Tissue engineering

Encyclopedia : T : TI : TIS : Tissue engineering

Tissue engineering can perhaps be best defined as the use of a combination of cells, engineering materials, and suitable biochemical factors to improve or replace biological functions in an effort to affect the advancement of medicine. Probably the first definition of tissue engineering was by Langer and Vacanti who stated it to be "an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ". MacArthur and Oreffo (as cited in "References") defined tissue engineering 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, and/or enhancement of tissue function." These more general definitions are driven in part by recent scientific progress with completely autologous approaches. That is, many groups (Nicolas L'Heureux at Cytograft Tissue Engineering, Julie Campbell at University of Queensland etc, Loex laboratories at the Universite of Laval etc.) are demonstrating functional tissue engineered devices/organs without using synthetic biomaterials/scaffolds. These recent approaches are clearly based more on an understanding of cell biology than materials science.

In 2003, the NSF published a report titled ["The Emergence of Tissue Engineering as a Research Field"], which gives a thorough description of the history of this field.

While the semi-official definition of tissue engineering covers a broad range of applications, in practice the term has come to represent applications that repair or replace structural tissues (i.e., bone, cartilage, blood vessels, bladder, etc...). These are tissues that function by virtue of their mechanical properties. A closely related (and older) field is cell transplantation. This field is concerned with the transplantation of cells that perform a specific biochemical function (e.g., an artificial pancreas, or an artificial liver). The term regenerative medicine is often used synonymously with tissue engineering, although those involved in regenerative medicine place more emphasis on the use of stem cells to produce tissues.

A typical tissue engineering solution consists of a number of parts as alluded to above. This article will discuss each part in turn, along with its implications.

Contents

1 Cells
2 Engineering materials
3 Synthesis of tissue engineering scaffolds
4 Assembly methods
5 Recent developments
6 See also
7 External links
8 Agencies that support tissue engineering research
9 Notes
10 References

Cells

Stained cells in culture

Tissue engineering solves problems by using living cells as engineering materials. These could be artificial skin that includes living fibroblasts, cartilage repaired with living chondrocytes, or other types of cells used in other ways.
Cells became available as engineering materials when scientists at Geron Corp. discovered how to extend telomeres in 1998, producing an immortalized cell line. Before this, laboratory cultures of healthy, noncancerous mammalian cells would only divide a fixed number of times, up to the Hayflick limit.
From fluid tissues such as blood, cells are extracted by bulk methods, usually centrifugation or apheresis.
From solid tissues, extraction is more difficult. Usually the tissue is minced, and then digested with the enzymes trypsin or collagenase to remove the extracellular matrix that holds the cells. After that, the cells are free floating, and extracted using centrifugation or aspheresis.
Digestion with trypsin is very dependent on temperature. Higher temperatures digest the matrix faster, but create more damage. Collagenase is less temperature dependent, and damages fewer cells, but takes longer and is a more expensive reagent.
Cells are often categorized by their source:

Autologous cells are obtained from the same individual to which they will be reimplanted. Autologous cells have the fewest problems with rejection and pathogen transmission, however in some cases might not be available. For example in genetic disease suitable autologous cells are not available. Also very ill or elderly persons, as well as patients suffering from severe burns, may not have sufficient quantities of autologous cells to establish useful cell lines. Moreover since this category of cells needs to be harvested from the patient, there are also some concerns related to the necessity of performing such surgical operations that might lead to donor site infection or chronic pain. Autologous cells also must be cultured from samples before they can be used: this takes time, so autologous solutions may not be very quick. Recently there as been a trend towards the use of mesenchymal stem cells from bone marrow and fat. These cells can differentiate into a variety of tissue types, including bone, cartilage, fat, and nerve. A large number of cells can be easily and quickly isolated from fat, thus opening the potential for large numbers of cells to be quickly and easily obtained. Several companies have been founded to capitalize on this technology, the most successful at this time being [Cytori Therapeutics].

Mouse embryonic stem cells. [More lab photos]

Allogenic cells come from the body of a donor of the same species. While there are some ethical constraints to the use of human cells for in vitro studies, the employment of dermal fibroblasts from human foreskin has been demonstrated to be immunologically safe and thus a viable choice for tissue engineering of skin.

Xenogenic cells are those isolated from individuals of another species. In particular animal cells have been used quite extensively in experiments aimed at the construction of cardiovascular implants.

Syngeneic or 'isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

Primary cells are from an organism.

Secondary cells are from a cell bank.

Stem cells (see main article: stem cell) are undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells. According to their source stem cells are divided into "adult" and "embryonic" stem cells, the first class being multipotent and the latter mostly pluripotent; some cells are totipotent, in the earliest stages of the embryo. While there is still a large ethical debate related with the use of embryonic stem cells, it is thought that stem cells may be useful for the repair of diseased or damaged tissues, or may be used to grow new organs.

Engineering materials

Cells as found above are generally implanted or 'seeded' into an artificial structure capable of supporting three-dimensional tissue formation. These scaffolds are often critical, both ex vivo as well as in vivo, to recapitulating the in vivo milieu and allowing cells to influence their own microenvironments. Such devices, usually referred to as scaffolds, 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

To achieve the goal of tissue reconstruction, scaffolds must meet some specific requirements. A high porosity and an adequate pore size are necessary to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients. Biodegradability is essential since scaffolds need to be absorbed by the surrounding tissues without the necessity of a 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 neotissue, newly formed tissue which will take over the mechanical load. Injectability is also important for clinical uses.

Many different materials (natural and synthetic, biodegradable and permanent) have been investigated. Most of these materials have been known in the medical field before the advent of tissue engineering as a research topic, being already employed as bioresorbable sutures. Examples of these materials are collagen or some linear aliphatic polyesters.

