foaming

To overcome the need to use organic solvents and solid porogens, a technique using gas as a porogen has been developed. First, disc-shaped structures made of the desired polymer are prepared by means of compression molding using a heated mold. The discs are then placed in a chamber where they are exposed to high pressure CO2 for several days. The pressure inside the chamber is gradually restored to atmospheric levels. During this procedure the pores are formed by the carbon dioxide molecules that abandon the polymer, resulting in a sponge-like structure. The main problems resulting from such a technique are caused by the excessive heat used during compression molding (which prohibits the incorporation of any temperature labile material into the polymer matrix) and by the fact that the pores do not form an interconnected structure.


Emulsification freeze-drying

This technique does not require the use of a solid porogen like SCPL. First, a synthetic polymer is dissolved into a suitable solvent (e.g. polylactic acid in dichloromethane) then water is added to the polymeric solution and the two liquids are mixed in order to obtain an
emulsion An emulsion is a mixture of two or more liquids that are normally immiscible (unmixable or unblendable) owing to liquid-liquid phase separation. Emulsions are part of a more general class of two-phase systems of matter called colloids. Altho ...
. Before the two phases can separate, the emulsion is cast into a mold and quickly frozen by means of immersion into liquid nitrogen. The frozen emulsion is subsequently
freeze-dried Freeze drying, also known as lyophilization or cryodesiccation, is a low temperature dehydration process that involves freezing the product and lowering pressure, removing the ice by sublimation. This is in contrast to dehydration by most conve ...
to remove the dispersed water and the solvent, thus leaving a solidified, porous polymeric structure. While emulsification and freeze-drying allow for a faster preparation when compared to SCPL (since it does not require a time-consuming leaching step), it still requires the use of solvents. Moreover, pore size is relatively small and porosity is often irregular. Freeze-drying by itself is also a commonly employed technique for the fabrication of scaffolds. In particular, it is used to prepare collagen sponges: collagen is dissolved into acidic solutions of acetic acid or
hydrochloric acid Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid Acid strength is the tendency of an acid, symbol ...
that are cast into a mold, frozen with liquid nitrogen and then lyophilized.


Thermally induced phase separation

Similar to the previous technique, the TIPS phase separation procedure requires the use of a solvent with a low melting point that is easy to sublime. For example,
dioxane 1,4-Dioxane () is a heterocyclic organic compound, classified as an ether. It is a colorless liquid with a faint sweet odor similar to that of diethyl ether. The compound is often called simply dioxane because the other dioxane isomers ( 1,2- ...
could be used to dissolve polylactic acid, then phase separation is induced through the addition of a small quantity of water: a polymer-rich and a polymer-poor phase are formed. Following cooling below the solvent melting point and some days of vacuum-drying to sublime the solvent, a porous scaffold is obtained. Liquid-liquid phase separation presents the same drawbacks of emulsification/freeze-drying.


Electrospinning

Electrospinning is a highly versatile technique that can be used to produce continuous fibers ranging in diameter from a few microns to a few nanometers. In a typical electrospinning set-up, the desired scaffold material is dissolved within a solvent and placed within a syringe. This solution is fed through a needle and a high voltage is applied to the tip and to a conductive collection surface. The buildup of electrostatic forces within the solution causes it to eject a thin fibrous stream towards the oppositely charged or grounded collection surface. During this process the solvent evaporates, leaving solid fibers leaving a highly porous network. This technique is highly tunable, with variation to solvent, voltage, working distance (distance from the needle to collection surface), flow rate of solution, solute concentration, and collection surface. This allows for precise control of fiber morphology. On a commercial level however, due to scalability reasons, there are 40 or sometimes 96 needles involved operating at once. The bottle-necks in such set-ups are: 1) Maintaining the aforementioned variables uniformly for all of the needles and 2) formation of "beads" in single fibers that we as engineers, want to be of a uniform diameter. By modifying variables such as the distance to collector, magnitude of applied voltage, or solution flow rateresearchers can dramatically change the overall scaffold architecture. Historically, research on electrospun fibrous scaffolds dates back to at least the late 1980s when Simon showed that electrospinning could be used to produced nano- and submicron-scale fibrous scaffolds from polymer solutions specifically intended for use as ''in vitro'' cell and tissue substrates. This early use of electrospun lattices for cell culture and tissue engineering showed that various cell types would adhere to and proliferate upon polycarbonate fibers. It was noted that as opposed to the flattened morphology typically seen in 2D culture, cells grown on the electrospun fibers exhibited a more rounded 3-dimensional morphology generally observed of tissues ''in vivo''.


