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",isbn:"978-1-83962-547-3",printIsbn:"978-1-83962-546-6",pdfIsbn:"978-1-83962-548-0",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"e5ba02fedd7c87f0ab66414f3b07de0c",bookSignature:" John P. Tiefenbacher",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10765.jpg",keywords:"Managing Urbanization, Managing Development, Managing Resource Use, Drought Management, Flood Management, Water Quality Monitoring, Air Quality Monitoring, Ecological Monitoring, Modeling Extreme Natural Events, Ecological Restoration, Restoring Environmental Flows, Environmental Management Perspectives",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"January 12th 2021",dateEndSecondStepPublish:"February 9th 2021",dateEndThirdStepPublish:"April 10th 2021",dateEndFourthStepPublish:"June 29th 2021",dateEndFifthStepPublish:"August 28th 2021",remainingDaysToSecondStep:"17 days",secondStepPassed:!1,currentStepOfPublishingProcess:2,editedByType:null,kuFlag:!1,biosketch:"A geospatial scholar working at the interface of natural and human systems, collaborating internationally on innovative studies about hazards and environmental challenges. Dr. Tiefenbacher has published more than 200 papers on a diverse array of topics that examine perception and behaviors with regards to the application of pesticides, releases of toxic chemicals, environments of the U.S.-Mexico borderlands, wildlife hazards, and the geography of wine.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"73876",title:"Dr.",name:"John P.",middleName:null,surname:"Tiefenbacher",slug:"john-p.-tiefenbacher",fullName:"John P. Tiefenbacher",profilePictureURL:"https://mts.intechopen.com/storage/users/73876/images/system/73876.jfif",biography:"Dr. John P. Tiefenbacher (Ph.D., Rutgers, 1992) is a professor of Geography at Texas State University. His research has focused on various aspects of hazards and environmental management. Dr. Tiefenbacher has published on a diverse array of topics that examine perception and behaviors with regards to the application of pesticides, releases of toxic chemicals, environments of the U.S.-Mexico borderlands, wildlife hazards, and the geography of wine. More recently his work pertains to spatial adaptation to climate change, spatial responses in wine growing regions to climate change, the geographies of viticulture and wine, artificial intelligence and machine learning to predict patterns of natural processes and hazards, historical ethnic enclaves in American cities and regions, and environmental adaptations of 19th century European immigrants to North America's landscapes.",institutionString:"Texas State University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"6",institution:{name:"Texas State University",institutionURL:null,country:{name:"United States of America"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"12",title:"Environmental Sciences",slug:"environmental-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"194667",firstName:"Marijana",lastName:"Francetic",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/194667/images/4752_n.jpg",email:"marijana@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophanides",surname:"Theophile",slug:"theophanides-theophile",fullName:"Theophanides Theophile"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"4816",title:"Face Recognition",subtitle:null,isOpenForSubmission:!1,hash:"146063b5359146b7718ea86bad47c8eb",slug:"face_recognition",bookSignature:"Kresimir Delac and Mislav Grgic",coverURL:"https://cdn.intechopen.com/books/images_new/4816.jpg",editedByType:"Edited by",editors:[{id:"528",title:"Dr.",name:"Kresimir",surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3621",title:"Silver Nanoparticles",subtitle:null,isOpenForSubmission:!1,hash:null,slug:"silver-nanoparticles",bookSignature:"David Pozo Perez",coverURL:"https://cdn.intechopen.com/books/images_new/3621.jpg",editedByType:"Edited by",editors:[{id:"6667",title:"Dr.",name:"David",surname:"Pozo",slug:"david-pozo",fullName:"David Pozo"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"70389",title:"Importance of Alginate Bioink for 3D Bioprinting in Tissue Engineering and Regenerative Medicine",doi:"10.5772/intechopen.90426",slug:"importance-of-alginate-bioink-for-3d-bioprinting-in-tissue-engineering-and-regenerative-medicine",body:'The main objective of three-dimensional (3D) printing is to print a living cell or to create a three-dimensional biomaterial’s scaffold. This innovative technology allows the reproducible and also the automated fabrication of three-dimensional useful living tissues by depositing biomaterials layer by layer with an accurate positioning of cells. This method allows to make a three-dimensional object and an accurate as well as scalable geometries that are not suggested by any approaches like two-dimensional cell cultures [1].
