Editing Tissue engineering (section)

=== Synthesis ===
{{More citations needed section|date=August 2024}}
[[File:Gefäßprothese.JPG|thumb|Tissue engineered vascular graft]]
[[File:Herzklappe.JPG|thumb|Tissue engineered heart valve]]

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.<ref name="cassidy">{{cite journal | vauthors = Cassidy JW | title = Nanotechnology in the Regeneration of Complex Tissues | journal = Bone and Tissue Regeneration Insights | volume = 5 | pages = 25–35 | date = November 2014 | pmid = 26097381 | pmc = 4471123 | doi = 10.4137/BTRI.S12331 | url = https://www.repository.cam.ac.uk/bitstream/1810/247086/1/Cassidy%202014%20Bone%20and%20Tissue%20Regeneration%20Insights.pdf }}</ref> Moreover, these hydrogel scaffolds have shown superiority in <!-- not a mistake -->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 textiles|non-woven meshes]] of different [[polymer]]s. 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====
[[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 wax|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.

====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 [[carbon dioxide|CO<sub>2</sub>]] 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]]. 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 drying|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]].

====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]] 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.<ref>{{cite journal | vauthors = Nam YS, Park TG | title = Biodegradable polymeric microcellular foams by modified thermally induced phase separation method | journal = Biomaterials | volume = 20 | issue = 19 | pages = 1783–90 | date = October 1999 | pmid = 10509188 | doi = 10.1016/S0142-9612(99)00073-3 }}</ref>

==== 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 rate{{snd}}researchers 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 produce 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''.<ref>{{cite web| vauthors = Simon EM |date=1988 |title=NIH Phase I Final Report: Fibrous Substrates for Cell Culture (R3RR03544A) |url=https://www.researchgate.net/publication/317053872|access-date=2017-05-22 |website=ResearchGate}}</ref>

====CAD/CAM technologies====
Because most of the above techniques are limited when it comes to the control of porosity and pore size, [[Computer-aided design|computer assisted design]] and [[Computer-aided manufacturing|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.<ref>{{cite journal |vauthors=Melchels F, Wiggenhauser PS, Warne D, Barry M, Ong FR, Chong WS, Hutmacher DW, Schantz JT |display-authors=6 |title=CAD/CAM-assisted breast reconstruction |journal=Biofabrication |volume=3 |issue=3 |pages=034114 |date=September 2011 |pmid=21900731 |doi=10.1088/1758-5082/3/3/034114 |url=https://eprints.qut.edu.au/46842/15/46842.pdf |bibcode=2011BioFa...3c4114M|s2cid=206108959 }}</ref> The scaffold is then realized by using ink-jet printing of polymer powders or through [[Fused Deposition Modeling]] of a polymer melt.<ref name=Elisseeff05>{{cite book| vauthors=Elisseeff J, Ma PX |title=Scaffolding in Tissue Engineering |publisher=CRC |location=Boca Raton |date=2005 |isbn=978-1-57444-521-3}}</ref>

A 2011 study by El-Ayoubi et al. investigated "3D-plotting technique to produce ([[biocompatible]] 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]] replacement as an "alternative to conventional tissue repair".<ref name="ReferenceA">{{cite journal | vauthors = Lee GY, Kenny PA, Lee EH, Bissell MJ | title = Three-dimensional culture models of normal and malignant breast epithelial cells | journal = Nature Methods | volume = 4 | issue = 4 | pages = 359–65 | date = April 2007 | pmid = 17396127 | pmc = 2933182 | doi = 10.1038/nmeth1015 }}</ref> 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.<ref name="ReferenceA"/>

====Laser-assisted bioprinting====
In a 2012 study,<ref name="Lai, Y 2011">{{cite journal | vauthors = Lai Y, Asthana A, Kisaalita WS | title = Biomarkers for simplifying HTS 3D cell culture platforms for drug discovery: the case for cytokines | journal = Drug Discovery Today | volume = 16 | issue = 7–8 | pages = 293–97 | date = April 2011 | pmid = 21277382 | doi = 10.1016/j.drudis.2011.01.009 }}</ref> 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.<ref name="Lai, Y 2011"/> The investigation was successful, the researchers foresee that "generated tissue constructs might be used for [[in vivo]] testing by implanting them into [[animal models]]" (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]], hair follicle cells) into the printed cell construct, the behavior of these cells in a 3D in vitro [[Microenvironment (biology)|microenvironment]] similar to their natural one can be analyzed", which is useful for drug discovery and [[toxicology]] studies.<ref name="Lai, Y 2011"/>