CAD/CAM technologies

Because most of the above techniques are limited when it comes to the control of porosity and pore size,
computer assisted design Computer-aided design (CAD) is the use of computers (or ) to aid in the creation, modification, analysis, or optimization of a design. This software is used to increase the productivity of the designer, improve the quality of design, improve co ...
and
manufacturing Manufacturing is the creation or production of goods with the help of equipment, labor, machines, tools, and chemical or biological processing or formulation. It is the essence of secondary sector of the economy. The term may refer to ...
techniques have been introduced to tissue engineering. First, a three-dimensional structure is designed using CAD software. The porosity can be tailored using algorithms within the software. The scaffold is then realized by using ink-jet printing of polymer powders or through
Fused Deposition Modeling Fused filament fabrication (FFF), also known as fused deposition modeling (with the trademarked acronym FDM), or called ''filament freeform fabrication'', is a 3D printing process that uses a continuous filament of a thermoplastic material. Filam ...
of a polymer melt. A 2011 study by El-Ayoubi et al. investigated "3D-plotting technique to produce (
biocompatible Biocompatibility is related to the behavior of biomaterials in various contexts. The term refers to the ability of a material to perform with an appropriate host response in a specific situation. The ambiguity of the term reflects the ongoing de ...
and biodegradable) poly-L-Lactide macroporous scaffolds with two different pore sizes" via solid free-form fabrication (SSF) with computer-aided-design (CAD), to explore therapeutic
articular cartilage Hyaline cartilage is the glass-like (hyaline) and translucent cartilage found on many joint surfaces. It is also most commonly found in the ribs, nose, larynx, and trachea. Hyaline cartilage is pearl-gray in color, with a firm consistency and has ...
replacement as an "alternative to conventional tissue repair". The study found the smaller the pore size paired with mechanical stress in a bioreactor (to induce in vivo-like conditions), the higher the cell viability in potential therapeutic functionality via decreasing recovery time and increasing transplant effectiveness.


Laser-assisted bioprinting

In a 2012 study, Koch et al. focused on whether Laser-assisted BioPrinting (LaBP) can be used to build multicellular 3D patterns in natural matrix, and whether the generated constructs are functioning and forming tissue. LaBP arranges small volumes of living cell suspensions in set high-resolution patterns. The investigation was successful, the researchers foresee that "generated tissue constructs might be used for
in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
testing by implanting them into
animal models A model organism (often shortened to model) is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the working ...
" (14). As of this study, only human skin tissue has been synthesized, though researchers project that "by integrating further cell types (e.g. melanocytes,
Schwann cells Schwann cells or neurolemmocytes (named after German physiologist Theodor Schwann) are the principal glia of the peripheral nervous system (PNS). Glial cells function to support neurons and in the PNS, also include satellite cells, olfactory en ...
, hair follicle cells) into the printed cell construct, the behavior of these cells in a 3D in vitro microenvironment similar to their natural one can be analyzed", which is useful for drug discovery and
toxicology Toxicology is a scientific discipline, overlapping with biology, chemistry, pharmacology, and medicine, that involves the study of the adverse effects of chemical substances on living organisms and the practice of diagnosing and treating e ...
studies.


Self-assembled recombinant spider silk nanomembranes

Gustafsson et al. demonstrated free‐standing, bioactive membranes of cm-sized area, but only 250 nm thin, that were formed by self‐assembly of spider silk at the interface of an aqueous solution. The membranes uniquely combine nanoscale thickness, biodegradability, ultrahigh strain and strength, permeability to proteins and promote rapid cell adherence and proliferation. They demonstrated growing a coherent layer of keratinocytes. These spider silk nanomembranes have also been used to create a static ''in-vitro'' model of a blood vessel.


Assembly methods

A persistent problem within tissue engineering is mass transport limitations. Engineered tissues generally lack an initial blood supply, thus making it difficult for any implanted cells to obtain sufficient oxygen and nutrients to survive, or function properly.


Self-assembly

Self-assembly methods have been shown to be promising methods for tissue engineering. Self-assembly methods have the advantage of allowing tissues to develop their own extracellular matrix, resulting in tissue that better recapitulates biochemical and biomechanical properties of native tissue. Self-assembling engineered articular cartilage was introduced by Jerry Hu and Kyriacos A. Athanasiou in 2006 and applications of the process have resulted in engineered cartilage approaching the strength of native tissue. Self-assembly is a prime technology to get cells grown in a lab to assemble into three-dimensional shapes. To break down tissues into cells, researchers first have to dissolve the extracellular matrix that normally binds them together. Once cells are isolated, they must form the complex structures that make up our natural tissues.


Liquid-based template assembly

The air-liquid surface established by Faraday waves is explored as a template to assemble biological entities for bottom-up tissue engineering. This liquid-based template can be dynamically reconfigured in a few seconds, and the assembly on the template can be achieved in a scalable and parallel manner. Assembly of microscale hydrogels, cells, neuron-seeded micro-carrier beads, cell spheroids into various symmetrical and periodic structures was demonstrated with good cell viability. Formation of 3-D neural network was achieved after 14-day tissue culture.