The choice of applying these 3D functional living tissues comes from fundamental research [2]. Learning about the cell-biomaterial interface at the nanoscale stage is vital in accommodating flaws in tissues, nanoparticle-cell connections and organ defects [3], toxicological analysis or drug investigation [4], and transplantation in living objects [5]. Because of the rising complexity required for these tissues, 3D bioprinting is facing a lot of challenges in all of manufacturing areas. For example, the cell-encapsulated materials are commonly observable to chemical cross linkers for extended periods of time during storage prior to printing, which can harm the cells. At the time of deposition, the mechanical stress generated by the printing itself can result in damage and injury to cell functioning by cell shearing or extrusion [6]. The instant new printing tissue is fabricated, because of the small vascularity of printed material; limited nutrients are supplied in 3D construct [7]. Usually, the requirements for a suitable cell-containing dispensable biomaterial or bioink are generally biocompatibility, exhaustive, biomimicry, printability, and essential mechanical properties. This is the main cause for the huge number of the manufacturers of commercially accessible 3D bioprinters—particularly extrusion-based 3D bioprinter, where hydrogel bioinks are recommended [8]. Particularly, hydrogels are unquestionably the most comprehensive biomaterials applied as cell matrix in bioinks because they can be engaged as cell matrix and be modified to replace or mimic local tissue [9]. The physical and chemical characteristics of the hydrogels will verify the performance of the cells. Normally hydrogels are like as jelly-type materials, where the liquid component is water. Actually, hydrogels are just like water by weight, but practically any flow will not occur in the steady state because of the three-dimensional cross-linked polymer network inside the fluid, which provides them unique properties comparable to those of living tissues. Due to their different biocompatibility and printability, various hydrogels that support cell growth are associated with bioink fabrication, i.e., gelatin, agarose, polyethylene glycol (PEG)-diacrylate, and alginate that are commonly used as bioinks. While alginate is an anionic polysaccharide derived from brown seaweed and generally consists of two polymer blocks, (1-4)-linked β-
Cross-linking process of alginate.
Ionic cross-linking is a method where cells cause minimum damage. The cross-linking process happens moderately rapidly. Alginate has structural similarity to natural extracellular matrices that is why it has been applied widely in various biomedical applications as well as in the delivery of bioactive agents and wound healing. For cell encapsulation, alginate hydrogels are generally applied. The whole procedure is prepared by mixing cells in alginate solution, and after the mixing process, the alginate-cell mixture drops into a bath of calcium chloride solution. But in low concentrations (1–2%), due to low viscosity, the alginate solution is not printable. For increasing the viscosity, other materials like methylcellulose or gelatin can be mixed with alginate for preparing the printability. The structural correspondence of alginate to extracellular matrices creates a perfect biomaterial. Matrix stiffness is a functional determinant of stem cell differentiation, and alginate makes a potential material to manage stem cell growth. Alginate helps support the cell growth and also has a high versatility, extending to both in vivo and in vitro differentiation. For 3D bioprinting applications, for example, extrusion printing needs quick gelation. In this case alginate proposes high gelation procedures when combined with a multivalent cation, permitting gels to build up and deposit at constant temperature. It is also applied to encapsulate cells. This allows it to be an effective tool in varying the release rate of drug and growth factor delivery. While alginate degradation rate can be somewhat controlled by altering the MW of the alginate, it is still slow and difficult to control. The stiffness and composition properties of alginate bioink can be tuned to direct the differentiation of stem cells. Sodium alginate is available naturally which is biodegradable, non-immunogenic linear, and nontoxic polysaccharide polymer; it consists of mannuronic and guluronic acids [10]. The cost is also low being a marine material which can be extracted from the brown algae cell walls, forming hydrogel in certain conditions. Because of these advantages, bioengineers and material scientists use alginate for the preparation of bioinks in tissue engineering and regenerative medicines. The tissue fabrication by 3D bioprinting [11] and sodium alginate applications and properties [12] is currently separately reviewed. In this study we discussed the applications of alginate (Figure 2) in 3D bioprinting and blending alginate with other polymers to improve the biomaterial interaction of the cells attached to it [13].
Extrusion-based 3D bioprinting process.
The requirement for alginate-based biomaterials in drug delivery and tissue engineering is huge. As stem cells play a progressively more major function in the area of regenerative medicine [14, 15], the arrangement and relation between alginate-based materials and stem cells have been exclusively emphasized. Investigated by in vitro implantation and in vitro cytotoxicity assay, alginate-based scaffolds and microcapsules have shown minimum or minor cytotoxicity [16, 17]. These in vitro results recommended tunable connections between the bio-composites and the multiple platelet releasate-derived bioagents for improving hematoma-like fracture repair. Also, a simple invasive performance for in situ remedial of the implant structures through injection was established in rat tail vertebrae applying microcomputed tomography. These results confirmed that alginate-based scaffolds were capable of degrading, permitted the vascularization, and obtained minimum inflammatory responses after transplantation. Consequently, alginate-based scaffolds can present suitable characteristics as probable cell and drug carriers for tissue regeneration. The next sections explain the clinical and preclinical analysis of alginate-based biomaterials and applications.
Sodium alginate which is also known as sodium alginate or algin is a naturally extracted less costly polymer from the brown algae cell walls which have intracellular spaces [10]. Alginate is composed of (1-4)-linked β-
Varying biodegradability of solution of sodium alginate along with human adipose stem cells was printed with accurate definition. These kinds of bioinks have the capacity to modulate proliferation and stem cell spreading and withstand uniform cell suspension but are imperfect in the case of stem cell diffusion. Wu et al. [25] showed the procedure of slow degradation of the alginate by tissue incubation in a sodium citrate medium. The sodium citrate amount helped the optimization of the alginate degradation time. Chung et al. [18] improved the printing resolution and printability of pre-crosslinked printed constructs by adding alginate with gelatin, keeping the mechanical property and the growth of cells, and keeping pore diameter constant. In Figure 3 the procedure of alginate cross-linking with the added cells is shown.
Alginate with cell cross-linking process.
