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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:"21 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:"46364",title:"Cadherin-Fc Chimeric Protein-Based Biomaterials: Advancing Stem Cell Technology and Regenerative Medicine Towards Application",doi:"10.5772/58287",slug:"cadherin-fc-chimeric-protein-based-biomaterials-advancing-stem-cell-technology-and-regenerative-medi",body:'‘Stem cell’ – the term was first coined by Russian histologist Alexander Maksimov in 1908 to herald the existence of special cells those have capacity to generate blood cell. Stem cells are the core materials of regenerative medicine and tissue engineering. Although there are multiple types of stem cells available based on their origin and functionality; however, scientifically they can be classified into four well-defined classes– (1) embryonic stem cell (ESC), (2) adult stem cells (ASC) for example, muscle satellite cells are muscle-specific adult stem cell, (3) induced pluripotent stem cell (iPSC), and (4) pathological stem cells (PSC) for example, cancer stem cells (CSC) [1]. Out of these 4 types, ESC and ASCs are true physiological stem cells, iPSCs are engineered stem cells and PSCs are conditional stem cells. Among them, ESC and iPSC are being considered true pluripotent stem cells, which have the capacity for unlimited self-renewal and differentiation into all the specialized cell types of the body. Therefore these cells have been considered the most favorable cells for using in regenerative medicine and tissue engineering [2,3,4,5,6,7,8].
Stem cells need a special environment for their survival, maintenance and growth. During the early stage of establishing the culture methodologies for stem cells, it was realized that they need support from other cells for example, mouse embryonic fibroblast (MEF). Co-culture methodology with gamma-irradiated MEF cells used as feeder-cells and enriched culture media with fetal bovine serum (FBS) were successfully utilized for establishing in vitro stem cell culture [9,10]. However, using a second non-related cell type (although growth restricted) is not suitable for differentiation studies – particularly, for 3D cell culture. Later, the MEF layer was successfully removed from the culture system by introducing MEF-conditioned media (MEF-CM) that has made the protocol more suitable for experimentation targeting regenerative medicine but not up to the desired standard due to the presence of xenogeneic agents in the system [11,12,13]. MEF-CM is enriched cell culture media with MEF-secreted molecules that functions as a depot for the necessary cytokines for the healthy maintenance of stem cells. However MEF-CM alone were not adequate to upkeep ESC and iPSC survival and growth thereby suggesting that MEF cells are not only providing necessary nutrients and cytokines, in addition they are also backing as physicochemical supports through the ECM to these cells. However, technically it remains elusive to point out the essential factors, required to maintain stem cell culture, present in the MEF-CM due to the inconsistency in expression and secretion of biological factors between experiments and batches. Moreover, it has been shown that not only proliferation of these cells but the secretion of necessary biomolecules and deposition of ECM components were also directly related to the gamma-irradiation [11,12,13]. Such factors directly influence properties of stem cells in culture, and instigate restriction for application of relevant protocols for regenerative medicine and tissue engineering. Therefore suitable cell-recognizable biomaterials are highly desired to overcome the dependency of cell-based basal supports for stem cell culture.
Matrigel was one of the first biomaterials that was effectively applied as plate-coating materials for in vitro culture of human ESC and iPSC with the aid of MEF-CM as culture medium [14,15]. This was a significant advancement in stem cell technology to make stem cells free from undesirable feeder-layer cells. Matrigel is a product from decellularization of Engelberth-Holm-Swarm (EHS) mouse sarcoma cells, and a cocktail of laminin, collagen IV, entactin, heparin sulfate proteoglycans, and known and unknown growth factors with variable compositions [16,17,18,19]. It closely resembles the embryonic basement membrane in consistency and activity as well as providing a biologically functional complex [17,19]. However, Matrigel is not a defined material with high purity and incorporated with substantial lot to lot variation in constituents both in qualitative and quantitative measures. It has also been reported contaminated with Lactate Dehydrogenage Elevating Virus, and has raised additional concerns for safe application of this material in stem cell culture [20]. Such kinds of issues are strongly demanding a more defined culture condition under good manufacturing practice (GMP) for safe application of stem cell protocols or methodologies if the ultimate objective is to employ stem cells in regenerative medicine or tissue engineering.
The individual components of Matrigel provide specific functional queues to ESCs and iPSCs. For example, ESC exhibits normal growth when cultured on laminin-coated plate, which was not observed on either fibronectin- or collagen IV-coated surface [21,22,23,24]. It was also reported that specific laminin isoforms have distinctive effects on stem cells; for instance, laminin-111, -332, -511 support adhesion and proliferation of stem cells but isoforms -211 and -411 of laminin do not [22]. The information suggested that designing a defined matrix for stem cell culture requires special biomaterials that can deliver concurrent supports for cell adhesion, proliferation and differentiation. In fact, effective stem cell culture condition with high pluripotency was occasionally achieved in spite of introducing several synthetic and semisynthetic biomaterials alone or as a blend as cell-culture substrate, and therefore, designing such a biomaterial remains a challenging but ultimately rewarding task.
Pioneering work from our laboratory introduced Fc-chimeric protein in stem cell technology approximately a decade ago, and over the years we and others have established multiple Fc-chimeric proteins as significantly favorable cell-recognizable biomaterials in stem cell technology. These works with varieties of Fc-chimeric proteins spanning from ECM component protein [for example, E-cadherin (ECad)] to cytokine [for example, hepatocyte growth factor (HGF)] have shown tremendous potential to overcome the major barriers in stem cell technology, namely defined condition for stem cell culture, selective differentiation to the target lineages, convenient purification of the desired cells etc., for the application of stem cell technology targeting to regenerative medicine. In this article we will focus on ECad-Fc and NCad-Fc chimeric proteins as novel cell-recognizable biomaterials in stem cell technology towards application in regenerative medicine.
An ideal chemically defined xenogeneic-agent free stem cell culture system might be consists of chemically known matrix for plate coating that would provide structural basal support to the stem cells and defined media that is supplemented with highly pure recombinant proteins as functional cytokines. The system should essentially be free from serum or feeder-cells or any other animal products. Even though it is very demanding however, designing and preparing a completely defined stem cell culture system is highly challenging. One worthwhile goal is to design a defined plate-coating material that can successfully replace Matrigel. Since stem cells are essentially dependent on cell-cell or cell-surface interaction for survival, which are mainly mediated by extracellular matrix protein (ECM), a cell-recognizable biomaterial should preferably mimic ECM protein(s).
Such kind of biomaterials can either be employed as a scaffolding molecule that may provide structural support of the growing cells, or as functional effector molecules that can target cellular signal recognition machineries like cell surface receptors or channels to trigger or maintain signaling cascades necessary for survival, proliferation, and differentiation of experimental cells [25]. To act as an artificial ECM the biomaterial under consideration should mimic the physicochemical and biological properties of native components of ECM to facilitate targeted functionalities of cell for example, adhesion, proliferation, differentiation, etc [26]. Similarly, the candidate effector molecules should have physicochemical signature of the comparable native molecules for recognition as functional substrate to endogenous receptors or channels of experimental cells. Synthetic biomaterials have limitations for providing perfect biochemical structural motif for effective recognition by the cellular recognition machineries to execute necessary cellular function, and therefore are generally not efficient enough for practical applications for in vivo condition. Moreover, many of these synthetic biomaterials are not biologically compatible at a desired level and may generate pathophysiological complications in the long term in the body.
