Editing Tissue engineering (section)

== 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 [[dimension|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: allowing cell attachment and migration, delivering and retaining cells and biochemical factors, enabling diffusion of vital cell nutrients and expressed products, and exerting certain mechanical and biological influences to modify the behaviour of the cell phase.{{cn|date=April 2025}}

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.<ref name="pmid19623015">{{cite journal |vauthors=Mertsching H, Schanz J, Steger V, Schandar M, Schenk M, Hansmann J, Dally I, Friedel G, Walles T |display-authors=6 |title=Generation and transplantation of an autologous vascularized bioartificial human tissue |journal=Transplantation |volume=88 |issue=2 |pages=203–210 |date=July 2009 |pmid=19623015 |doi=10.1097/TP.0b013e3181ac15e1 |s2cid=46083673 |author-link9=Thorsten Walles|doi-access=free }}</ref>
[[File:Kohlenstoffnanoroehre Animation.gif|thumb|left|This animation of a rotating [[carbon nanotube]] shows its 3D structure. Carbon nanotubes are among the numerous candidates for tissue engineering scaffolds since they are [[biocompatible]], resistant to [[biodegrade|biodegradation]] and can be functionalized with [[biomolecule]]s. However, the possibility of toxicity with non-biodegradable nano-materials is not fully understood.<ref>{{cite journal |vauthors=Newman P, Minett A, Ellis-Behnke R, Zreiqat H |title=Carbon nanotubes: their potential and pitfalls for bone tissue regeneration and engineering |journal=Nanomedicine |volume=9 |issue=8 |pages=1139–1158 |date=November 2013 |pmid=23770067 |doi=10.1016/j.nano.2013.06.001}}</ref>]]
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. [[Biodegradation|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.{{cn|date=April 2025}}

=== Materials ===
Material selection is an essential aspect of producing a scaffold.&nbsp; The materials utilized can be natural or synthetic and can be biodegradable or non-biodegradable. Additionally, they must be biocompatible, meaning that they do not cause any adverse effects to cells.<ref>{{cite book|title=Frontiers in tissue engineering|date=1998|publisher=Pergamon| vauthors = Patrick CW, Mikos AG, McIntire LV |author3-link= Larry McIntire |isbn=978-0-08-042689-1 |edition=1st|location=Oxford, UK|oclc=162130841}}</ref> Silicone, for example, is a synthetic, non-biodegradable material commonly used as a drug delivery material,<ref>{{cite journal | vauthors = Stewart SA, Domínguez-Robles J, Donnelly RF, Larrañeta E | title = Implantable Polymeric Drug Delivery Devices: Classification, Manufacture, Materials, and Clinical Applications | journal = Polymers | volume = 10 | issue = 12 | pages = 1379 | date = December 2018 | pmid = 30961303 | pmc = 6401754 | doi = 10.3390/polym10121379 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Kajihara M, Sugie T, Maeda H, Sano A, Fujioka K, Urabe Y, Tanihara M, Imanishi Y | display-authors = 6 | title = Novel drug delivery device using silicone: controlled release of insoluble drugs or two kinds of water-soluble drugs | journal = Chemical & Pharmaceutical Bulletin | volume = 51 | issue = 1 | pages = 15–19 | date = January 2003 | pmid = 12520121 | doi = 10.1248/cpb.51.15 | doi-access = free }}</ref> while gelatin is a biodegradable, natural material commonly used in cell-culture scaffolds<ref>{{cite journal | vauthors = Afewerki S, Sheikhi A, Kannan S, Ahadian S, Khademhosseini A | title = Gelatin-polysaccharide composite scaffolds for 3D cell culture and tissue engineering: Towards natural therapeutics | journal = Bioengineering & Translational Medicine | volume = 4 | issue = 1 | pages = 96–115 | date = January 2019 | pmid = 30680322 | pmc = 6336672 | doi = 10.1002/btm2.10124 }}</ref><ref>{{cite journal| vauthors = Martin CA, Radhakrishnan S, Nagarajan S, Muthukoori S, Dueñas JM, Ribelles JL, Lakshmi BS, Nivethaa EA, Gómez-Tejedor JA, Reddy MS, Sellathamby S | display-authors = 6 |date=2019|title=An innovative bioresorbable gelatin based 3D scaffold that maintains the stemness of adipose tissue derived stem cells and the plasticity of differentiated neurons|journal=RSC Advances|language=en|volume=9|issue=25|pages=14452–64|doi=10.1039/C8RA09688K| pmid = 35519343 | pmc = 9064131 | bibcode = 2019RSCAd...914452M |issn=2046-2069|doi-access=free}}</ref><ref>{{cite journal| vauthors = Takagi Y, Tanaka S, Tomita S, Akiyama S, Maki Y, Yamamoto T, Uehara M, Dobashi T |date=2017|title=Preparation of gelatin scaffold and fibroblast cell culture |journal=Journal of Biorheology |volume=31|issue=1|pages=2–5|doi=10.17106/jbr.31.2|issn=1867-0466|doi-access=free |url=https://www.jstage.jst.go.jp/article/jbr/31/1/31_2/_article}}</ref>