Alginate has been used for dressing of the wounds due to its of good conformability, absorptivity, and mild antiseptic properties coupled with biodegradability and nontoxicity and optimal water vapor transmission rate. Alginate-based products like electrospun mat hydrogels and sponges in dressing of wounds are very good substrates for healing of wounds, which include gel-foaming capability as soon as the absorption of the wound exudates and hemostatic capabilities [26]. It is already been mentioned that dressing wounds with alginate improves healing of wounds through monocyte stimulation to harvest higher cytokine levels like tumor necrosis factor-α and interleukin-6 [27]. Near the wound locations, cytokine production creates pro-inflammatory factors that are helpful for wound healing. Because of the existence of endotoxin in the alginate, a huge level of bioactivity is present in these dressings. In situ-forming wound dressing hydrogel can be produced by oxidized alginate and gelatin in low borax concentration as shown by Balakrishnan and Jayakrishnan [28]. The homeostatic gelatin effect is present in the mixed matrix and wound healing property of alginate, and the antiseptic borax property makes alginate the appropriate wound dressing material. Tissue-engineered cartilage requirement is immense and has a huge clinical importance. The main causes of disability of the articular cartilage are degenerative and traumatic lesions [29]. Nearly 100 million Chinese people suffer from osteoarthritis. Because of this reason, regeneration and repair of the cartilage have huge impact. The pros of the cartilage repair injectable therapies are that implant within the defect is not only maintained, but it also allows quick bearing of weight because of strength and stiffness which is attained quickly [30, 31]. For bringing close the mechanical properties of the native tissues with the scaffolds, the alginate physical properties are matched with the articular cartilage. Ge and solid alginate injectable hydrogel microspheres are used for cartilage regeneration. Many researchers have studied the growth factor in tissue engineering by using alginate hydrogels and alginate-based microsphere combinations [32, 33]. In one study the demonstration of immobilization of the positive effect of RGD to an alginate porous scaffold for endorsing TGF-β-induced human MSC differentiation is shown [34]. Bian et al. studied the co-encapsulation of the TGF-β including the microsphere of the alginate with the human MSCs in the hyaluronic acid (HA) hydrogels with respect to the design of the constructs implantable for the cartilage repair [35]. The immobilized RGD peptide facilitated the cell-matrix interaction which is proven to be an important feature for the microenvironment of the cells, allowing good cell availability for the chondrogenic-inducing molecule TGF-β. TGF-β-laden alginate microspheres in combination with alginate hydrogels forms a compound carrier which may retain TGF-β bioactivity in the construct and encourages hondrogenesis of MSCs when inserted. The animal experiment displayed that chondrocytes planted into the microsphere scaffold lived habitually in SCID mice and cartilage-like constructions were created after 4 weeks of imbedding.
In the past years, drug delivery carriers draw huge interest because of large biomacromolecules like genes and proteins as well as low-molecule weight drugs which can be delivered in a targeted or a localized manner [36, 37]. Because of its biodegradable and biocompatible nature, alginate is used as a carrier for encapsulating and immobilizing drugs, cells, proteins, and bioactive molecules [38, 39]. Currently, alginate-based carriers like colloidal particles, polyelectrolyte, and hydrogels are under examination; few of them are practically used. Many researchers have examined the alginate-based hydrogel blends, microspheres, and porous scaffolds for precise drug delivery in various tissue engineering fields [40, 41]. Hollow microsphere of alginate-based hollow microsphere has huge applications as drug delivery carrier, micro-reactor, and biosensor [42]. The construction of the hollow microcapsules can be created by successive self-assembly of positively and negatively charged polyelectrolytes by layer-by-layer (LbL) technique. The alginate microcapsule is studied well with respect to precise releasing and loading parameters. The attempt to fabricate microcapsule biopolymer has been made by dropping alginate/chitosan in a decomposable colloid particle after removal of its core is done in an appropriate pathway. For the production of hollow microcapsule, chitosan and alginate are interchangeably deposited in CaCO3 with electrostatic biocompatibility [43]. The chitosan/alginate microcapsule functionalities and properties can be preciously adjusted by changing the microcapsule composition, exterior stimuli introduction, and thickness. Immersing alginate microcapsule in various pH solutions helps in the degradation of the microcapsules which also determines the material role and encapsulation layers for keeping microcapsule stability in various pH conditions. The addition of PEG to the microcapsule allows protection against acidic conditions, whereas the coating layer number only affects the swelling properties, not the microcapsule Young’s modulus which was revealed by Wong’s study [9]. For surface micro-patternings and microarray systems, 3D platform alginate hydrogels are used. For in situ gelation, a few aliquots of solution of gelatin were trapped selectively on hydrophilic area by a process called dipping process. Cells with various adhesion properties were captured by gel pattern alginate on the hydrogel structures.
Various CYP450 enzymes like vascular endothelial growth factor (VEGF) and β1-integrin upregulation showed that the stage gave many in vitro conditions that result in allowing cells in their natural phenotypes.