Proteins are native elements of cells and natural ECM scaffolds [27] and therefore recombinant proteins could be one of the best candidates to design superior biomaterial for application in regenerative medicine and tissue engineering. Recent progress in biochemistry, molecular biology, bioinformatics, and engineering provides the prospect of expressing and purifying desired recombinant protein with high yield (g/L is achievable) in large scale [28], which can eventually be applied (directly or with modification) as novel, simplified, and bio-active macromolecules in regenerative medicine and tissue engineering [29,30,31]. Such proteins can be generated from a genetic template by natural cellular read-out process namely, DNA>RNA>protein that ensures excellent uniformity and reproducibility of the designed biomaterial depending on cellular conditions, where the production is executed. The native biological production process confirms high degree of reproducibility, which is not realistic by traditional chemosynthetic or mechanosynthetic processes. On the contrary, protein science has its own negative issues for example, highly efficient expression system for the desired protein, convenient purification of the target protein, proper folding of the purified protein, stability of the functional protein, mode of application of experimental protein etc. Chimeric protein technology has long been considered one of the potential methodologies to overcome many of these issues including higher productivity, better stability, and efficient purification of a target protein for bulk scale. Fc-chimeric protein is one such engineered protein that was introduced in 1989, and has been showing great promise for comparatively convenient production efficiency of chimeric protein with functional integrity and long-term stability, and therefore successful applcation in diverse fields of biomedical sciences [32,33,34]. An illustration of Fc-chimeric protein is shown in Fig. 1 with ECad-Fc as a model.
Schematics of Fc-chimeric protein, and its molecular function. (A) Functional domain of target protein is fused as N-terminal with the Fc domain of IgG. ECad is shown here as an example. (B) Plasma-membrane localized ECad dimer can interact with apposing ECad dimer and form high affinity binding that makes cell-cell and cell-surface adhesion.
The cadherins is a large family of single transmembrane proteins with more than 100 members. Out of these we will be focusing on epithelial cadherin (ECad) and neural cadherin (NCad) in this report. They are the member of classical cadherin family, and both of them are glycosylated in their extracellular domain. They have the ability to function as adhesion molecules for the relevant protein-expressing cells. Generally cadherin forms homophilic dimer, and the dimeric forms of cadherins take part in Ca+2-dependent coupling from apposing cells that mediates cell-cell adhesion. These single transmembrane-domain plasma membrane-resident proteins are not only necessary for cell-cell adhesion but also involved in indispensible signaling cascades, which are critical for the development-to-homeostasis-to-demise of cells and organisms.
The extracellular N terminal region of ECad consists of 5 structural domains, which are the signature motifs for ECad and are responsible for the homophilic binding between two neighboring as well as apposing molecules, while the C-terminal intracellular region of ECad interacts with several intracellular proteins such as β-catenin/Armadillo and p-120 catenin [35,36,37]. The p-120 catenin is associated with the targeted transport and stabilization of the adhesion complexes on the plasma membrane. Beside, β-catenin interacts with α-catenin, which in turn initiates actin filament formation via interaction with formin at the adherens junction [38,39,40,41,42]. However, how cadherin-catenin complexes are connected with cytoskeletal components e.g., actin is not clearly known.
ECad has been shown linked with many early-to-late developmental and differentiation processes in vivo and in vitro systems including ESCs, MSCs, iPSCs, and whole embryo [43,44,45,46,47]. ECad knock out mouse was reported embryonic lethal [48,49], which is a direct evidence of its critical importance in stem cell biology and regenerative medicine. Our lab first envisioned the application of ECad as a novel cell-recognizable biomaterial little over a decade ago while Nagaoka et al. endeavored to improve the differentiation and maturation efficiency of hepatocyte in an in vitro system [50]. The idea was conceived from the fact that Fc domain of IgG can bind directionally with an appropriate surface via hydrophobic interaction, and the fused protein stretches out directionally to offer interaction with a suitable partner [51]. At that period, several reports suggested that ECad is indispensable for tissue morphogenesis, and is also required for maintenance of matured tissues. Awata et al. showed that ECad-mediated cell-cell interaction is necessary for hepatocytes to maintain their differentiated phenotypes by forming 3D spheroid structure, or multi-layer cell aggregates [52]. Further it was reported that high cell density culture of fetal liver cells [53,54], which most likely is an ECad-dependent characteristics, enhanced hepatocyte maturation in culture. These findings suggested that cell-cell interaction may directly influence hepatocyte maturation as well as maintenance of differentiated phenotypes. There was, however, no substantial information regarding the role of ECad in the relevant processes, and to reveal the answer it was essential to have a suitable tool or methodology that can expedite cell-cell interaction analysis in a controlled manner. ECad-Fc was designed and deployed as a novel biomaterial in the regenerative medicine field to address this issue; after a decade, it has been proven to be a suitable material for stem cell technology and regenerative medicine.
As a biomaterial, ECad-Fc was first applied as plate-coating materials for hepatocyte differentiation experiments [50]. It was observed that differentiated hepatocytes can efficiently adhere with the cell culture plate coated with ECad-Fc. The adhered cells demonstrated comparable molecular characteristics e.g., low DNA synthesizing activity and maintenance of tryptophan oxygenase (TO) expression like those of spheroid-form hepatocytes. As well, the hepatocyte cultured on ECad-Fc-coated plate supported the differentiation of hepatocytes in culture. These results suggested important roles of ECad-Fc matrix for the maintenance of differentiating hepatocytes. This was the first report of ECad-mediated matrix dependability, as a biomaterial, for any cell type in regenerative medicine. After a while, Nagaoka et al. published the landmark report regarding the application of ECad-Fc cell-cooking plate (since target cell can be obtained on such type of biomaterial-coated plate without additional cell purification method therefore named so) as a defined matrix for successful maintenance of murine stem cells without any feeder layer in 2006 [55]. This report signified the alluring potential of ECad-Fc as a biomaterial for practical application in stem cell technology and regenerative medicine.
Xenogeneic-agent free stem cell culture method is extremely critical if the objective of the relevant protocol is to apply the relevant products in regenerative medicine. Since MEF secrets many unidentified molecules, which are potential xenogeneic elements for human subject therefore feeder-cell-based early methodologies are not considerable for applying in regenerative medicine. Matrigel is also produced from mouse carcinoma tissue and ill-defined therefore causing serious known and unknown hazards of xenogeneic contamination in experimentations. An immunogenic sialic acid (NeuGc) has been identified in a co-culture experiment for human ESCs applying MEF and animal derivatives as serum replacement [24,56]. This is specifically worrying as such kind of non-human sialic acid can initiate immunogenic processes in human triggering complete graft rejection and consequential complexities. Non-human animal-derived products also can be a possible cause for mycoplasma contamination, which can directly infect the cells in culture and either damage them totally or can change their properties, and thereby directly or indirectly initiate complicacies for regenerative medicine protocols. Human feeder-cells and serum have been recommended for culturing human ESCs to evade xenogeneic compound in experimental system for regenerative medicine. However, this is associated with a high risk of microbial contamination, for example retroviral components, and hence are not as suitable for in vivo application. Therefore it is a prime importance to establish completely defined human stem cell culture system for safe application of relevant products in regenerative medicine.
The study of Nagaoka et al. [55] revealed that murine ESCs can maintain their pluripotency on ECad-Fc-coated surface for extended culture periods (Fig. 2). Cells cultured on such type of substratum were later successfully used to generate germline-competent chimeric mouse [57]. Consistent with the findings, a separate study using mouse mesenchymal cell lines STO and NIH3T3 stably expressed with ECad as feeder-cell showed higher level of stem cell marker expression with standard colony-forming phenotype compare to the cells cultured on normal MEF-feeder-cell layer [58]. A number of feeder-free culture methods for ESCs have been reported where ESCs grow with their standard tightly-bound colony phenotype [4,11,13,22,24,56,59]. This type of tight colony formation generates heterogeneous cell population within a colony, which potentially affects homogenous accessibility of cytokines to these cells as well as creates heterogeneous niches. As a result stem cells in a colony differentiate heterogeneously and produce various kinds of cells as contamination with the desired type of cells, a major drawback that regenerative medicine has to overcome. In this respect, ECad-Fc matrix drives murine stem cells out of the colony to form a normal monolayer of cells, where stem cell resides as single cell condition [55]. This is a ground breaking technology that provides an exciting solution for overcoming the inherent colony forming phenotype-linked cellular heterogeneity. Biochemical analyses revealed that these cells bear all the signatures of pluripotent stem cells, and can form all three germ layers in a teratoma forming assay, and as mentioned earlier can generate germline-competent chimeric mouse. Additionally, they require lower amounts of LIF for maintenance of pluripotency, reducing costs related to ESCs culture. The monolayer-type single cell ESCs was also associated with higher proliferation ability and greater transfection efficiency compared to the colony-forming cells cultured on other substratum. Such improved proliferation ability could be extremely helpful for quick amplification of iPSCs on ECad-Fc substratum, which could mean shorter waiting periods for patients to receive cell therapy. The higher transfection efficiency of stem cells on ECad-Fc cooking plate could be exploited for targeted delivery of desired extracellular cargo for example, transgene products or drug molecules, into these cells for better outcomes.