Additive manufacturing

It might be possible to print organs, or possibly entire organisms using
additive manufacturing 3D printing or additive manufacturing is the construction of a three-dimensional object from a CAD model or a digital 3D model. It can be done in a variety of processes in which material is deposited, joined or solidified under computer co ...
techniques. A recent innovative method of construction uses an ink-jet mechanism to print precise layers of cells in a matrix of thermo-reversible gel. Endothelial cells, the cells that line blood vessels, have been printed in a set of stacked rings. When incubated, these fused into a tube. This technique has been referred to as "bioprinting" within the field as it involves the printing of biological components in a structure resembling the organ of focus. The field of three-dimensional and highly accurate models of biological systems is pioneered by multiple projects and technologies including a rapid method for creating tissues and even whole organs involve a 3-D printer that can bio-print the scaffolding and cells layer by layer into a working tissue sample or organ. The device is presented in a
TED talk TED Conferences, LLC (Technology, Entertainment, Design) is an American-Canadian non-profit media organization that posts international talks online for free distribution under the slogan "ideas worth spreading". TED was founded by Richard Sau ...
by Dr. Anthony Atala, M.D. the Director of the Wake Forest Institute for
Regenerative Medicine Regenerative medicine deals with the "process of replacing, engineering or regenerating human or animal cells, tissues or organs to restore or establish normal function". This field holds the promise of engineering damaged tissues and organs by st ...
, and the W.H. Boyce Professor and Chair of the Department of
Urology Urology (from Greek οὖρον ''ouron'' "urine" and '' -logia'' "study of"), also known as genitourinary surgery, is the branch of medicine that focuses on surgical and medical diseases of the urinary-tract system and the reproductive org ...
at Wake Forest University, in which a kidney is printed on stage during the seminar and then presented to the crowd. It is anticipated that this technology will enable the production of livers in the future for transplantation and theoretically for
toxicology Toxicology is a scientific discipline, overlapping with biology, chemistry, pharmacology, and medicine, that involves the study of the adverse effects of chemical substances on living organisms and the practice of diagnosing and treating e ...
and other biological studies as well. Recently Multi-Photon Processing (MPP) was employed for in vivo experiments by engineering artificial cartilage constructs. An ex vivo histological examination showed that certain pore geometry and the pre-growing of chondrocytes (Cho) prior to implantation significantly improves the performance of the created 3-D scaffolds. The achieved biocompatibility was comparable to the commercially available collagen membranes. The successful outcome of this study supports the idea that hexagonal-pore-shaped hybrid organic-inorganic micro-structured scaffolds in combination with Cho seeding may be successfully implemented for cartilage tissue engineering.


Scaffolding

In 2013, using a 3-D scaffolding of
Matrigel Matrigel is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel resembles the laminin/collagen IV-rich basement membrane extracellular ...
in various configurations, substantial pancreatic
organoid An organoid is a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. They are derived from one or a few cells from a tissue, embryonic stem cells or induced pluripotent stem ...
s was produced in vitro. Clusters of small numbers of cells proliferated into 40,000 cells within one week. The clusters transform into cells that make either digestive
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products ...
s or
hormone A hormone (from the Greek participle , "setting in motion") is a class of signaling molecules in multicellular organisms that are sent to distant organs by complex biological processes to regulate physiology and behavior. Hormones are require ...
s like insulin, self-organizing into branched pancreatic organoids that resemble the pancreas.
Lay summary:
The cells are sensitive to the environment, such as gel stiffness and contact with other cells. Individual cells do not thrive; a minimum of four proximate cells was required for subsequent organoid development. Modifications to the medium composition produced either hollow spheres mainly composed of pancreatic progenitors, or complex organoids that spontaneously undergo pancreatic morphogenesis and differentiation. Maintenance and expansion of pancreatic progenitors require active Notch and FGF signaling, recapitulating in vivo niche signaling interactions. The organoids were seen as potentially offering mini-organs for drug testing and for spare insulin-producing cells. Aside from Matrigel 3-D scaffolds, other collagen gel systems have been developed. Collagen/hyaluronic acid scaffolds have been used for modeling the mammary gland In Vitro while co-coculturing epithelial and adipocyte cells. The HyStem kit is another 3-D platform containing ECM components and hyaluronic acid that has been used for cancer research. Additionally, hydrogel constituents can be chemically modified to assist in crosslinking and enhance their mechanical properties.