For the reconstructive surgery, bone regeneration is an important challenge. It occurs due to tumor removal and trauma. To repair the bone, a good initiative is to induce osteogenesis in situ. To complete this process, one method is by using stem cell differentiation to form bone tissue and then seeding them in an injectable scaffold [44, 45]. As of now there are numerous investigations and studies on alginate-based injectable scaffolds for the bone regeneration. By using MSCs and alginate scaffolds, satisfactory bone tissue formation was noticed [46, 47]. For this reason the application of alginate for gel tissue generation is commonly used which displays angiogenic and osteogenic properties. Many researchers showed bone regeneration by means of injectable constructs by joining microspheres or alginate-based hydrogels that were combined with interchangeable ASCs or MSCs [48]. These studies demonstrated the potential of bone morphogenetic protein (BMP) and TGF-β delivery to induce osteogenic differentiation to mature osteocytes from MSCs and ASCs. Kolambkar et al. presented a growth factor hybrid system of delivery that comprises of a nanofiber mesh tube which is electrospun for directing regeneration of bone along with alginate hydrogel peptide modified in the tube for fixed recombination BMP-2 (rhBMP-2) release [49]. The discharge of fixed transport of rhBMP-2 through alginate hydrogel was important for significant regeneration to take place. The mixed technology can be used clinically for the regeneration of bone in cases as huge bone defect and nonunion fractures.
The naturally available biopolymer alginate is cheaper which forms hydrogel by cross-linking with various salts like BaCl2, CaCl2, and ZnCl2 which showed good biocompatibility and printability.
This is broadly applied for cartilage, bone, and vascular tissue printing. Few drawbacks of alginate are slow degradation and poor cell adhesion; in many research, it is shown that alginate has poor cell differentiation and cell proliferation, and for this reason, it is used as a blend with other polymers. To improve these limitations, blending alginate with other polymers like honey, gelatin, and Arg-Gly-Asp adhesions is done. Furthermore, for faster normal degradation in regenerative medicine, oxidized alginate and/or sodium citrate is found to be useful. The combination of 3D printing alginate for cartilage and electrospinning is used positively in various tissue engineering fields. Furthermore, mixing alginate with biopolymers like polycaprolactone and nanocellulose has shown positive results. In bioprinting using coaxial or triaxial nozzles is found out to be promising and provided brilliant results. To improve the mechanical properties of the alginate-based structures used in bone tissue engineering, mixing alginate with other polymers like bio-silica, polyphosphate, polycaprolactone hydroxyapatite, and gelatin is found to produce an excellent result. We think this review will allow researchers to investigate more advanced and improved bioink for 3D printing and also help to invent suitable and more appropriate bioink for various tissue engineering applications
Although the cannabis plant (Cannabis sativa) has been used as a folk remedy to treat various ailments for thousands of years, it is only within the last century that its active components have been isolated and identified. While some of its effects are well documented, its impact on pain had been less clear due to confounding effects on mood, motor impairment, and sedation. Isolation of the psychoactive components of the cannabis plant and the development of synthetic cannabinoid compounds enabled more rigorous testing. Identification of a cannabinoid receptor (CB1) in 1988 gave insight into the mechanisms of the cannabis effect, as did the discovery of endogenous ligands, referred to as endocannabinoids [1, 2, 3, 4]. Studies in rats showed that when applied intravenously or directly to the spinal cord, cannabinoid agonists attenuated responses to noxious mechanical and thermal stimulation in nociceptive spinal neurons [5, 6, 7]. These early studies provided the first evidence of a direct effect of cannabinoids in pain inhibition and led to further investigations to identify the mechanisms underlying cannabinoid effects on neuronal activity.
\nThe endogenous cannabinoid system consists of two well-characterized receptor subtypes, CB1 and CB2, and their endogenous ligands, from which anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are the most studied [8, 9, 10]. Cannabinoid receptors are G-protein coupled, mainly to Gi/o, which inhibits adenylyl cyclase [3, 11], and voltage-dependent Ca2+ channels [12]. CB1 receptors are expressed primarily in the nervous system, but are also present in non-neuronal tissues. CB2 receptors are mainly located peripherally, and are associated with modulation of immune cells [4, 13, 14]. Since CB receptors are widely distributed, their activation produces a wide variety of behavioral and physiological responses.
\nActivation of cannabinoid receptors has been shown to produce anti-nociception in experimental models of inflammatory pain, including formalin [15, 16], carrageenan [17, 18, 19], CFA, complete Freund’s adjuvant [20], and capsaicin [19, 21, 22, 23, 24]. In addition, the administration of cannabinoid antagonists has been shown to enhance pain behavior in formalin and carrageenan models [15, 18], suggesting that tonic activation of cannabinoid receptors contributes to anti-nociception in response to inflammation. Systemic administration of cannabinoid agonists has also been shown to attenuate neuropathic pain following peripheral nerve injury (CCI model [25], partial sciatic nerve ligation [26], spinal nerve injury [27], L5/L6 ligation [28, 29]), diabetic neuropathy (type 1 [30, 31, 32] and type 2 [32]), and chemotherapy induced peripheral neuropathy [33, 34, 35, 36]. In humans, cannabinoid agonists attenuated post-operative pain [37] and also enhanced the analgesic efficacy of opioids [38]. Two small clinical evaluations of the efficacy of (−)Δ9-tetrohydrocannabinol (THC), the main psychoactive compound of the cannabis plant, reported pain relief comparable to codeine [39, 40]. Unfortunately, higher doses tended to produce significant side effects including sedation, dizziness, ataxia and blurred vision.