====Self-assembled recombinant spider silk nanomembranes====
Gustafsson et al.<ref>{{cite journal | vauthors = Gustafsson L, Tasiopoulos CP, Jansson R, Kvick M, Duursma T, Gasser TC, van der Wijngaart W, Hedhammar M |title=Recombinant Spider Silk Forms Tough and Elastic Nanomembranes that are Protein-Permeable and Support Cell Attachment and Growth |journal=Advanced Functional Materials |date=16 August 2020 |volume=30 |issue=40 |pages=2002982 |doi=10.1002/adfm.202002982 |doi-access=free }}</ref> demonstrated free‐standing, bioactive membranes of cm-sized area, but only 250&nbsp;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.<ref>{{cite journal | vauthors = Tasiopoulos CP, Gustafsson L, van der Wijngaart W, Hedhammar M | title = Fibrillar Nanomembranes of Recombinant Spider Silk Protein Support Cell Co-culture in an ''In Vitro'' Blood Vessel Wall Model | journal = ACS Biomaterials Science & Engineering | volume = 7 | issue = 7 | pages = 3332–3339 | date = July 2021 | pmid = 34169711 | pmc = 8290846 | doi = 10.1021/acsbiomaterials.1c00612 | doi-access = free }}</ref>

==== Tissue engineering ''in situ'' ====
''In situ'' tissue regeneration is defined as the implantation of biomaterials (alone or in combination with cells and/or biomolecules) into the tissue defect, using the surrounding microenvironment of the organism as a natural bioreactor.<ref>{{Cite journal |last1=Dias |first1=Juliana R. |last2=Ribeiro |first2=Nilza |last3=Baptista-Silva |first3=Sara |last4=Costa-Pinto |first4=Ana Rita |last5=Alves |first5=Nuno |last6=Oliveira |first6=Ana L. |date=2020 |title=In situ Enabling Approaches for Tissue Regeneration: Current Challenges and New Developments |journal=Frontiers in Bioengineering and Biotechnology |volume=8 |pages=85 |doi=10.3389/fbioe.2020.00085 |issn=2296-4185 |pmc=7039825 |pmid=32133354 |doi-access=free }}</ref> This approach has found application in bone regeneration,<ref>{{Cite journal |last1=Malek-Khatabi |first1=A. |last2=Javar |first2=H.A. |last3=Dashtimoghadam |first3=E. |last4=Ansari |first4=S. |last5=Hasani-Sadrabadi |first5=M.M. |last6=Moshaverinia |first6=A. |date=2020 |title=In situ bone tissue engineering using gene delivery nanocomplexes |url=https://pubmed.ncbi.nlm.nih.gov/32160962 |journal=Acta Biomaterialia |volume=108 |pages=326–336 |doi=10.1016/j.actbio.2020.03.008 |issn=1878-7568 |pmid=32160962|s2cid=212679291 }}</ref> allowing the formation of cell-seeded constructs directly in the operating room.<ref>{{Cite journal |last1=Krasilnikova |first1=O.A. |last2=Baranovskii |first2=D.S. |last3=Yakimova |first3=A.O. |last4=Arguchinskaya |first4=N. |last5=Kisel |first5=A. |last6=Sosin |first6=D. |last7=Sulina |first7=Y. |last8=Ivanov |first8=S.A. |last9=Shegay |first9=P.V. |last10=Kaprin |first10=A.D. |last11=Klabukov |first11=I.D. |date=2022 |title=Intraoperative Creation of Tissue-Engineered Grafts with Minimally Manipulated Cells: New Concept of Bone Tissue Engineering In Situ |journal=Bioengineering  |volume=9 |issue=11 |pages=704 |doi=10.3390/bioengineering9110704 |issn=2306-5354 |pmc=9687730 |pmid=36421105 |doi-access=free }}</ref>