Tissue culture

In many cases, creation of functional tissues and biological structures ''in vitro'' requires extensive culturing to promote survival, growth and inducement of functionality. In general, the basic requirements of cells must be maintained in culture, which include
oxygen Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements as ...
, pH,
humidity Humidity is the concentration of water vapor present in the air. Water vapor, the gaseous state of water, is generally invisible to the human eye. Humidity indicates the likelihood for precipitation, dew, or fog to be present. Humidity dep ...
,
temperature Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measurement, measured with a thermometer. Thermometers are calibrated in various Conversion of units of temperature, temp ...
, nutrients and osmotic pressure maintenance. Tissue engineered cultures also present additional problems in maintaining culture conditions. In standard cell culture,
diffusion Diffusion is the net movement of anything (for example, atoms, ions, molecules, energy) generally from a region of higher concentration to a region of lower concentration. Diffusion is driven by a gradient in Gibbs free energy or chemica ...
is often the sole means of nutrient and metabolite transport. However, as a culture becomes larger and more complex, such as the case with engineered organs and whole tissues, other mechanisms must be employed to maintain the culture, such as the creation of capillary networks within the tissue. Another issue with tissue culture is introducing the proper factors or stimuli required to induce functionality. In many cases, simple maintenance culture is not sufficient.
Growth factor A growth factor is a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. Usually it is a secreted protein or a steroid hormone. Growth factors are important for regul ...
s,
hormone A hormone (from the Greek participle , "setting in motion") is a class of signaling molecules in multicellular organisms that are sent to distant organs by complex biological processes to regulate physiology and behavior. Hormones are require ...
s, specific metabolites or nutrients, chemical and physical stimuli are sometimes required. For example, certain cells respond to changes in oxygen tension as part of their normal development, such as chondrocytes, which must adapt to low oxygen conditions or hypoxia during skeletal development. Others, such as endothelial cells, respond to
shear stress Shear stress, often denoted by (Greek: tau), is the component of stress coplanar with a material cross section. It arises from the shear force, the component of force vector parallel to the material cross section. ''Normal stress'', on the ...
from fluid flow, which is encountered in
blood vessel The blood vessels are the components of the circulatory system that transport blood throughout the human body. These vessels transport blood cells, nutrients, and oxygen to the tissues of the body. They also take waste and carbon dioxide away ...
s. Mechanical stimuli, such as pressure pulses seem to be beneficial to all kind of cardiovascular tissue such as heart valves, blood vessels or pericardium.


Bioreactors

In tissue engineering, a bioreactor is a device that attempts to simulate a physiological environment in order to promote cell or tissue growth in vitro. A physiological environment can consist of many different parameters such as temperature, pressure, oxygen or carbon dioxide concentration, or osmolality of fluid environment, and it can extend to all kinds of biological, chemical or mechanical stimuli. Therefore, there are systems that may include the application of forces such as electromagnetic forces, mechanical pressures, or fluid pressures to the tissue. These systems can be two- or three-dimensional setups. Bioreactors can be used in both academic and industry applications. General-use and application-specific bioreactors are also commercially available, which may provide static chemical stimulation or a combination of chemical and mechanical stimulation. Cell proliferation and differentiation are largely influenced by mechanical and biochemical cues in the surrounding
extracellular matrix In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide s ...
environment. Bioreactors are typically developed to replicate the specific physiological environment of the tissue being grown (e.g., flex and fluid shearing for heart tissue growth). This can allow specialized cell lines to thrive in cultures replicating their native environments, but it also makes bioreactors attractive tools for culturing stem cells. A successful stem-cell-based bioreactor is effective at expanding stem cells with uniform properties and/or promoting controlled, reproducible differentiation into selected mature cell types. There are a variety of
bioreactors A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical reaction, chemical process is carried out which involves organisms or biochemistry, ...
designed for 3D cell cultures. There are small plastic cylindrical chambers, as well as glass chambers, with regulated internal humidity and moisture specifically engineered for the purpose of growing cells in three dimensions. The bioreactor uses bioactive synthetic materials such as polyethylene terephthalate membranes to surround the spheroid cells in an environment that maintains high levels of nutrients. They are easy to open and close, so that cell spheroids can be removed for testing, yet the chamber is able to maintain 100% humidity throughout. This humidity is important to achieve maximum cell growth and function. The bioreactor chamber is part of a larger device that rotates to ensure equal cell growth in each direction across three dimensions. QuinXell Technologies now unde
Quintech Life Sciences
from
Singapore Singapore (), officially the Republic of Singapore, is a sovereign island country and city-state in maritime Southeast Asia. It lies about one degree of latitude () north of the equator, off the southern tip of the Malay Peninsula, bor ...
has developed a bioreactor known as th
TisXell Biaxial Bioreactor
which is specially designed for the purpose of tissue engineering. It is the first bioreactor in the world to have a spherical glass chamber with
biaxial In crystal optics, the index ellipsoid (also known as the ''optical indicatrix'' or sometimes as the ''dielectric ellipsoid'') is a geometric construction which concisely represents the refractive index, refractive indices and as ...
rotation; specifically to mimic the rotation of the fetus in the womb; which provides a conducive environment for the growth of tissues. Multiple forms of mechanical stimulation have also been combined into a single bioreactor. Using gene expression analysis, one academic study found that applying a combination of cyclic strain and ultrasound stimulation to pre-osteoblast cells in a bioreactor accelerated matrix maturation and differentiation. The technology of this combined stimulation bioreactor could be used to grow bone cells more quickly and effectively in future clinical stem cell therapies. MC2 Biotek has also developed a bioreactor known as ProtoTissue that uses
gas exchange Gas exchange is the physical process by which gases move passively by diffusion across a surface. For example, this surface might be the air/water interface of a water body, the surface of a gas bubble in a liquid, a gas-permeable membrane, or a ...
to maintain high oxygen levels within the cell chamber; improving upon previous bioreactors, since the higher oxygen levels help the cell grow and undergo normal cell respiration. Active areas of research on bioreactors includes increasing production scale and refining the physiological environment, both of which could improve the efficiency and efficacy of bioreactors in research or clinical use. Bioreactors are currently used to study, among other things, cell and tissue level therapies, cell and tissue response to specific physiological environment changes, and development of disease and injury.