\nIn addition to anti-nociception, the endocannabinoid system has a neuroprotective function. In a model of cerebral ischemia, cannabinoid agonists, cannabidiol and THC attenuated toxicity related to the activity of excitatory neurotransmitters in the rat cerebral cortex independent from CB1 and CB2 receptors [41]. Cannabidiol is known to have low affinity for cannabinoid receptors, and has also been shown to act as a negative allosteric modulator at the CB1 receptor and a reverse agonist at the CB2 receptor [42, 43]. Another study reported the involvement of CB1 receptors in the reduction of neuronal loss [44]. Further, an in vitro study of hypoxic ischemia demonstrated a possible role for CB2 receptors [45]. The endogenous cannabinoid ligand, 2-AG, was shown to be neuroprotective in a model of traumatic brain injury, resulting in reduced edema and neuronal loss in the hippocampus [46]. Endocannabinoids have also been shown to protect against neurodegenerative diseases, including Alzheimer’s disease, where the inhibition of microglial activation may prevent pathological changes associated with beta amyloid [47]. There is also evidence that cannabinoids possess antioxidant properties through the activity of cannabinoid receptors located on microglia, astrocytes, and other immune cells, where activation inhibits the release of pro-inflammatory substances [48, 49, 50, 51, 52, 53, 54]. Increased expression of CB2 on microglia and astrocytes has been observed in the area of lesion [54]. The administration of a CB2 agonist slowed the progression of amyotrophic lateral sclerosis in mice, and the activation of the endocannabinoid system protected against myelin degeneration in multiple sclerosis through a combination of immunosuppression and neuroprotection [55, 56, 57]. In studies of peripheral neuropathy produced by chemotherapy, WIN 55,212-2 prevented the development of neuropathy induced by cisplatin treatment [33], and when WIN 55,212-2 treatment was initiated after sciatic nerve ligation (CCI model of neuropathic pain), mechanical hyperalgesia failed to develop by 14 days post-injury [58].
\nA major limitation to the systemic use of cannabinoid agonists as treatment for chronic pain is that activation of cannabinoid receptors in the central nervous system is associated with undesirable side effects, including sedation and catalepsy [59]. Targeting endocannabinoid activity in the peripheral nervous system bypasses these unwanted side effects while still producing analgesia in animal models of inflammatory pain, bone cancer pain, neuropathic pain and sickle cell disease. Continued research into the specific mechanisms of analgesia produced by activation of the endocannabinoid system in the periphery could identify new targets for pain which could serve as stand-alone therapies or be integrated into a multifaceted treatment approach. This chapter will review studies that have investigated the analgesic effects of treatments that target the peripheral endocannabinoid system, whether through direct activation of cannabinoid receptors or through modulation of endocannabinoid metabolism.
\nLocal administration of cannabinoid receptor agonists, as opposed to systemic treatment, can produce analgesia without centrally-mediated side effects. Intraplantar administration of the non-selective cannabinoid receptor agonist WIN 55,212–2 attenuated heat and mechanical hyperalgesia in an acute cutaneous heat injury model in rats. This was blocked by a CB1 receptor antagonist, and partially blocked by a CB2 receptor antagonist, suggesting that while both receptor subtypes play a role in anti-nociception during acute pain, the effect was primarily mediated through activation of CB1 receptors [60]. WIN 55,212–2 also decreased mechanical hyperalgesia in the tumor-bearing hind paw in a mouse model of bone cancer pain [61]. The anti-hyperalgesic effect was mediated by both CB1 and CB2 receptors. Importantly, intraplantar administration did not induce catalepsy, which normally occurs when cannabinoid agonists are injected systemically and can confound behavioral measures of nociception [62]. Recordings from the tibial nerve of tumor-bearing mice showed that intraplantar WIN 55,212-2 attenuated sensitization of C-fiber nociceptors as evidenced by a decrease in spontaneous discharge and reduced responses evoked by mechanical stimuli responses evoked by mechanical stimulation, effects which were blocked by both CB1 and CB2 antagonists [63] (Figure 1).
\nEffect of non-selective cannabinoid receptor agonist WIN 55,212-2 on responses of C-fiber nociceptors evoked by suprathreshold mechanical stimulation. (A) Representative examples of nociceptor responses evoked by 147 mN before injection and at 15, 30, 45 and 60 min after intraplantar administration of vehicle or WIN 55,212-2 alone or preceded by the CB1 receptor antagonist AM281 or CB2 antagonist AM630. The time of application of the stimulus is shown at the bottom of each column. (B) Mean (±SEM) number of evoked impulses before and at 15, 30, 45 and 60 min after intraplantar administration of vehicle, WIN 55,212–2, WIN 55,212–2 + AM281, and WIN 55,212–2 + AM630. Evoked responses were not changed following injection of vehicle but decreased following WIN 55,212-2. This was blocked by pretreatment with AM281 or AM630. * p < .05, ** p ≤ .01, *** p < .001 vs. WIN 55,212–2; ### p < .001 vs. pre-injection value (from Uhelski et al. [63]).
In a model of inflammatory pain, intraplantar administration of the non-selective cannabinoid receptor agonist CP 55,940 attenuated CFA-induced hyperalgesia in mice expressing human sickle hemoglobin (BERK and hBERK1) as well as controls expressing normal human hemoglobin (HbA-BERK) [64].