ECad-Fc is a defined matrix for culturing monolayer of iPS cells. Mouse EB3 cells were successfully cultured on ECad-Fc-coated surface that showed monolayer phenotype (C and D) compare with compact colony phenotype (A and B) for general protocol, which was significantly advantageous for faster growth (E), and higher transfection eficiency (F).
This type of cooking-plate technology, where ECad-Fc provides basal support to the cells, and other immobilized factors for example, LIF-Fc [57] which satisfy specific needs, can be very advantageous for (1) ensuring undifferentiated state of stem cell in culture, (2) cost reduction associated with cytokines, and (3) hassle-free working condition without the necessity of regular media change, which is a standard time-consuming practice for stem cell culture.
The single-cell phenotype seen for ESCs was also observed for other stem cells for example, mouse embryonal carcinoma cells F9 and P19 but not for differentiated cells for example, NMuMG mouse mammary gland cells, MDCK kidney epithelial cells and isolated mouse primary hepatocytes [60]. This result indicated that ECad-Fc-mediated cellular migratory behaviors are most likely specific for embryonic stem cells. Reportable that ECad-facilitated cell-cell adhesion is often rearranged during initial stages of embryogenesis to control cell migration, cell sorting, and tissue function, which is suggesting a close cooperativity of stem cell maintenance, proliferation, and differentiation with ECad [39,48,49,61,62]. However, there is no such suitable system to explore the necessary signaling pathways to address these questions. Nevertheless, since ESC does not form colony on ECad-Fc cell-cooking plate therefore this can be a perfect tool for obtaining single cell model system of stem cells to investigate relevant signaling pathways necessary for stem cell maintenance, proliferation, and differentiation. Our recent study successfully exploited this single-cell phenotype for monitoring cell cycle properties of stem cells on cell-cooking plate (unpublished), indicating the importance of this system for cell biology experiments designed to reveal their individual characteristics. The findings could be invaluable for regulating stem cells for desired application in regenerative medicine.
Most of the stem cell innovations, comprising generation of ESCs and iPSCs, were primarily established in mouse model, and then applied in human models. Similarly, ECad-Fc cell-cooking plate technology was first developed and established for murine stem cells [55,57]. Thereafter, ECad-Fc cooking-plate was successfully applied for human ESC culture following similar methodologies with additional consideration for mild enzymatic treatment during the cell dissociation and seeding steps [56]. A strong protease cocktail Accutase (Millipore) was used for murine ESC culture; however, Accutase treatment was found detrimental to human ESCs, which was recuperated by using enzyme-free proprietary preparation named, Cell Dissociation Buffer (Life Technologies). It is reportable that the human ESCs were cultured on ECad-Fc cooking plate with a completely defined media named mTeSR1 (Stemcell Technologies), and that made the culture method completely defined and xenogeneic-agent free, which is a significant achievement in regenerative medicine. The stem cells cultured on ECad-Fc cooking-plate were practically identical to those cultured on Matrigel-coated plate including cell morphology, proliferation rate, preservation of undifferentiated phenotype, and ability of differentiation into multiple cell types in embryoid bodies as well as in teratoma assay [56]. Interestingly, contrasting with the single-cell phenotype for mouse ESCs, human ESCs produced normal colony forming phenotype on ECad-Fc cooking-plate. The mechanism underlying the difference for this observation was not completely understood though.
Human and mouse ESCs have been shown to demonstrate significant disparities in expression of cell surface markers, transcription factors, cytokines, and proteins in them. The difference was evidently recognized by the fact that mouse ESC can be maintained in undifferentiated state with the addition of LIF devoid of feeder-cell but human ESC cannot [14]. It has been shown that the inhibition of Rho-ROCK signaling pathway generates cell scattering in human ESCs suggesting direct connection between cell scattering and signaling pathways [63]. While both mouse and human ESCs express ECad, however, it appears there are diverse additional factors involved to define ECad-mediated activities in these cells and additional investigations are required to reveal the complete molecular circuitry associated to this phenomenon.
MSC is a type of ASCs, and can be collected from donor by satisfying approved ethical issues. These cells have been considered as potential starting materials for regenerative medicine and tissue engineering. They must be expanded in vitro before dispensing for specific applications to accomplish anticipated therapeutic effects. MSCs also need xenogeneic agent-free culture method for maintaining their differentiation potency over the culture period. ECad-Fc cooking-plate technology was effectively applied for this reason as well [43]. The cultured MSCs on human ECad-Fc (hECad-Fc) matrix exhibited superior attachment on culture plate compare with standard tissue culture plate and gelatin-coated plate. The MSCs cultured on hECad-Fc showed comparable level of CD 105 and significantly greater level of β-catenin and ECad expression. It has been reported that β-catenin enhances the activity of Oct-4, which is one of the principal Yamanaka factors that plays critical function during the regulation of self-renewal of ESC [45,64], on conjecture it can be suggested that MSCs maintained on ECad-FC cooking-plate might preserve superior stem-ness compare to the MSCs maintained on tissue culture-treated plate and gelatin-coated plate, and therefore possess greater applicability for regenerative medicine.
Targeted differentiation of stem cells and enrichment of desired cell for example, hepatocytes, from the pool of differentiated cells are very important steps towards use of the cells for regenerative medicine. Functionally matured hepatocytes derived from stem cells can be a potential remedy for various hepatic diseases. There have been several hepatic differentiation protocols reported from ESCs using orthodox techniques including embryonic body (EB) formation, and clustered colony formation on gelatin- or feeder-cell-coated plates [52,54]. However, these protocols come with many drawbacks, for example, heterogeneous cell population, spontaneous differentiation, xenogeneic contamination, inefficient conversion to hepatocytes, requirement for enrichment of target cell population etc. Our group has effectively applied ECad-Fc as a cell-recognizable plate-coating materials that facilitated good quality mouse ESCs in culture with superior proliferative activities and single-cell phenotype. Similarly, the cell-recognition property of such Cadherin-Fc chimeric protein was exploited for the possibility of facilitated differentiation of ESCs to specific cells for example, hepatocytes and neural cells [29,30,50,65]. Remarkably, ECad-Fc substratum favored progressive differentiation of ESCs to cells with features of definitive endoderm, hepatic progenitor cells, and finally phenotypical as well as functional hepatocytes-like cells [30,50]. The ECad-Fc-coated substratum stimulated selective hepatocyte differentiation in association with ectopic hepatocyte-producing cocktail resulting around 55% hepatic endoderm cells devoid of neuroectoderm and mesoderm markers [30]. High level of (approximately 98%) ECad and developing-hepatocyte marker α-fetoprotein (FTP) were co-expressed in these cells. Since these differentiating hepatocytes express high level of ECad on the plasma membrane therefore ECad-Fc was employed for on-site one-step enrichment of de novo hepatocyte-like cells. Practically, 92% albumin expressing cells were successfully harvested on ECad-Fc cooking-plate without any harsh enzymatic treatment or mechanical cell sorting, which are usually detrimental for cells [30]. Therefore the technology can be successfully applied for quick and stress-free cell purification, which will be useful in regenerative medicine.