Long fiber generation

In 2013, a group from the University of Tokyo developed cell laden fibers up to a meter in length and on the order of 100  µm in size. These fibers were created using a microfluidic device that forms a double coaxial laminar flow. Each 'layer' of the microfluidic device (cells seeded in ECM, a hydrogel sheath, and finally a calcium chloride solution). The seeded cells culture within the hydrogel sheath for several days, and then the sheath is removed with viable cell fibers. Various cell types were inserted into the ECM core, including
myocytes A muscle cell is also known as a myocyte when referring to either a cardiac muscle cell (cardiomyocyte), or a smooth muscle cell as these are both small cells. A skeletal muscle cell is long and threadlike with many nuclei and is called a muscl ...
,
endothelial cells The endothelium is a single layer of squamous endothelial cells that line the interior surface of blood vessels and lymphatic vessels. The endothelium forms an interface between circulating blood or lymph in the lumen and the rest of the vessel ...
, nerve cell fibers, and epithelial cell fibers. This group then showed that these fibers can be woven together to fabricate tissues or organs in a mechanism similar to textile
weaving Weaving is a method of textile production in which two distinct sets of yarns or threads are interlaced at right angles to form a fabric or cloth. Other methods are knitting, crocheting, felting, and braiding or plaiting. The longitudinal ...
. Fibrous morphologies are advantageous in that they provide an alternative to traditional scaffold design, and many organs (such as muscle) are composed of fibrous cells.


Bioartificial organs

An artificial organ is an engineered device that can be extra corporeal or implanted to support impaired or failing organ systems. Bioartificial organs are typically created with the intent to restore critical biological functions like in the replacement of diseased hearts and lungs, or provide drastic quality of life improvements like in the use of engineered skin on burn victims. While some examples of bioartificial organs are still in the research stage of development due to the limitations involved with creating functional organs, others are currently being used in clinical settings experimentally and commercially.


Lung

Extracorporeal membrane oxygenation (ECMO) machines, otherwise known as heart and lung machines, are an adaptation of
cardiopulmonary bypass Cardiopulmonary bypass (CPB) is a technique in which a machine temporarily takes over the function of the heart and lungs during surgery, maintaining the circulation of blood and oxygen to the body. The CPB pump itself is often referred to as a ...
techniques that provide heart and lung support. It is used primarily to support the lungs for a prolonged but still temporary timeframe (1–30 days) and allow for recovery from reversible diseases. Robert Bartlett is known as the father of ECMO and performed the first treatment of a newborn using an EMCO machine in 1975. Skin Tissue-engineered skin is a type of bioartificial organ that is often used to treat burns, diabetic foot ulcers, or other large wounds that cannot heal well on their own. Artificial skin can be made from autografts, allografts, and xenografts. Autografted skin comes from a patient's own skin, which allows the dermis to have a faster healing rate, and the donor site can be re-harvested a few times. Allograft skin often comes from cadaver skin and is mostly used to treat burn victims. Lastly, xenografted skin comes from animals and provides a temporary healing structure for the skin. They assist in dermal regeneration, but cannot become part of the host skin. Tissue-engineered skin is now available in commercial products. Integra, originally used to only treat burns, consists of a collagen matrix and chondroitin sulfate that can be used as a skin replacement. The chondroitin sulfate functions as a component of proteoglycans, which helps to form the extracellular matrix. Integra can be repopulated and revascularized while maintaining its dermal collagen architecture, making it a bioartificial organ Dermagraft, another commercial-made tissue-engineered skin product, is made out of living fibroblasts. These fibroblasts proliferate and produce growth factors, collagen, and ECM proteins, that help build granulation tissue.