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.<ref>{{cite journal | vauthors = Roohani-Esfahani SI, Newman P, Zreiqat H | title = Design and Fabrication of 3D printed Scaffolds with a Mechanical Strength Comparable to Cortical Bone to Repair Large Bone Defects | journal = Scientific Reports | volume = 6 | issue = 1 | pages = 19468 | date = January 2016 | pmid = 26782020 | pmc = 4726111 | doi = 10.1038/srep19468 | bibcode = 2016NatSR...619468R }}</ref><ref>{{cite journal | vauthors = Nokoorani YD, Shamloo A, Bahadoran M, Moravvej H | title = Fabrication and characterization of scaffolds containing different amounts of allantoin for skin tissue engineering | journal = Scientific Reports | volume = 11 | issue = 1 | pages = 16164 | date = August 2021 | pmid = 34373593 | pmc = 8352935 | doi = 10.1038/s41598-021-95763-4 | bibcode = 2021NatSR..1116164N }}</ref>

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. [[Polylactide|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 [[Polyglycolide|polyglycolic acid]] (PGA) and [[polycaprolactone]] (PCL): their degradation mechanism is similar to that of PLA, &nbsp;but PCL degrades slower and PGA degrades faster.{{citation needed|date=December 2020}} 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.<ref>{{cite journal | vauthors = Gentile P, Chiono V, Carmagnola I, Hatton PV | title = An overview of poly(lactic-co-glycolic) acid (PLGA)-based biomaterials for bone tissue engineering | journal = International Journal of Molecular Sciences | volume = 15 | issue = 3 | pages = 3640–59 | date = February 2014 | pmid = 24590126 | pmc = 3975359 | doi = 10.3390/ijms15033640 | doi-access = free }}</ref>