New biomaterials have been engineered to have ideal properties and functional customization: injectability, synthetic manufacture, biocompatability, non-immunogenicity, transparency, nano-scale fibers, low concentration, resorption rates, etc. PuraMatrix, originating from the MIT labs of Zhang, Rich, Grodzinsky and Langer is one of these new biomimetic scaffold families which has now been commercialized and is impacting clinical tissue engineering.

A commonly used synthetic material is PLA - polylactic acid. This is a polyester which degrades within the human body to form lactic acid, a naturally occurring chemical which is easily removed from the body. Similar materials are polyglycolic acid (PGA) and polycaprolactone (PCL): their degradation mechanism is similar to that of PLA, but they exhibit respectively a faster and a slower rate of degradation compared to PLA.

Scaffolds may also be constructed from natural materials: in particular different derivatives of the extracellular matrix have been studied to evaluate their ability to support cell growth. Proteic materials, such as collagen or fibrin, and polysaccharidic materials, like chitosan or glycosaminoglycans (GAGs), have all proved suitable in terms of cell compatibility, but some issues with potential immunogenicity still remains. Among GAGs hyaluronic acid, possibly in combination with cross linking agents (e.g. glutaraldehyde, water soluble carbodiimide, etc...), is one of the possible choices as scaffold material. Functionalized groups of scaffolds may be useful in the delivery of small molecules (drugs) to specific tissues.

Synthesis of tissue engineering scaffolds

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

Nanofiber Self-Assembly: Molecular self-assembly is one of the few methods to create biomaterials with properties similar in scale and chemistry to that of the natural in vivo extracellular matrix (ECM). Moreover, these hydrogel scaffolds have shown superior in vivo toxicology and biocompatibility compared with traditional macroscaffolds and animal-derived materials. PuraMatrix synthetic peptide hydrogels exemplify this category.

Textile technologies: these techniques include all the approaches that have been successfully employed for the preparation of non-woven meshes of different polymers. In particular non-woven polyglycolide 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 of obtaining high porosity and regular pore size.

Solvent Casting & Particulate Leaching (SCPL): this approach allows the preparation of porous structures with regular porosity, but with a 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 case of sodium chloride, saccharose and gelatin or an aliphatic solvent like hexane for 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 necessity 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 meas of compression molding using a heated mold. The discs are then placed in a chamber where 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 related to 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. 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 to remove the dispersed water and the solvent, thus leaving a solidified, porous polymeric structure. While emulsification and freeze-drying allows a faster preparation if 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 that are cast into a mold, frozen with liquid nitrogen then liophylized.

Liquid-liquid phase separation: similar to the previous technique, this procedure requires the use of a solvent with a low melting point that is easy to sublime. For example dioxane 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.

CAD/CAM Technologies: since most of the above described approaches are limited when it comes to the control of porosity and pore size, computer assisted design and manufacturing techniques have been introduced to tissue engineering. First a three-dimensional structure is designed using CAD software, then the scaffold is realized by using ink-jet printing of polymer powders or through Fused Deposition Modeling of a polymer melt.

Assembly methods

One of the continuing, persistent problems with 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, and/or function properly.

Self-assembly may play an important role here, both from the perspective of encapsulating cells and proteins, as well as creating scaffolds on the right physical scale for engineered tissue constructs and cellular ingrowth.

It might be possible to print organs, or possibly entire organisms. A recent innovative method of construction uses an ink-jet mechanism to print precise layers of cells in a matrix of thermoreversable 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.Mironov et al., 2003

Recent developments

Dr. Anthony Atala of Wake Forest University has successfully implanted artificially grown bladders into seven human test subjects as part of a long-term experiment, with demonstrated positive benefits to the recipients thus far. [Doctors grow organs from patients' own cells], CNN, April 3, 2006

Lab-grown tissue was successfully used to repair knee cartilage.[link]

External links

[Tissue Engineering], an authoritative peer-reviewed journal that jointly applies the principles of engineering and life sciences.
[Institute for Chemical Process and Environmental Technology] Tissue engineered (TE) corneas
[Organ Printing] Multi-site NSF-funded initiative
[Pittsburgh Tissue Engineering Initiative]*[Tissue Engineering] A peer-reviewed journal focusing on the engineering of new biologic tissues
[Tissue engineering]
[Tissue Engineering Pages]
[Self-Assembled Nanofiber Scaffolds: peer reviewed articles]
[Clinical Tissue Engineering Center] State of Ohio Initiative for Tissue Engineering -- Part of the National Center for Regenerative Medicine

Agencies that support tissue engineering research

Notes

References

Langer, R & Vacanti JP, Tissue engineering. Science 260, 920-6; 1993 (cited more than 1000 times!)
Davis, M.E., et al., Injectable Self-Assembling Peptide Nanofibers Create Intramyocardial Microenvironments for Endothelial Cells. Circulation 111:442-450 (2005).
Ma, Peter X.: Scaffolds for tissue fabrication - Materials Today, May 2004, 30-40.
Holmes, T.C., et al., Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. PNAS USA 97 :6728 (2000).
Semino, C.E., et al., Entrapment of migrating hippocampal neural cells in 3D peptide nanofiber scaffold. Tissue Engineering 10:643 (2004).
URL accessed on April 28, 2006
Mironov, V., Boland, T., Trusk, T., Forgacs, G. & Markwald, R.R., Organ printing: computer-aided jet-based 3D tissue engineering. Trends in Biotechnology 21, 157-61; 2003.
URL accessed on April 28, 2006
Nerem, R.M., in Principles of Tissue Engineering. Lanza, Langer and J Vacanti (eds), 2000

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