The enhanced differentiation and cell-recognizable properties were also observed with ECad-Fc and NCad-Fc-based mixed biomaterial cooking-plate for neural cells [65], and is discussed in detail under NCad-Fc section. Such kind of ECad-Fc and NCad-Fc hybrid cooking-plate can be applied for either generation of large number of homogeneous cell population, which can be applied for therapeutic evaluation, or for analyzing the signaling pathways related to nerve generation at a single cell level.
iPSCs are commonly derived from somatic cells by ectopic and forced expression of common transcription factors Oct4, Sox2, and Nanog along with protocol-dependent treatments with cocktails of some other transcription factors, and even miRNA or small molecules [10,66,67,68,69,70,71,72,73]. Despite the existence of many protocols for generating iPSCs, the required time and efficiency of iPSC generation is still not practical for application of the technology to a mass scale. As per recent published information, depending on protocol, it may take somewhere between 2~4 weeks to get a 1% conversion of cells to iPSCs. During the reprograming process, starting cells experience mesenchymal-to-epithelial transitions (METs) as a natural requirement [74]. This fact was further proved by the findings that MET happens during the initial stage of reprograming process [71,74,75]. Recent evidence further suggested significant functional roles of ECad and other cell adhesion molecules in METs.
ECad interacts with cytoskeletal components via various intracellular molecules for example, α-catenin, β-catenin, and p-120 [38]. ECad-mediated signaling was found associated with cytoskeletal remodeling processes through Rho activation [41,63,76]. ECad has been established as an essential factor for maintaining typical colony-forming phenotype of ESCs and iPSCs. Recent studies, remarkably, revealed that forced expression of ECad can significantly enhance the effectiveness of relevant iPSCs-generation protocol [45]. A separate study revealed that ECad expression was enhanced upon treatment with small molecules resulting in enhanced efficiency for the relevant iPSC-generation protocol [77]. This enhanced productivity for iPSCs was successfully reproduced by the application of N terminal extracellular domains of ECad, which suggested that the phenomenon is mainly mediated by the extracellular functional domains of this protein [77]. Most importantly, ECad was sufficient to generate iPSCs with only three Yamanaka factors –KLF4, SOX2, and c-MYC from murine fibroblasts without OCT4 [45]. This study indicated that the spatial and mechanical input exerted by ECad has a critical role in driving cell fate. However, it is not clearly understood how ECad can compensate for OCT4. Since many studies showed that where it was possible to skip other factors of Yamanaka-cocktail for reprograming of somatic cells to iPSCs but OCT4 was hardly indispensable [10,70,78], further studies are warranted to determine the underlying mechanism. One potential explanation might be that ECad and KLF4 together initiated an early MET process of the experimental cells, and then SOX2 and KLF4 operated co-operatively to propel pluripotency genes to induce initiation of reprogramming [74]. The hypothesis is favored by the fact that cells those already express ECad, for example keratinocytes, can be reprogrammed more effectively and quicker because the MET process is not required [71]. Since the extracellular domain of ECad is adequate to produce ECad-mediated influences related to the reprogramming of somatic cells to iPSCs we have therefore assumed that ECad-Fc could significantly enhance the reprogramming efficiency. Our preliminary observation suggested that indeed co-transfection of ECad-Fc-expressing plasmid with Yamanaka factors enhanced reprograming efficiency of mouse fibroblast (unpublished). Enhanced reprogramming efficiency was further witnessed while the Yamanaka-cocktail-transfected starting cells were cultured on ECad-Fc-coated plate compare to gelatin-coated plate. However, further experiments are necessary for providing detail quantitative and qualitative information for these observations. Nonetheless this finding is highly promising regarding enhanced and efficient generation of iPSCs using a biomaterial as substratum.
The protocols for generating ESCs or iPSCs as well as differentiation to target cells from these cells require cell isolation step either by mechanical process or in combination with enzymatic treatment [79]. These types of methodologies require skilled labor, specialized instrumentation, additional time and cost, and distinct morphologic and phenotypic features. Several protocols have been described recently for enzyme-selective passage of specific cells; however, they are not globally applicable and very often appeared with unwanted cells. Enzymatic treatment also caused karyotypic anomalies compared with manual passaging [66,80]. FACS protocol has been applied for cell sorting based on surface marker recognition. However, relevant protocols need enzymatic treatment, application of foreign molecules, and mechanical processes involving severe stress on experimental cells [81,82], which are highly unfavorable for cells. ECad-Fc cooking-plate, advantageously, neither needs any kind of mechanical sorting nor any harsh chemical or enzymatic treatment. The experimental cells can selectively and strongly make homophilic binding with ECad-Fc matrix in a Ca+2-dependent manner subjected to the differential expression pattern of ECad in them during the transformation process. The cells with no or low level expression of ECad cannot and does not firmly bind with ECad-Fc substratum and can be washed off with suitable buffer thus offering a unique, robust, and stress-free cell enrichment system. Such a protocol ensures quicker, cheaper and convenient cell enrichment system for in vitro culture without risk of additional contamination and cellular alteration, and therefore, is highly advantageous for application in regenerative medicine and tissue engineering to achieve desired therapeutic effect with minimal adverse consequences.
N-cadherin (NCad) or neural cadherin is also known as Cadherin-2, which is encoded in human by CADH2 gene [83,84]. Like ECad, it is also a cell-cell adhesion molecule composed of five extracellular cadherin domains, a transmembrane domain and a highly conserved cytoplasmic region. NCad can exist either as strand dimers or in an alternate monomeric form [85]. NCad typically forms homotypic homophilic interactions between two neighbouring cells for example, Sertoli cells and spermatides, and also heterotypic homophilic and heterophilic interactions, such as interaction between N- and R-cadherin in transfected L cells [86]; such interactions are Ca+2 dependent [87], and can be reversed by withdrawing Ca+2 from the system.
During embryogenesis cells undergo an epithelial-mesenchymal transition (EMT) initiating upregulation of NCad and the downregulation of ECad in the mesoderm [88]. It has been suggested that NCad expression is essential for morphogenesis of the mesodermal germ layer during gastrulation [89]. NCad expression pattern has been found complementary to that of ECad in epidermal ectoderm [88,90,91]. NCad expression has been detected in mesoderm and notochord in the early phase of embryonic development, which is later also evident in neural tissue, lens placode [92], some epithelial tissues, myocardium of heart [93], epiblast of skeletal muscle [94], endothelial cells, osteoblasts, mesothelium, limb cartilage, and primordial germ cells [95,96].
NCad is found to be present in the early hematopoietic progenitor CD34+CD19+ cells, and it was proposed that NCad plays critical role for the hematopoietic cell differentiation as well as the early retention of this subpopulation in bone marrow [97]. During skeletal muscle formation mesodermal precursors exit from the cell cycle, and differentiate into myoblasts that terminally differentiates into multinucleate myofibers [98]. Cell cycle arrest and the expression of skeletal muscle–specific genes are the critical checkpoints for this developmental process [99]. All the epiblast cells undergoing skeletal myogenesis express the skeletal muscle-specific transcription factor MyoD, among them only the cells expressing NCad but not ECad can differentiate into skeletal muscle [94]. NCad function-perturbing antibodies showed that it plays a significant role in interaction between myoblasts in myotube formation and in myofibrillogenesis [100,101,102]. NCad is also found to be involved in myoblast migration in limb bud [103].
Cartilage is formed from the vertebrate embryonic limb by a highly synchronized and systematic event of cell commitment, condensation and chondrogenic differentiation of mesenchymal cells to chondrogens, and by the production of cartilaginous matrix. SOX9, an essential transcription factor for chondrocyte differentiation and cartilage formation, binds to the SOX9-binding motif in NCad promoter [104] that facilitates expressing of NCad gene products to play necessary roles in cellular condensation [105]. Prolonged expression of NCad due to the missexpression of wnt7a stabilizes NCad-mediated cell-cell adhesion resulting inhibition of chondrogenesis from mesenchymal chondrogenic culture [106]. The level of NCad mRNA was found increases during osteoblast differentiation and decreased during adipogenic differentiation thus suggesting their involvement in relevant differentiation processes [107]. NCad expression is increased in osteoblasts by BMP-2, FGF-2 and phorbol ester (e.g., PMA) in PKC-dependent manner, whereas factors like TNFα and IL-1 reduce the expression of NCad [108].