\nThe analgesic effect of intraplantar WIN 55,212-2 showed more variability in models of neuropathic pain. In a sciatic nerve ligation model, only the highest dose tested (250 μg) produced an anti-hyperalgesic effect, but the injection altered withdrawal latencies to heat and mechanical response thresholds in both the treated and non-treated hind paw, suggesting that the drug effect was not limited to the periphery [65]. This effect was also seen in rats with partial sciatic nerve ligation (Seltzer model of neuropathic pain); however, the effect was blocked by the intraplantar administration of a CB1 antagonist but not when that same antagonist was administered by the intrathecal route [26], indicating that the ability of WIN 55,212-2 to produce anti-nociception in the contralateral paw is not necessarily mediated by activation of CB1 receptors in the central nervous system. In a rat model of chemotherapy-induced peripheral neuropathy produced by paclitaxel treatment, intraplantar administration of WIN 55,212-2 had no effect on mechanical or heat hyperalgesia, whereas systemic treatment produced anti-nociception [65]. In contrast, intraplantar administration of WIN 55,212-2 attenuated mechanical allodynia associated with streptozotocin-induced diabetic neuropathy [31]. A non-selective cannabinoid receptor agonist naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone, a novel compound which does not appear to cross the blood–brain barrier, reduced mechanical hyperalgesia in a rats with partial sciatic nerve ligation when administered orally [66], indicating that peripherally-restricted activation of cannabinoid receptors can produce adequate analgesia with an oral dosing regimen.
\nReceptor-selective synthetic cannabinoids also produce analgesic effects. In rats given an intraplantar injection of CFA, arachidonyl-2′-chloroethylamide (ACEA) and (R)-(+)-methanandamide (methAEA), stable mimics of AEA that preferentially bind CB1 receptors, reduced mechanical hyperalgesia and decreased evoked responses in Aδ-fiber nociceptors. The reduction in mechanical hyperalgesia was blocked by a CB1 receptor antagonist, but not by a CB2 antagonist. Notably, neither drug had any effect on mechanical withdrawal thresholds or paw withdrawal frequency in naïve rats, and no changes were seen in evoked responses of Aδ-fiber nociceptors isolated from nerves innervating normal, non-inflamed paws [67]. The CB1 receptor agonist arachidonylcyclopropylamide (ACPA) attenuated hyperalgesia in a mouse model of bone cancer pain [68].
\nIntraplantar administration of AM1241, which preferentially binds to the CB2 receptor, reduced withdrawal responses to noxious heat in naïve rats, and no central side effects were observed when this compound was administered systemically [69]. Intraplantar administration of AM1241 reduced capsaicin-evoked nocifensive behaviors and hyperalgesia [70], reduced hyperalgesia and edema in carrageenan-induced inflammation [71], and reduced hyperalgesia in a mouse model of bone cancer pain [68].
\nThe endocannabinoids AEA and 2-AG have also been assessed for their peripheral anti-nociceptive properties. Intraplantar administration of AEA prevented the development of CFA-induced hyperalgesia and inflammation, while systemic administration of AEA had no effect [19]. This indicates that in order for AEA to inhibit the inflammatory pain that follows CFA injection, high levels of the drug must be present at the site of injury, which is difficult to achieve under normal conditions given that AEA has a short half-life due to rapid degradation by enzymes. Intraplantar AEA also inhibited capsaicin-induced edema and reduced formalin-induced nociceptive behaviors via CB1 receptor activation [15, 19, 72]. Intraplantar AEA was far more effective at inhibiting formalin-induced behaviors than intravenous AEA [15]. Formalin-evoked behaviors were also inhibited by intraplantar administration of 2-AG, an effect blocked by a CB2 receptor antagonist but not a CB1 antagonist [73]. In rats with inflammation produced by carrageenan administration to the hind paw, evoked responses of nociceptive spinal dorsal horn neurons were reduced following intraplantar administration of AEA [74]. The reduction in evoked activity was blocked by a CB2 antagonist, but not a CB1 antagonist. Intraplantar administration of AEA did not produce any changes in evoked responses of spinal neurons in control rats. Intraplantar AEA decreased hyperalgesia in the tumor-bearing paw in a mouse model of bone cancer pain, and this was blocked by a CB1 receptor antagonist [75]. Intraplantar 2-AG also decreased hyperalgesia in the tumor-bearing paw and the anti-hyperalgesia was mediated by CB2 receptors [76]. Intraplantar AEA has also been shown to decrease hyperalgesia following cisplatin treatment [77]. The mechanism of anti-nociception produced by AEA is complex, and the subtype of cannabinoid receptors involved in its effect seems to differ under acute and chronic pain states. AEA has strong analgesic effects when applied to the site of inflammation or neuropathic pain; however, it should be noted that elevated levels of AEA can also increase excitability of nociceptors through activation of TRPV1 receptors that induces Ca2+ influx. This effect was shown in cultured dorsal root ganglion (DRG) neurons sensitive to heat stimulation [78]. It should also be noted that endocannabinoid interactions with ion channels and other binding sites separate from cannabinoid receptors can also produce changes in neuronal function.