Heart

Since the number of patients awaiting a heart transplant is continuously increasing over time, and the number of patients on the waiting list surpasses the organ availability, artificial organs used as replacement therapy for terminal heart failure would help alleviate this difficulty. Artificial hearts are usually used to bridge the heart transplantation or can be applied as replacement therapy for terminal heart malfunction. The total artificial heart (TAH), first introduced by Dr. Vladimir P. Demikhov in 1937, emerged as an ideal alternative. Since then it has been developed and improved as a mechanical pump that provides long-term circulatory support and replaces diseased or damaged heart ventricles that cannot properly pump the blood, restoring thus the pulmonary and systemic flow. Some of the current TAHs include AbioCor, an FDA-approved device that comprises two artificial ventricles and their valves, and does not require subcutaneous connections, and is indicated for patients with biventricular heart failure. In 2010 SynCardia released the portable freedom driver that allows patients to have a portable device without being confined to the hospital.


Kidney

While kidney transplants are possible, renal failure is more often treated using an artificial kidney. The first artificial kidneys and the majority of those currently in use are extracorporeal, such as with hemodialysis, which filters blood directly, or peritoneal dialysis, which filters via a fluid in the abdomen. In order to contribute to the biological functions of a kidney such as producing metabolic factors or hormones, some artificial kidneys incorporate renal cells. There has been progress in the way of making these devices smaller and more transportable, or eve
implantable
One challenge still to be faced in these smaller devices is countering the limited volume and therefore limited filtering capabilities. Bioscaffolds have also been introduced to provide a framework upon which normal kidney tissue can be regenerated. These scaffolds encompass natural scaffolds (e.g., decellularized kidneys, collagen hydrogel, or silk fibroin), synthetic scaffolds (e.g., poly actic-co-glycolic acidor other polymers), or a combination of two or more natural and synthetic scaffolds. These scaffolds can be implanted into the body either without cell treatment or after a period of stem cell seeding and incubation. In vitro and In vivo studies are being conducted to compare and optimize the type of scaffold and to assess whether cell seeding prior to implantation adds to the viability, regeneration and effective function of the kidneys. A recent systematic review and meta-analysis compared the results of published animal studies and identified that improved outcomes are reported with the use of hybrid (mixed) scaffolds and cell seeding; however, the meta-analysis of these results were not in agreeement with the evaluation of descriptive results from the review. Therefore, further studies involving larger animals and novel scaffolds, and more transparent reproduction of previous studies are advisable.


Biomimetics

Biomimetics is a field that aims to produce materials and systems that replicate those present in nature. In the context of tissue engineering, this is a common approach used by engineers to create materials for these applications that are comparable to native tissues in terms of their structure, properties, and biocompatibility. Material properties are largely dependent on physical, structural, and chemical characteristics of that material. Subsequently, a biomimetic approach to system design will become significant in material integration, and a sufficient understanding of biological processes and interactions will be necessary. Replication of biological systems and processes may also be used in the synthesis of bio-inspired materials to achieve conditions that produce the desired biological material. Therefore, if a material is synthesized having the same characteristics of biological tissues both structurally and chemically, then ideally the synthesized material will have similar properties. This technique has an extensive history originating from the idea of using natural phenomenon as design inspiration for solutions to human problems. Many modern advancements in technology have been inspired by nature and natural systems, including aircraft, automobiles, architecture, and even industrial systems. Advancements in nanotechnology initiated the application of this technique to micro- and nano-scale problems, including tissue engineering. This technique has been used to develop synthetic bone tissues, vascular technologies, scaffolding materials and integration techniques, and functionalized nanoparticles.


Constructing neural networks in soft material

In 2018, scientists at Brandeis University reported their research on soft material embedded with chemical networks which can mimic the smooth and coordinated behavior of neural tissue. This research was funded by the U.S. Army Research Laboratory. The researchers presented an experimental system of neural networks, theoretically modeled as reaction-diffusion systems. Within the networks was an array of patterned reactors, each performing the Belousov-Zhabotinsky (BZ) reaction. These reactors could function on a nanoliter scale. The researchers state that the inspiration for their project was the movement of the blue ribbon eel. The eel's movements are controlled by electrical impulses determined by a class of neural networks called the
central pattern generator Central pattern generators (CPGs) are self-organizing biological neural circuits that produce rhythmic outputs in the absence of rhythmic input. They are the source of the tightly-coupled patterns of neural activity that drive rhythmic and stereo ...
.  Central Pattern Generators function within the autonomic nervous system to control bodily functions such as respiration, movement, and
peristalsis Peristalsis ( , ) is a radially symmetrical contraction and relaxation of muscles that propagate in a wave down a tube, in an anterograde direction. Peristalsis is progression of coordinated contraction of involuntary circular muscles, whic ...
. Qualities of the reactor that were designed were the network topology, boundary conditions, initial conditions, reactor volume, coupling strength, and the synaptic polarity of the reactor (whether its behavior is inhibitory or excitatory). A BZ emulsion system with a solid elastomer
polydimethylsiloxane Polydimethylsiloxane (PDMS), also known as dimethylpolysiloxane or dimethicone, belongs to a group of polymeric organosilicon compounds that are commonly referred to as silicones. PDMS is the most widely used silicon-based organic polymer, as its ...
(PDMS) was designed. Both light and bromine permeable PDMS have been reported as viable methods to create a pacemaker for neural networks.