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. Protein based materials – such as collagen, or [[fibrin]], and polysaccharidic materials- like [[chitosan]]<ref>{{cite journal | vauthors = Park JH, Schwartz Z, Olivares-Navarrete R, Boyan BD, Tannenbaum R | title = Enhancement of surface wettability via the modification of microtextured titanium implant surfaces with polyelectrolytes | journal = Langmuir | volume = 27 | issue = 10 | pages = 5976–85 | date = May 2011 | pmid = 21513319 | pmc = 4287413 | doi = 10.1021/la2000415 }}</ref> or [[glycosaminoglycan]]s (GAGs), have all proved suitable in terms of cell compatibility. Among GAGs, [[Hyaluronan|hyaluronic acid]], possibly in combination with cross linking agents (e.g. [[glutaraldehyde]], [[Ethyl(dimethylaminopropyl) carbodiimide|water-soluble carbodiimide]], etc.), is one of the possible choices as scaffold material.
Due to the covalent attachment of thiol groups to these polymers, they can crosslink via disulfide bond formation.<ref>{{cite journal | vauthors = Leichner C, Jelkmann M, Bernkop-Schnürch A | title = Thiolated polymers: Bioinspired polymers utilizing one of the most important bridging structures in nature | journal = Advanced Drug Delivery Reviews | volume = 151-152 | pages = 191–221 | date = 2019 | pmid = 31028759 | doi = 10.1016/j.addr.2019.04.007 | s2cid = 135464452 }}</ref> The use of thiolated polymers ([[thiomer]]s) as scaffold material for tissue engineering was initially introduced at the 4th Central European Symposium on Pharmaceutical Technology in [[Vienna]] 2001.<ref>{{cite journal | vauthors = Kast CE, Frick W, Losert U, Bernkop-Schnürch A | title = Chitosan-thioglycolic acid conjugate: a new scaffold material for tissue engineering? | journal = International Journal of Pharmaceutics | volume = 256 | issue = 1–2 | pages = 183–189 | date = April 2003 | pmid = 12695025 | doi = 10.1016/S0378-5173(03)00076-0 }}</ref> As thiomers are biocompatible, exhibit cellular mimicking properties and efficiently support proliferation and differentiation of various cell types, they are extensively used as scaffolds for tissue engineering.<ref>{{cite journal | vauthors = Bae IH, Jeong BC, Kook MS, Kim SH, Koh JT | title = Evaluation of a thiolated chitosan scaffold for local delivery of BMP-2 for osteogenic differentiation and ectopic bone formation | journal = BioMed Research International | volume = 2013 | pages = 878930 | date = 2013 | pmid = 24024213 | pmc = 3760211 | doi = 10.1155/2013/878930 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Bian S, He M, Sui J, Cai H, Sun Y, Liang J, Fan Y, Zhang X | display-authors = 6 | title = The self-crosslinking smart hyaluronic acid hydrogels as injectable three-dimensional scaffolds for cells culture | journal = Colloids and Surfaces B: Biointerfaces | volume = 140 | pages = 392–402 | date = April 2016 | pmid = 26780252 | doi = 10.1016/j.colsurfb.2016.01.008 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Gajendiran M, Rhee JS, Kim K | title = Recent Developments in Thiolated Polymeric Hydrogels for Tissue Engineering Applications | journal = Tissue Engineering. Part B, Reviews | volume = 24 | issue = 1 | pages = 66–74 | date = February 2018 | pmid = 28726576 | doi = 10.1089/ten.TEB.2016.0442 }}</ref> Furthermore thiomers such as thiolated hyaluronic acid<ref>{{cite journal | vauthors = Bauer C, Jeyakumar V, Niculescu-Morzsa E, Kern D, Nehrer S | title = Hyaluronan thiomer gel/matrix mediated healing of articular cartilage defects in New Zealand White rabbits-a pilot study | journal = Journal of Experimental Orthopaedics | volume = 4 | issue = 1 | pages = 14 | date = December 2017 | pmid = 28470629 | pmc = 5415448 | doi = 10.1186/s40634-017-0089-1 | doi-access = free }}</ref> and thiolated [[chitosan]]<ref>{{cite journal | vauthors = Zahir-Jouzdani F, Mahbod M, Soleimani M, Vakhshiteh F, Arefian E, Shahosseini S, Dinarvand R, Atyabi F | display-authors = 6 | title = Chitosan and thiolated chitosan: Novel therapeutic approach for preventing corneal haze after chemical injuries | journal = Carbohydrate Polymers | volume = 179 | pages = 42–49 | date = January 2018 | pmid = 29111069 | doi = 10.1016/j.carbpol.2017.09.062 }}</ref> were shown to exhibit [[wound healing]] properties and are subject of numerous [[clinical trials]].<ref>{{Cite web|url=https://www.uibk.ac.at/pharmazie/phtech/drugdelivery/neue-bilder-2022/studies-in-humans---clinical-trials.pdf|title=Studies in humans clinical trials}}</ref> Additionally, a fragment of an extracellular matrix protein, such as the [[Arginylglycylaspartic acid|RGD peptide]], can be coupled to a non-bioactive material to promote cell attachment.<ref>{{cite journal | vauthors = Pomeroy JE, Helfer A, Bursac N | title = Biomaterializing the promise of cardiac tissue engineering | journal = Biotechnology Advances | volume = 42 | pages = 107353 | date = 2020-09-01 | pmid = 30794878 | pmc = 6702110 | doi = 10.1016/j.biotechadv.2019.02.009 }}</ref> 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.{{cn|date=April 2025}}

=== 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>