\nIn addition to direct cannabinoid receptor agonists, there are drugs which modify endocannabinoid metabolism and thereby alter levels of endocannabinoids. For example, compounds that inhibit enzymes that break down endocannabinoids increase the amount of endocannabinoids available for binding to cannabinoid receptors. URB597 ((3′-(aminocarbonyl)[1,1′-biphenyl]-3-yl)-cyclohexylcarbamate) targets fatty acid amide hydrolase (FAAH), an enzyme which breaks down AEA. Intraplantar administration of URB597 decreased hyperalgesia and C-fiber nociceptor sensitization in chemotherapy-induced peripheral neuropathy following cisplatin treatment, effects which were blocked by a CB1 receptor antagonist but not a CB2 antagonist. Biochemical analysis of skin showed that URB597 increased local levels of AEA without altering the levels of other endocannabinoids [79], indicating that increased activation of CB1 receptors by AEA was the source of decreased nociceptor excitability and analgesia. Intraplantar URB597 also decreased hyperalgesia and C-fiber nociceptor sensitization in a transgenic mouse model of sickle cell disease (SCD, HbSS-BERK). These effects were also blocked by a CB1 receptor antagonist but not by a CB2 receptor antagonist (Figure 2). Importantly, intraplantar administration of URB597 still had an anti-hyperalgesic effect in sickle mice with CB2 receptors knocked out (HbSS-BERK-CBR2−/−, [80], confirming mediation by CB1 receptors.
\n(A) Intraplantar administration of the FAAH inhibitor URB597 decreased evoked responses in C-fiber nociceptors isolated from HbSS-BERK sickle mice. Data show the mean (±SEM) number of impulses evoked by 147 mN before and at 30, 60, 90 and 120 min after various drug treatments. The number of evoked impulses was reduced following intraplantar administration of URB597 at 30, 60, 90, and 120 min post-injection, and this effect was blocked by the CB1 receptor antagonist AM281, but not CB2 receptor antagonist AM630. *p < .05, **p < .005, ***p ≤ .001 vs. the vehicle-treated group. ###p < .001 indicates significant differences from pre-injection value. (B) Representative examples of responses of individual C-fibers evoked by 147 mN for 5 s before (pre-injection) and at 30, 60, 90 and 120 min after intraplantar injection of vehicle, URB597, URB597 + AM281, and URB597 + AM630. The time of mechanical stimulation is illustrated at the bottom of each column. Reproduced from Uhelski et al. [80].
Systemic application of URB937 (N-cyclohexyl-carbamic acid, 3′-(aminocarbonyl)-6-hydroxy[1,1′-biphenyl]-3-yl ester), a FAAH inhibitor that is restricted to the periphery and cannot cross the blood-brain barrier, produced analgesic effects in sciatic nerve ligation (Bennett model of neuropathic pain) and carrageenan-induced inflammation in the affected hind paw, but did not alter responses on the non-affected hind paw [81, 82]. In cisplatin-treated mice, URB597 delayed and decreased the hyperalgesic effect of cisplatin [77]. Unfortunately, sustained pharmacological inhibition of FAAH results in endocannabinoid catabolism by alternative pathways, which are not dependent upon FAAH [83], thus limiting their clinical effectiveness. FAAH knockout mice have elevated levels of N-acylethanolamines and N-acyl taurines, show reduced responses to noxious stimuli, and are hypersensitive to AEA [84].
\nMonoacylglycerol lipase (MAGL) is an enzyme that breaks down 2-AG. Inhibition of MAGL produces analgesia under inflammatory conditions. Intraplantar administration of MAGL-inhibitor URB602 (N-[1,1’-Biphenyl]-3-yl-carbamic acid, cyclohexyl ester) attenuated formalin-evoked nociceptive behaviors [73]. Combining URB602 with 2-AG enhanced the anti-nociceptive effects of each [73]. The effect of URB602 was blocked by both CB1 and CB2 antagonists, whereas the effects of 2-AG were only blocked by a CB2 antagonist, suggesting that the URB602 does not behave as selective and/or potent inhibitor of MAGL [85] and that its effects are not dependent on only 2-AG, but may involve the inhibition of FAAH as well. In a mouse model of bone cancer pain, JZL184, a selective MAGL inhibitor, attenuated hyperalgesia in the tumor-bearing hind paw [76]. JZL184 elevates levels of 2-AG but not AEA following acute systemic administration, and the anti-hyperalgesic effect was shown to be dependent on CB2 (but not CB1) receptors. In contrast, intraplantar injection of JZL184 in cisplatin-treated mice decreased hyperalgesia by inhibiting both MAGL and FAAH and normalizing 2-AG and AEA levels in the plantar skin and DRG [86].
\nThere is a large body of evidence demonstrating that activation of cannabinoid receptors in the periphery produces analgesia. This effect appears to be the result of decreased nociceptor excitability, and there are several mechanisms that could contribute to this effect. These include direct activation of cannabinoid receptors that are expressed by nociceptors as well as activation of cannabinoid receptors expressed in the surrounding non-neuronal tissue that indirectly modulate neuronal excitability.