Market

The history of the tissue engineering market can be divided into three major parts. The time before the crash of the biotech market in the early 2000s, the crash and the time afterward.


Beginning

Most early progress in tissue engineering research was done in the US. This is due to less strict regulations regarding stem cell research and more available funding than in other countries. This leads to the creation of academic startups many of them coming from Harvard or
MIT The Massachusetts Institute of Technology (MIT) is a private land-grant research university in Cambridge, Massachusetts. Established in 1861, MIT has played a key role in the development of modern technology and science, and is one of the m ...
. Examples are BioHybrid Technologies whose founder, Bill Chick, went to
Harvard Medical School Harvard Medical School (HMS) is the graduate medical school of Harvard University and is located in the Longwood Medical Area of Boston, Massachusetts. Founded in 1782, HMS is one of the oldest medical schools in the United States and is consi ...
and focused on the creation of artificial pancreas. Another example would be Organogenesis Inc. whose founder went to MIT and worked on skin engineering products. Other companies with links to the MIT are TEI Biosciences, Therics and Guilford Pharmaceuticals. The renewed interest in biotechnologies in the 1980s leads to many private investors investing in these new technologies even though the business models of these early startups were often not very clear and did not present a path to long term profitability. Government sponsors were more restrained in their funding as tissue engineering was considered a high-risk investment. In the UK the market got off to a slower start even though the regulations on stem cell research were not strict as well. This is mainly due to more investors being less willing to invest in these new technologies which were considered to be high-risk investments. Another problem faced by British companies was getting the
NHS The National Health Service (NHS) is the umbrella term for the publicly funded healthcare systems of the United Kingdom (UK). Since 1948, they have been funded out of general taxation. There are three systems which are referred to using the " ...
to pay for their products. This especially because the NHS runs a cost-effectiveness analysis on all supported products. Novel technologies often do not do well in this respect. In Japan, the regulatory situation was quite different. First cell cultivation was only allowed in a hospital setting and second academic scientists employed by state-owned universities were not allowed outside employment until 1998. Moreover, the Japanese authorities took longer to approve new drugs and treatments than there US and European counterparts. For these reasons in the early days of the Japanese market, the focus was mainly on getting products that were already approved elsewhere in Japan and selling them. Contrary to the US market the early actors in Japan were mainly big firms or sub-companies of such big firms, such as J-TEC, Menicon and Terumo, and not small startups. After regulatory changes in 2014, which allowed cell cultivation outside of a hospital setting, the speed of research in Japan increased and Japanese companies also started to develop their own products. Japanese companies, such a
REPROCELL
and iPS Academia Japan, are currently working on iPS cell-related products.


Crash

Soon after the big boom, the first problems started to appear. There were problems getting products approved by the FDA and if they got approved there were often difficulties in getting insurance providers to pay for the products and getting it accepted by health care providers. For example, organogenesis ran into problems marketing its product and integrating its product in the health system. This partially due to the difficulties of handling living cells and the increased difficulties faced by physicians in using these products over conventional methods. Another example would be Advanced Tissue Sciences Dermagraft skin product which could not create a high enough demand without reimbursements from insurance providers. Reasons for this were $4000 price-tag and the circumstance that Additionally Advanced Tissue Sciences struggled to get their product known by physicians. The above examples demonstrate how companies struggled to make profit. This, in turn, lead investors to lose patience and stopping further funding. In consequence, several Tissue Engineering companies such as Organogenesis and Advanced Tissue Sciences filed for bankruptcy in the early 2000s. At this time, these were the only ones having commercial skin products on the market.


Reemergence

The technologies of the bankrupt or struggling companies were often bought by other companies which continued the development under more conservative business models. Examples of companies who sold their products after folding were Curis and Intercytex. Many of the companies abandoned their long-term goals of developing fully functional organs in favor of products and technologies that could turn a profit in the short run. Examples of these kinds of products are products in the cosmetic and testing industry. In other cases such as in the case of Advanced Tissue Sciences, the founders started new companies. In the 2010s the regulatory framework also started to facilitate faster time to market especially in the US as new centres and pathways were created by the FDA specifically aimed at products coming from living cells such as the
Center for Biologics Evaluation and Research The Center for Biologics Evaluation and Research (CBER) is one of six main centers for the U.S. Food and Drug Administration (FDA), which is a part of the U.S. Department of Health and Human Services. The current Director of CBER is Peter Marks, ...
. The first tissue engineering products started to get commercially profitable in the 2010s.