Migratory cell populations, also known as neural crest cells, are pluripotent cells those originate from dorsal part of neural tube and play important roles in embryonic development and pathophysiological conditions. These cells express NCad when they are associated with neural tube; however, NCad expression is down-regulated after EMT process and the relevant cells started to migrate over long distance, and finally transform into different types of tissues and cell populations, such as peripheral nervous system, cartilage, bone and melanocytes. Slug plays here important roles in down-regulating NCad that leads to a loss of cell-cell adhesion and allowing the cells to migrate. The dorso-ventral migratory cells re-express NCad during dorsal root and sympathetic ganglia developmental steps and promotes cell aggregation; thereafter, only dermal melanocytes express NCad [109,110]. This observation is suggesting critical involvement of NCad in the development of relevant tissues.
Several proteins can interact with NCad via intracellular and extracellular domains and influence subsequent signaling pathways. The functions of NCad in controlling neurite outgrowth, synaptic plasticity and guidance in synapse formation have been proposed [111]. These functions may involve interaction with other membrane bound molecules, such as fibroblast growth factor receptor (FGFR), which was confirmed by blocking the FGFR by pharmacological inhibitor [112]. NCad directly interacts with FGFR via HAV epitope of FGFR with IDPVNGQ epitope of EC4 of NCad [112], and this interaction between NCad and FGFR can be of both ligand dependent and independent [113] suggesting wider cooperative functional significance of this duo in relevant development and physiology.
EMT of squamous epithelial cells ectopically expressed specific amino acid sequences of EC4 of NCad induces motility. The cell motility behavior and adhesion is independent to each other, as antibody against the aforementioned relevant amino acid sequence of NCad inhibits cell motility but the cell-cell adhesion phenomena was uninterrupted [114]. The influence of NCad mediated cell migration is cell type specific, as it was found that NCad can inhibit LM8 mouse osteosarcoma cell migration but it did not have any significant effect on the movement of MDA-MB-435 cells [115]. The cytoplasmic domain of NCad form complexes with various types of molecules, such as p120, β-catenin, α-catenin and GAP-43, and regulate various cytoskeletal dynamics. All of these interactions are critically involved in tissue-to-animal development, morphogenesis and maturation, and is suggesting the possibility of exploiting this gene product for regenerative medicine.
NCad-Fc was introduced by Lambert et al. in 2000, and the study revealed that NCad-Fc not only induced the recruitment of NCad on the plasma membrane but also other components of the cadherin/catenin complex. This work for the first time demonstrated that NCad-Fc can mimic natural cell-cell contact formation and signal transduction [116]. Pioneering work from our lab has introduced NCad-Fc as cell-coating biomaterials for stem cell culture. NCad-Fc protein was collected from ‘pRC-NCFC’ plasmid, which was constructed by inserting the N terminal extracellular domain of mouse NCad into pRC/CMV (Invitrogen) plasmid [29]. The expression and purification methodologies of NCad-Fc are similar like ECad-Fc and have been described in details in relevant publications [50,55,117]. Over recent years our laboratory work revealed significant advantages of NCad-Fc in neural differentiation from stem cells. Early work was performed with mouse embryonic carcinoma cell P19 and neural stem cell MEB5 because of their easy management over the ESCs. It was observed that culturing these cell lines on NCad-Fc substratum can maintain the undifferentiated state and scattering morphology compare with other control substratum such as gelatin, fibronectin, laminin or poly-L- ornithine. P19 and MEB5 cells were differentiated effectively to neural lineage on this defined matrix in presence of retinoic acid supplemented with insulin-transferrin-selenium commercial preparation (ITS, Invitrogen). Interestingly, P19 cells showed higher level of Neurog1 expression on NCad-Fc-coated surface compare with gelatin-coated surface. Additionally, MEB5 differentiated on NCad-Fc matrix, compared to fibronectin-coated surface, showed complete neuronal differentiation phenomena and significantly higher expression levels of neural markers, such as Neurog1 and MAP2. These results clearly suggested the superiority of NCad-Fc substratum over the other experimental substratum for neuronal differentiation process.
Later, the findings were extrapolated to MEF-dependent mouse embryonic stem cell ST1 and mouse iPSCs to evaluate whether the effect is restricted to specific pre-committed cell lines or it is globally applicable [65]. Since during EMT conversion ECad is downregulated and NCad is upregulated therefore a hybrid matrix of ECad-Fc and N-Cad-Fc was designed to exploit the stage-specific cadherin switching phenomenon. The concept was that, initially the ESCs and iPSCs would bind to ECad-Fc through cell-resident ECad, however, during and after neuroectoderm formation cadherin switching will cater for cellular NCad in place of ECad that would bind to NCad-Fc. The cadherin switching was experimentally confirmed in house during neural differentiation protocol (Fig. 3A), where Dkk-1, a Wnt signaling pathway antagonist, and LeftyA, a Nodal signaling pathway antagonist were used for triggering neural differentiation. Specific markers for primitive ectoderm, primitive neural stem cells, neural stem and progenitor cells were checked. Along with, promisingly, the efficiency of neural progenitor differentiation from mouse ESCs on cadherin-Fc chimeric matrix was significantly higher compare to the cells cultured on other standard substratum as evaluated by the higher level of expression of neural progenitor marker Nestin gene products. Furthermore, the differentiated cells exhibited greater levels expression of βIII-tubulin (Tuj1) (Fig. 3B), microtubule associated protein 2 (MAP2), Pax6, and tyrosine hydroxylase but not GFAP, which is a marker of glial cell, signifying the presence of a lineage confined to neural cells.
NCad-Fc, and ECad-Fc promote directed differentiation of target lineage from iPSCs. (A) Western blot data revealed ECad to NCad switching occurs during neuronal differentiation. The expression level was normalized using house-keeping gene, β-actin. (B) βIII-tubulin expression was significantly higher on E/NCad-Fc matrix compared to gelatin.
Culturing of ESCs and iPSCs on ECad-Fc and NCad-Fc hybrid substratum not only developed scattered cell morphology as reported for ECad-Fc substratum but higher cell proliferation rate and enhanced differentiation efficiency were also noted. Along with these phenomena significant higher degree of homogeneity and enhanced differentiation efficiency were also observed, which is a remarkable advantage for harvesting target neuronal cells from in vitro system that can later be applied for regenerative medicine protocol. Although EB-based protocols are being relatively well-practiced for neural differentiation, however, the inconsistency of the embryoid body (EB) size and shape, and the asynchronous distribution of growth factors throughout the EBs give rise to heterogeneous products. Besides, monitoring cell morphology during differentiation process for EB-based differentiation protocols is inconvenient. Culturing ESCs or iPSCs in scattered single cell condition, on the contrary, can effectively overcome these issues. Interestingly, this blend of Cadherin-Fc matrices maintained a complete homogeneous cell population for murine ESCs and iPSCs for several passages. Highly homogeneous population of primitive ectoderm and neural progenitor cells were routinely generated on such a hybrid-type cooking-plate [65]. Enriched population of neuroectoderm progenitor cells can be obtained within 4 to 6 days by using E/NCad-Fc based monolayer-forming ESCs and iPSCs culture protocol and standard neurogenic cocktail treatment, which is a great advantage for quick generation of the target cells for application in regenerative medicine.
Some cells release 90 kDa fragment of soluble NCad (sNCad), and NCad-Fc was used to mimic sNCad response on neurite development [118]. Application of NCad-Fc by Doherty et al. with cerebral neurons showed that NCad-Fc initiated neurite outgrowth in a FGF receptor depended manner [111,119] suggesting that NCad-Fc can be utilized for controlling FGF receptor signaling pathway to facilitate relevant neuronal development events. Using mouse E12.5 ventral spinal cord explants as a convenient model Marthiens et al. showed that the axons formed contacts along the axon-shaft by long filopodia-like processes on NCad-Fc matrix [120]. They further showed that growth cones preferentially interact with cad-11 or NCad-Fc when progressing on this substratum whereas it differs on laminin. This study proved direct involvement of cadherin-11 and NCad in peripheral nervous system establishment from embryonic tissues [120].