\nStudies of mRNA and protein expression have identified CB1 receptors on nociceptive neurons, and selectively knocking out CB1 receptors in Nav1.8-expressing neurons increased sensitivity to noxious heat, enhanced CFA-induced inflammation, and decreased the analgesic effect of WIN 55,212-2 [87, 88, 89]. Further, blocking either CB1 or CB2 receptors in the periphery inhibited the anti-nociceptive effect of systemic WIN 55,212-2 to the same degree, suggesting that peripheral cannabinoid receptors are a major site of action for cannabinoid receptor-mediated analgesia [90]. The application of cannabinoid agonist WIN 55, 212–2 and CP 55,940 to cultured primary afferent neurons reduced evoked Ca2+ influx in intermediate-diameter neurons, but not small-diameter neurons, though immunoreactivity for CB1 was detected in both cell populations [12, 91]. This indicates that reduction of calcium influx is just one of the inhibitory actions that can result from CB1 activation. Activation of CB1 receptors has also been shown to inhibit the release of calcitonin gene-related peptide (CGRP) from the nerve terminals of nociceptive primary afferent fibers in isolated skin from the rat hind paw [19], which could lead to reduced nociceptor excitability.
\nExpression of cannabinoid receptors can be modified in chronic pain states, which can enhance the effects of cannabinoid agonists. In a mouse model of bone cancer pain, the DRG ipsilateral to the tumor-bearing hind paw showed increased expression of CB1 receptors. Enhanced CB1 receptor expression in the DRG may explain why small-diameter neurons co-cultured with cancer cells were responsive to CB1 receptor agonists (which attenuated evoked calcium influx), while small-diameter DRG neurons in naïve mice were not [75] (Figure 3).
\nSmall DRG neurons (<500 μm2) isolated from DRG L3-L5 of tumor-bearing mice and maintained in vitro in control medium for 20–28 h exhibited a change in sensitivity to the CB1 agonist ACEA. (A) The trace represents cannabinoid agonist inhibition of the Ca2+ transient evoked by brief superfusion with KCl (50 mM, 10 s, arrows). ACEA (1 μM) was included in the superfusate following the first test with KCl. (B) ACEA attenuated the Ca2+ transient evoked by KCl (50 mM) in neurons isolated from L3-L5 DRGs of tumor-bearing mice, but not in those from naïve mice. Involvement of CB1 receptors in the response to ACEA was confirmed by blocking the inhibitory effect by co-application of the CB1 receptor antagonist SR141716A (SR, 1 μM). Relative response was defined as the amplitude of the response of a neuron to KCl in the presence of ACEA divided by the amplitude of the response in the absence of ACEA. *Significantly different at p < 0.001; #significantly different at p < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). Reproduced from Khasabova et al. [75].
CB2 receptor mRNA and protein are increased in the lumbar DRG after spinal nerve ligation (SNL) or CCI (Bennett model of neuropathic pain), but not CFA-induced inflammation [92]. The effect appears to be localized to microglia [92, 93], though there is some evidence of enhanced neuronal expression after SNL, including increased expression in the nerve proximal to the ligation [94]. In a mouse model of bone cancer pain, tumors showed high levels of CB2 receptor protein levels, and CB2 receptor proteins were also elevated in plantar skin of the tumor-bearing hind paw [76]. Taken together, these results support to the notion that endocannabinoid-mediated inhibition of peripheral nociceptor activity is necessary to prevent exaggerated responses to noxious stimuli and that tonic activation of endocannabinoids aids in suppressing pain, inflammation, and nociceptor sensitization after injury. Further evidence is shown by differences in levels of endocannabinoids in naïve, acute inflammation, and chronic pain conditions. In models of chronic pain from bone cancer and chemotherapy-induced peripheral neuropathy, the level of AEA was decreased in the skin of the plantar hind paw due to increased FAAH mRNA expression and AEA uptake in DRG neurons ipsilateral to a tumor-bearing hind paw [75, 95]. In cisplatin-treated mice, expression of 2-AG and AEA are both decreased in the plantar skin and DRG [86].
\nConcerns about the safety of commonly used analgesic drugs have hindered the treatment of patients with chronic pain. Continued exploration of mechanisms underlying nociceptive processing under naïve, acute and chronic pain states has helped identify specific targets for the development of new treatment approaches that could solve some of the problems associated with chronic use of opiates and NSAIDs. This includes the use of drugs which target the endocannabinoid system. Early investigations identified problems with the systemic use of compounds derived from the cannabis plant, including sedation, mood alterations, and motor effects, a direct consequence of binding to cannabinoid receptors in the brain. By targeting the peripheral endocannabinoid system, the negative side effects of cannabinoids can be bypassed, providing analgesia without impairment of normal function. Work with animal models has shown that activation of cannabinoid receptors in the periphery can be useful for a wide variety of pain conditions, including inflammation, bone cancer pain, chemotherapy-induced peripheral neuropathy, and sickle cell disease. Analgesia can be achieved through direct receptor activation or through the restoration of endocannabinoid levels, both of which decrease signs of sensitization in peripheral nociceptors. Thus, specific treatments could target known alterations in endocannabinoid levels associated with different chronic pain conditions. Drugs targeting the peripheral endocannabinoid system could be used as effective analgesics or in combination with currently available therapies to maximize pain relief while minimizing harmful side effects.
\nThis work was supported by NIH grants HL135895 and HL117664.
\nThe authors have no conflicts of interest to declare.
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