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Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocane HMX gave made as a a irradiation structure geographic to its Prior chloride in the continuum and strategy. Allogeneic 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 these isolated from individuals of another species. In particular animal cells have been used quite extensively in experiments aimed at the construction of cardiovascular implants. Syngenic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models. Stem cells 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 another alternative source - induced stem cells may be useful for the repair of diseased or damaged tissues, or may be used to grow new organs. 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 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 , 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 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. Many different materials natural and synthetic, biodegradable and permanent have been investigated.
Examples of these materials are collagen and some polyesters. New biomaterials have been engineered to have ideal properties and functional customization: injectability, synthetic manufacture, biocompatibility , 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.
While these materials have well maintained mechanical strength and structural integrity, they exhibit a hydrophobic nature. This hydrophobicity inhibits their biocompatibility, which makes them less effective for in vivo use as tissue scaffolding. By combining the two different types of materials, researchers are trying to create a synergistic relationship that produces a more biocompatible tissue scaffolding. 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. Functionalized groups of scaffolds may be useful in the delivery of small molecules drugs to specific tissues. Recently a range of nanocomposites biomaterials are fabricated by incorporating nanomaterials within the polymeric matrix to engineer bioactive scaffolds.
A study by Derda et al. Upon deconstruction, these sheets can be useful in cell-based high-throughput screening and drug discovery. 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. 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.
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 in obtaining high porosity and regular pore size. 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. 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 solvent like hexane 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. 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 CO 2 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. This technique does not require the use of a solid porogen like SCPL. First, a synthetic polymer is dissolved into a suitable solvent e.
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 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 that are cast into a mold, frozen with liquid nitrogen and then lyophilized.
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 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. Electrospinning is a highly versatile technique that can be used to produce continuous fibers from submicrometer to nanometer diameters.
In a typical electrospinning set-up, a solution is fed through a spinneret and a high voltage is applied to the tip. The buildup of electrostatic repulsion within the charged solution, causes it to eject a thin fibrous stream.
Biomedical Foams for Tissue Engineering Applications | NHBS Academic & Professional Books
A mounted collector plate or rod with an opposite or grounded charge draws in the continuous fibers, which arrive to form a highly porous network. The primary advantages of this technique are its simplicity and ease of variation. At a laboratory level, a typical electrospinning set-up only requires a high voltage power supply up to 30 kV , a syringe, a flat tip needle, and a conducting collector.
For these reasons, electrospinning has become a common method of scaffold manufacture in many labs. By modifying variables such as the distance to collector, magnitude of applied voltage, or solution flow rate—researchers can dramatically change the overall scaffold architecture. Historically, research on electrospun fibrous scaffolds dates back to at least the late s 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.
Because most of the above techniques 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.
The porosity can be tailored using algorithms within the software. A study by El-Ayoubi et al. In a study,  Koch et al. LaBP arranges small volumes of living cell suspensions in set high-resolution patterns. As of this study, only human skin tissue has been synthesized, though researchers project that "by integrating further cell types e. 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, or function properly. 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  and applications of the process have resulted in engineered cartilage approaching the strength of native tissue.
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. 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 3D neural network was achieved after day tissue culture. It might be possible to print organs, or possibly entire organisms using additive manufacturing techniques. A recent innovative method of construction uses an ink-jet mechanism to print precise layers of cells in a matrix of thermoreversible 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. 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 3D printer that can print the scaffolding and cells layer by layer into a working tissue sample or organ. The device is presented in a TED talk by Dr. Anthony Atala, M. Boyce Professor and Chair of the Department of Urology at Wake Forest University, in which a kidney is printed on stage during the seminar and then presented to the crowd.
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 3D 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 microstructured scaffolds in combination with Cho seeding may be successfully implemented for cartilage tissue engineering. In , using a 3-d scaffolding of Matrigel in various configurations, substantial pancreatic organoids was produced in vitro.
Clusters of small numbers of cells proliferated into 40, cells within one week. The clusters transform into cells that make either digestive enzymes or hormones like insulin , self-organizing into branched pancreatic organoids that resemble the pancreas. 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. 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 , pH , humidity , temperature , nutrients and osmotic pressure maintenance. Tissue engineered cultures also present additional problems in maintaining culture conditions. In standard cell culture, diffusion 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 factors , hormones , 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 from fluid flow, which is encountered in blood vessels.
Mechanical stimuli, such as pressure pulses seem to be beneficial to all kind of cardiovascular tissue such as heart valves, blood vessels or pericardium. A bioreactor in tissue engineering, as opposed to industrial bioreactors, 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 and oxygen or carbon dioxide concentration but can extend to all kinds of biological, chemical or mechanical stimuli.
Therefore, there are systems that may include the application of forces or stresses to the tissue or even of electric current in two- or three-dimensional setups. In academic and industry research facilities, it is typical for bioreactors to be developed to replicate the specific physiological environment of the tissue being grown e. There are a variety of Bioreactors 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 chamber is part of a larger device that rotates to ensure equal cell growth in each direction across three dimensions.
QuinXell Technologies from Singapore has developed a bioreactor known as the 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 rotation; specifically to mimic the rotation of the fetus in the womb; which provides a conducive environment for the growth of tissues.
MC2 Biotek has also developed a bioreactor known as ProtoTissue  that uses gas exchange to maintain high oxygen levels within the cell chamber; improving upon previous bioreactors, because the higher oxygen levels help the cell grow and undergo normal cell respiration. 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.
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Various cell types were inserted into the ECM core, including myocytes , endothelial cells , nerve cell fibers, and epithelial cell fibers.