Not only for neuronal population related regenerative medicine, NCad-Fc also showed potentials for application in other tissues as well, for example myogenesis related issues. Charrasse et al. used NCad-Fc to mimic NCad binding effect for myogenic differentiation [121]. They showed that NCad-Fc based NCad–dependent cell–cell adhesion triggers RhoA GTPase activity, which is essential for myogenic differentiation. Activity and expression of SRF, a transcription factor that binds to the promoter regions of muscle-specific genes [122,123] and controls the expression of MyoD, is controlled by RhoA. In turn, MyoD binds to the promoter region of skeletal muscle activating genes in mesenchymal cells and convert them to skeletal myoblasts [124,125,126]. These findings demonstrated that N-cadherin–dependent adhesion event that regulates the RhoA/SRF pathway to trigger myogenesis can be harnessed by NCad-Fc matrix and therefore such technology is holding great promises for using in relevant regenerative medicine protocols.
To design an efficient biomaterial capable of maintaining and stewarding specific cell phenotypes critical for the development, homeostasis, differentiation, and regeneration of tissues, the material must have a high degree of selective recognition property to the desired cells. As well, such a biomaterial should be devoid of unexpected stimulation characteristics to the cells that can be hazardous to them or to the desired results of the protocols. Being the intrinsic component of cellular milieu, proteins are highly desirable molecules to be used in regenerative medicine and tissue engineering technology. Their 3D conformation made them perfectly fit in the cell-biology and ensuring that only specific function to the experimental cells has been achieved. The natural homeostasis properties of cells can adequately remove these proteins once they are used up without exerting any unnatural effect or stress to the cells. Expressing and purifying large protein with proper 3D conformation is extremely challenging therefore mimetic peptide technology has been becoming popular. These small peptide sequences represent small functional domain of the relevant proteins, albeit not with the native 3D structure of the parent protein molecule. While most cases they are being generated using artificial synthetic technology in test tubes, however, their purity, reproducibility and yield are major concerns for their confident application in stem cell technology. Additional limitations for mimetic peptides are (1) the restricted size of desired peptides, and (2) inability to provide native post-translational modifications, most of which are critical for proper bio-functionality of the relevant molecule. Therefore mimetic peptides cannot and do not behave identically as their natural parent protein. On the contrary, Fc-chimeric proteins can be generated with high degree of reproducibility with identical molecular properties using the natural cellular readout process from the DNA template. The additional stability of the target protein instigated by the presence of Fc domain is significantly advantageous for higher yield of the tailored chimeric protein. The intrinsic property of Fc domain to form homodimer is beneficial to keep the target chimeric protein in soluble form. On the other hand, the natural affinity of Fc domain to bind with Protein A or Protein G is a technical boon for convenient purification of the target protein without fusion of any secondary bait to the amino acid sequence, which often create complex situation for getting rid of them at the later stage of the processing to harvest only the desired designed protein. Directional binding of Fc domain with the polystyrene or hydrophobic surface and catering the functional protein outwards is also an intrinsic benefit for using this class of chimeric proteins for obtaining higher functional efficacy of the applied biomaterials. Since the specific homophilic interactions between cadherins mediate cell attachment therefore specific cadherin isoform-expressing cells can be purified by using the relevant cadherin-Fc biomaterial as surface-coating materials. For example, iPS cells express high level of ECad and neuronal cells express NCad therefore, by employing these matrices in different time points of differentiation protocol, the target cells can be purified in situ without the necessity of any harsh enzymatic or mechanical treatments. Some of these chimeric proteins are commercially available for application and some are in pipeline, which can be obtained from our laboratory under proper regulatory affairs. Collectively, Fc-chimeric protein-based biomaterials provide distinct advantages for overcoming many existing challenges in stem cell technology and significantly advancing the regenerative medicine and tissue engineering field towards practical application.
The temporal bone is a dense complex bone that constitutes the lower lateral aspect of the skull and has complex anatomy because of the three-dimensional relationships between neurovascular structures. The petrous portion of the temporal bone has a role as the partition between the middle and posterior cranial fossae. It articulates with the occipital bone (occipitomastoid suture) posteriorly, the parietal bone (squamous suture) superiorly, the sphenoid bone (spheno-squamosal suture) and the zygomatic bone (arcus zygomaticus) anteriorly, and the mandible (temporomandibular joint) inferiorly [1, 2]. It contains multiple intrinsic channels, along with the internal carotid artery (ICA), cranial nerves, and sigmoid sinus (SS), all within intricate spatial architecture. Owing to a complex web of foramina and neurovascular structures of the temporal bone, the lateral skull base is a technically difficult region for surgeons. Because the middle and inner ear structures of hearing and equilibrium are preserved in the temporal bone, a surgical dissection of it requires thorough understanding of three-dimensional (3D) map of the topographic anatomy to avoid iatrogenic risks. The relationship between the surface landmarks and expected internal structures and the segmentation of the temporal structures by using key surgical lines and spaces allow a better understanding of its anatomic architecture. Each temporal bone consists of five distinct osseous segments including the squamous, tympanic, petrous, mastoid, and styloid portions [3, 4].
The anterosuperior part of the temporal bone is a large flattened scale-like plate that forms the lateral boundary of the middle cranial fossa. It has three borders and two surfaces [1].
Superiorly, it overlaps the sculpted squamous margin of the middle third of the parietal bone and constructs the squamosal suture. Posteriorly, it forms the occipitomastoid suture with the squamous part of the occipital bone. Also, there is an angle, parietal notch, between the squamous and mastoid portions of the temporal bone (Figure 1). Antero-inferiorly, its thick serrated margin takes part in pterion formation and articulates with the greater wing of the sphenoid bone to form the spheno-squamosal suture. Inferiorly, it fuses and forms the petro-squamous suture with the superior surface of the petrous portion by extending medially as tegmen tympany [5, 6].
The surface landmarks on the squamous portion: 1, temporal fossa; 2, supra-meatal crest; 3, temporal line; 4, external acoustic meatus; 5, supra-meatal triangle (Macewen’s triangle); 6, middle temporal artery; 7, squamo-mastoid suture; 8, mandibular fossa (glenoid fossa); 9, articular eminence; 10, zygomatic process; 11, petrotympanic fissure (Glaserian fissure); 12, mastoid foramen; 13, parietal notch; 14, mastoid process; 15, mastoid notch (digastric fossa); 16, occipital sulcus; 17, tympano-mastoid suture; 18, vaginal process; 19, styloid process.
External surface, the greater part of the temporal fossa, provides origin to the temporalis muscle and is limited below by the curved line, the temporal line, that lies from the supra-meatal crest to the mastoid cortex posteriorly. Below this line, just above and behind the external acoustic meatus (EAM), the supra-meatal triangle (Macewen’s triangle) contains the supra-meatal spine, spine of Henle and the cribriform area (Figure 1). Also, the squamo-mastoid suture is located approximately 1 cm below the temporal line [5, 6, 7]. On this smooth surface, there is a sulcus for the middle temporal artery, which is the medial branch of the superficial temporal artery (STA). Antero-inferiorly, the zygomatic process projects by two roots: the upper border of the posterior root forms the supra-meatal crest and the lower border forms a laterally based projection, known as post-glenoid tubercle or process (PGP). Inferiorly, the concavity along the surface of the anterior root is called the glenoid fossa (GF), which is bounded by the articular eminence (ArE) anteriorly and the PGP posteriorly [5, 6, 7].
Internal surface is rough and concave in shape, and the anterior and posterior divisions of middle meningeal artery (MMA) run in a groove on this surface that defines the boundary of middle cranial fossa with impressions for the gyri of the temporal lobe. Inferiorly, it forms the petro-squamosal suture with the anterior surface of the petrous part [5, 6].
The Macewen’s triangle, a surgical surface marking for the mastoid antrum (MA), is formed between the temporal line superiorly, the posterosuperior wall of the EAM antero-inferiorly, and the opening of the mastoid emissary vein or sinodural angle posteriorly (Figure 1). The temporal line corresponds to the tegmen tympani (TT), which is a bony plate below the middle cranial fossa dura and over the mastoid air cells. The mastoid cortex posterior to the spine of Henle is a guide to the lateral wall of the MA and located 15 mm deep to it in adults but in new born about 2 mm [5, 6, 8]. The cribriform area in Macewen’s triangle is perforated by numerous small holes that serve as a passage for the vessels of the mucosa of the antrum. The dissection along the margins of this triangle is safer because the vital neurovascular structures are absent. Peris-Celda et al. reported that the temporal line is supratentorial and infratentorial in 93% and 7% of the cases, respectively [9]. During retro-auricular mastoidectomy, the MA may be exposured by drilling the cribriform area and provides a safer surgical approach to the tympanic cavity. The tympanic portion and the styloid process may show variations depending on the shape and the position of the spine of Henle. The MA is located in the same line of the spine of Henle at about 10 years; then the MA is enlarged and placed 1 cm behind it [6, 9].