Regulation

In Europe, regulation is currently split into three areas of regulation:
medical device A medical device is any device intended to be used for medical purposes. Significant potential for hazards are inherent when using a device for medical purposes and thus medical devices must be proved safe and effective with reasonable assura ...
s,
medicinal product A medication (also called medicament, medicine, pharmaceutical drug, medicinal drug or simply drug) is a drug used to diagnose, cure, treat, or prevent disease. Drug therapy (pharmacotherapy) is an important part of the medical field and rel ...
s, and
biologics A biopharmaceutical, also known as a biological medical product, or biologic, is any pharmaceutical drug product manufactured in, extracted from, or semisynthesized from biological sources. Different from totally synthesized pharmaceuticals, th ...
. Tissue engineering products are often of hybrid nature, as they are often composed of cells and a supporting structure. While some products can be approved as medicinal products, others need to gain approval as medical devices. Derksen explains in her thesis that tissue engineering researchers are sometimes confronted with regulation that does not fit the characteristics of tissue engineering. New regulatory regimes have been observed in Europe that tackle these issues. An explanation for the difficulties in finding regulatory consensus in this matter is given by a survey conducted in the UK. The authors attribute these problems to the close relatedness and overlap with other technologies such as
xenotransplantation Xenotransplantation (''xenos-'' from the Greek meaning "foreign" or strange), or heterologous transplant, is the transplantation of living cells, tissues or organs from one species to another. Such cells, tissues or organs are called xenograft ...
. It can therefore not be handled separately by regulatory bodies. Regulation is further complicated by the ethical controversies associated with this and related fields of research (e.g. stem cells controversy, ethics of organ transplantation). The same survey as mentioned above shows on the example of autologous cartilage transplantation that a specific technology can be regarded as 'pure' or 'polluted' by the same social actor. Two regulatory movements are most relevant to tissue engineering in the
European Union The European Union (EU) is a supranational political and economic union of member states that are located primarily in Europe. The union has a total area of and an estimated total population of about 447million. The EU has often been de ...
. These are the Directive on standards of quality and safety for the sourcing and processing of human tissues which was adopted by the European Parliament in 2004 and a proposed cells and the Human Tissue- Engineered Products regulation. The latter was developed under the abscise of the European Commission DG Enterprise and presented in Brussels in 2004.


See also

*
Biomedical engineering Biomedical engineering (BME) or medical engineering is the application of engineering principles and design concepts to medicine and biology for healthcare purposes (e.g., diagnostic or therapeutic). BME is also traditionally logical sciences ...
* Biological engineering *
Biomolecular engineering Biomolecular engineering is the application of engineering principles and practices to the purposeful manipulation of molecules of biological origin. Biomolecular engineers integrate knowledge of biological processes with the core knowledge of chemi ...
*
Biochemical engineering Biochemical engineering, also known as bioprocess engineering, is a field of study with roots stemming from chemical engineering and biological engineering. It mainly deals with the design, construction, and advancement of unit processes that inv ...
* Cell engineering *
Chemical engineering Chemical engineering is an engineering field which deals with the study of operation and design of chemical plants as well as methods of improving production. Chemical engineers develop economical commercial processes to convert raw materials int ...
* ECM Biomaterial * In vivo bioreactor *
Induced stem cells Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotent – ...
* Molecular processor * Molecular self-assembly * Muscle tissue engineering *
National Institutes of Health The National Institutes of Health, commonly referred to as NIH (with each letter pronounced individually), is the primary agency of the United States government responsible for biomedical and public health research. It was founded in the late ...
*
National Science Foundation The National Science Foundation (NSF) is an independent agency of the United States government that supports fundamental research and education in all the non-medical fields of science and engineering. Its medical counterpart is the National ...
* Quality control in tissue engineering *
Regeneration in humans Regeneration in humans is the regrowth of lost tissues or organs in response to injury. This is in contrast to wound healing, or partial regeneration, which involves closing up the injury site with some gradation of scar tissue. Some tissues such ...
* Soft tissues *
Tissue Engineering and Regenerative Medicine International Society Tissue Engineering and Regenerative Medicine International Society is an international learned society dedicated to tissue engineering and regenerative medicine Regenerative medicine deals with the "process of replacing, engineering or regenera ...
* Tissue engineering of heart valves *
Xenotransplantation Xenotransplantation (''xenos-'' from the Greek meaning "foreign" or strange), or heterologous transplant, is the transplantation of living cells, tissues or organs from one species to another. Such cells, tissues or organs are called xenograft ...


Notes


References

* * * * * * * * * * * *


External links


Cell-Based Bone Tissue Engineering

Clinical Tissue Engineering Center
State of Ohio Initiative for Tissue Engineering (National Center for Regenerative Medicine)
Organ Printing
Multi-site NSF-funded initiative
LOEX Center
Université Laval Initiative for Tissue Engineering {{Authority control Cell culture techniques Biomedical engineering