The MMA lies underneath the pterion which is a common junction between the temporal, parietal, frontal, and sphenoid bones. The fracture of this weakest bony part may result in an epidural bleeding. Between the temporal muscle and fascia, the STA and the superficial temporal vein (STV) courses in close proximity with the zygomaticotemporal (ZTN) and the auriculotemporal (ATN) nerves, branches of the trigeminal nerve (TN). Because of a vessel running superficial to the nerve (80% STA), the underlying nerve may be compressed and results in temporal migraine headache. Lee et al. reported that the intersection (compression) point among the ATN, STA, and STV was at an average of 40 mm superior and 10 mm anterior to the tragus, which is a significant surface landmark at the most anterosuperior point of the EAM. The applications of surgical decompression of the ATN in these compression points improve migraine headache [10].
The anterior articular part of the GF is formed by a gentle sloped area of the squamous portion, which facilitates the movement of the temporomandibular joint (TMJ) during wide mouth opening. At the lateral aspect of the ArE, a small bony ridge, articular tubercle (AT), serves as an attachment for the lateral collateral ligament. The PGP inhibits backward displacement of mandibular head and participates to the superior wall of the EAM [8]. The posterior nonarticular part of the GP is formed by the tympanic portion and the squamo-tympanic suture intervenes between them. The inferior edge of the TT (petrous part) divides this suture into two: a petro-squamosal fissure in front and a petrotympanic fissure (Glaserian fissure) behind (Figure 1). The chorda tympani nerve, a branch of the facial nerve, exits the temporal bone through the Glaserian fissure and joins the lingual nerve as the parasympathetic input to start the submandibular and sublingual gland secretions [2, 4, 5].
The articulation between the GF and the condyle of the mandible is called TMJ, which plays an essential role in speech, respiration, swallowing, and specially mastication. Because the TMJ is in close proximity with the MMA, some surgical landmarks around the TMJ and foramen spinosum (FS) play a critical role in surgical approaches. Miller et al. reported that researchers measured the distances from the zygomatic root (first projection of the zygomatic arch = PGP) to some surgical landmarks such as the arcuate eminence (AE), the head of the malleus (HM) under the TT, and the FS to identify the location of the internal auditory meatus (IAM) or the superior semicircular canal (SSC). Also, they described the superior petrosal triangle as a consistent triangle between the zygomatic root, the FR, and the HM to localize the bony tegmen over the tympanic cavity [11]. Baur et al. offered simply identifiable reference landmarks including the AE, the most lateral aspect of the Glaserian fissure and the FS and measured the distances between them to predict the location of the MMA [12]. According to these researchers, the internal landmarks including the HM and Bill’s bar (the vertical crest in the fundus of the internal auditory canal) are in a single plane with the zygomatic root [11].
After the ArE forming the anterior limit of the GF, the anterior root continues in front as a bony ridge that forms the posterior boundary of the infratemporal fossa, which is a small triangular area transmitting the neurovascular structures between the pterygopalatine fossa and temporal fossa. Then, a serrated anterior end of the zygomatic process passes straight forward and articulates with the temporal process of the zygomatic bone and completes the zygomatic arch. The temporal fascia inserts to this arch and the temporal line superiorly and also the masseter muscle origins from the arch inferiorly. The lateral temporomandibular ligament attaches to the AT, and the GF is covered with an articular disc to construct the synovial TMJ with the condyle of the mandible [5, 6, 7].
Anteriorly, the small part of squamous portion takes part in the infratemporal fossa formation with the zygomatic bone and the greater wing of the sphenoid bone. Below the zygomatic bone, the branches of the first and second mandibular parts of the MA with veins and the pterygoid plexus of veins, the mandibular and lingual nerves pass through the infratemporal fossa. During the infratemporal fossa approaching for surgical removal of tumors localized in the orbit, the maxillary and sphenoid sinuses, the detailed anatomical knowledge of these neurovascular structures is needed. Depending on the position of the infratemporal fossa below the floor of the middle cranial fossa and posterior to the maxilla, it is in close proximity with the parapharyngeal and masticator spaces. The parapharyngeal carotid artery enters the carotid canal (CC) behind the FS and foramen ovale. During transpterygoid infratemporal fossa approach, the positions of these surgical landmarks can be used to prevent ICA injury [13].
Ossification of the squamous portion starts intramembranously from one center around the zygomatic process at the 2nd month. At birth it fuses with the other membranous bone, tympanic portion. Normally, at birth the temporal bone consists of three parts; the petrous, squamous, and the tympanic [1].
The mastoid portion forms the pneumatized thick posterior part of the temporal bone. It fuses with the squamous portion antero-superiorly and the tympanic portion anteriorly and the petrous portion anteromedially. It has three borders and two surfaces [5, 6].
Posteriorly, it articulates with the squamous part of the occipital bone between lateral angle and the jugular process and constructs the occipitomastoid suture. Inferiorly, the mastoid process extends as a rough and conical shaped projection and filled with mastoid cells variable in shape and size. Anteriorly, it associates with the tympanic portions of the temporal bone to form the tympano-mastoid suture, and the inferior auricular branch of the vagus nerve (Arnold’s nerve) exits through this suture [5, 14, 15].
Near the squamo-mastoid suture, the occipital belly of occipito-frontalis and auricularis posterior muscles attach on the external surface that is perforated by numerous small foramina. At the posterior border of the mastoid portion or the occipitomastoid suture, the largest one, mastoid foramen is located and transmits an emissary vein connecting the SS with the posterior auricular vein and a branch of occipital artery to the dura mater (Figure 1). The mastoid process serves for the attachment of the sternocleidomastoid, splenius capitis, and longissimus capitis muscles and shows variations in shape and size with respect to sex. The posterior belly of the digastric muscle is originated from the mastoid notch (digastric fossa), which is a depression on the inferomedial margin of the mastoid process (Figure 1). More medial to the notch lies a sulcus, the occipital sulcus, forming a groove for the occipital artery [4, 6].
The internal surface includes a well-defined and curved sigmoid sulcus lying along its junction with the posterior surface of petrous part and lodges the SS, partially the transverse sinus, which are separated from mastoid air cells by a thin plate of bone. The mastoid foramen transmitting the mastoid emissary vein may be open to this sulcus. The SS begins as the continuation of the transverse sinus and lies downward in a S-shaped groove and opens into the superior jugular bulb. There is a sinodural angle between the dura plates of the SS and middle and posterior cranial fossae [2, 5, 9, 16].
The mastoid process shows tree types of pneumatization patterns including pneumatic (full air cell), sclerotic (solid mass of bone), and mixed (air cells and bone marrow) types. Especially, in the anterosuperior part of the mastoid process, there is an irregular cavity that is larger than other mastoid cells and called MA, which corresponds to the cribriform area. It is covered with the mucous membrane of the tympanic cavity and communicates anteriorly with the epitympanic recess of the middle ear via the aditus ad antrum. The tegmen antri, a roof of the MA, separates it from the middle cranial fossa. During embryonic period, the squamous and petrous portions fused each other and forms the petro-squamous suture. In adults, it forms a thin bony septum, the Körner’s septum, by extending into the mastoid process [1, 4, 6, 9, 17]. Körner’s septum divides the mastoid air cells in the mastoid process into a deep petrous part medially and a superficial squamous part laterally. The petro-squamosal sinus or the mastoid emissary vein may infrequently be observed along this septum. During mastoidectomy or transmastoid approaches, awareness of this crucial landmark and its variations is essential to avoid iatrogenic complications. The squamous part starts to develop at 8th week, whereas the petrous part develops later at 6th months during embryogenesis, and each part opens into the MA separately [1]. Also, the mastoid cells are separated by bony plates from the adjacent structures such as the posterior wall of the EAM anteriorly, tegmen plate superiorly, SS posteriorly, digastric ridge inferiorly, and the lateral semicircular canal (LSC) or solid triangle medially. The solid triangle is a compact bony angle between three SCs. During the mastoidectomy, all the air cells around this septum and adjacent bony structures should be removed without damaging the bony plates. To avoid iatrogenic injury to the adjacent structures, the MA must be open superiorly toward TT. The tympano-mastoid suture at the posterior wall of the MA is surface marking of the course of the vertical portion of the facial nerve (FN) [9, 16, 18]. Peris-Celda et al. reported that the parietal notch corresponds to the posterior petrosal point and the SS (the transverse-SS junction) in 66 and 34% of the cases, respectively [9].
Ossification of the mastoid portion is endochondral which is identical to the petrous and styloid portions. At birth, the mastoid process is absent, and the MA is invisible and covered by a thin bony plate that is extension of the squamous portion. At the first year, the mastoid process becomes prominent and the petro-squamous suture arises. The antrum can be seen obviously at about the fifth year. During puberty, the thickness of the process increases, and it becomes pneumatic that is lined by mucous membrane. In adults, the mastoid process may not contain air cells in 20% cases [1, 2, 17].
An annular shaped part of the temporal bone forms the tympano-mastoid suture posteriorly and the squamo-tympanic suture superiorly (Figure 1). Medially, it fuses with the petrous portion, whereas a free lateral part of it constructs the major part of the EAM and also serves an attachment for the cartilaginous part of the external auditory canal (EAC). Its inferior margin is free, and it has two parts on the lateral surface; posterosuperior part forms the EAM, and anteroinferior part limits the mandibular fossa posteriorly [5, 19].
Medially, just above the GF, this suture is subdivided by a thin tegmen part of the petrous portion into two: the petrotympanic fissure posteriorly and the petro-squamosal fissure anteriorly. Lateral part of this upper margin fuses with the back of the PGP to form the nonarticular part of the GF. Inferiorly, the lateral part of the margin gives an attachment for the deep part of the parotid fascia and forms the vaginal process, which wraps the root of the styloid process laterally [2, 4].
Laterally, external surface is bounded by the cartilaginous part of the EAC which extends from the auricle to the tympanic membrane. The EAC is an S-shaped tube, about 2.5 cm in long, that is composed of the lateral third cartilaginous part and the medial two-thirds osseous part [14, 15, 18]. The tympanic part constructs the anterior wall and floor and the lower part of posterior wall of the EAM, whereas the squamous part forms the superior and upper part of the posterior wall of it (Figure 1). The tympanic part grows from the tympanic ring, which is open U-shaped possessing two edge anterior and posterior. The anterior edge forms the tympano-squamous fissure within the anterosuperior part of the EAM and the petrotympanic fissure within the middle ear, whereas the posterior edge forms the tympano-mastoid fissure within the posteroinferior part of the EAM near the stylomastoid foramen (SMF) [2, 4, 19].
The internal surface fuses with the petrous portion and forms the tympanic sulcus for the lodgement of the tympanic membrane, which forms an angle about 55° with the floor of the EAM and separates the external and middle ear (ME). At the upper part, the tympanic sulcus does not fuse each other by forming the greater and lesser tympanic spines and a notch called Rivinus between them. This notch is closed by the pars flaccida of the tympanic membrane. The notch of Rivinus corresponds to the junction between the squamous and tympanic portions [1, 4, 14, 20].
Ossification starts from the four centers around the tympanic ring at the end of the embryonic period (8th week) via intramembranous ossification of the EAM. The tympanic ring at first is nearly straight and then turns into horseshoe shape (annular) and then, the open arms extending upwards terminate in a notch for the location of the tympanic membrane between them. After birth, the upper segment of the tympanic bone grows rapidly but because of the gradual development of the lower segment, a deep notch (tympanic foramen) is left in the anterior part of the bony EAM. Normally, the tympanic ring fuses until the age of 5 year but a dehiscence may persist (range 4.6−22.7%) at the anteroinferior aspect of the EAM, called foramen of Huschke (foramen tympanicum). This fusion defect is not a true foramen, but it may cause a connection between the EAM and the posteromedial part of the TMJ and results in TMJ herniation and the secretion of the parotid gland and also the dissemination of tumor and infections into the EAM [1, 14, 19, 20]. Anteriorly, the EAM may communicate with the retromandibular part of the parotid gland via the fissures of Santorini within the anterior cartilage. Peris-Celda et al. reported that the SSC dehiscence can be observed approximately 1.5 cm posterior to the middle point of the EAM in 86% of the cases [9]. In newborn, the tympanic membrane is infiltrated with air and the tympanic ring forms a bony plate, which may cause the development of a cleft, the auricular fissure, posteriorly and a cleft, the tympano-squamous fissure, anteriorly [19, 20].
The petrous portion is a dense pyramid-shaped bone and composed of the labyrinth of the internal ear, the tympanic cavity of the middle ear and a bony part of the auditory Eustachian tube (ET), and canals for the passage of the ICA and the FN. It is ossified from the otic capsule by forming a 45° angle with the horizontal axis. It has a base, an apex, and three surfaces and three borders [3, 4, 21].
Superiorly, the petrous ridge is the longest border and a boundary between the posterior part of the middle cranial fossa (the anterior surface of the petrous part) and the anterior part of the posterior cranial fossa (the posterior surface of the petrous part). It contains a groove that lodges the superior petrosal sinus (SPS) and the lateral margin of tentorium cerebelli attaches to this margin (Figure 2). Posteriorly, the medial part of the posterior margin articulates with the basilar part of occipital bone along the petro-clival fissure and forms a groove that lodges the inferior petrosal sinus (IPS) that extends from the posteroinferior part of the cavernous sinus to the internal jugular vein (IJV). The lateral part of the posterior margin is free and limits the jugular foramen (JF) supero-laterally and has a triangular notch for the lodgement of the inferior ganglion of the glossopharyngeal nerve (Jacobson’s nerve = GPN). Anterolateral border is formed by the ET extending from the anteroinferior wall of the tympanic cavity to the nasopharynx [3, 4, 9].
The surface landmarks on the anterior surface of the petrous portion: a, petrous ridge (sulcus of the superior petrosal sinus); b, arcuate eminence; c, tegmen tympani; d, sulcus of the lesser petrosal nerve; e, sulcus of the greater petrosal nerve; f, trigeminal impression; g, petrous apex; ıocc, internal opening of carotid canal.
The base is integrated with the inner surface of the squamous and mastoid portions, whereas the apex forms the posterolateral margin of the foramen lacerum (FL) and faces the Meckel’s cave medially. There is a fibrocartilage connection between the apex and the clivus. The internal opening of the carotid canal (IOCC) is observed at the apex for the intracranial entry of the ICA. At the anterolateral part of the FL, the petro-sphenoid ligament connects the tip of the apex to the dorsum sellae of the sphenoid and the abducent nerve lies below this ligament and enters the cavernous sinus adjoining the ICA [1, 7, 16].
Anterior surface describes a triangular area, between the linear lines as follows: a horizontal line that starts from the preauricular burrhole in front of the tragus to petrous apex at the FL and passes through the FS anteriorly, the petrous ridge posteriorly and the petro-squamous suture, which lies along the junction of the petrous pyramid with the vertical part of the squamous portion laterally [3, 16, 22]. It consists of some marking landmarks (Figure 2).
The anteromedial two-third of the musculotubal canal is cartilaginous, whereas the posterolateral third is bony. The bony part consists of two small canals that are separated by a thin bony septum at the lateral part the petrous portion. The tensor tympani muscle passes through the superior semicanal, whereas the inferior semicanal forms the bony portion of the ET. The tensor tympani muscle originates from the greater wing of the sphenoid and inserts into the upper part of the medial surface of the handle of malleus after making a bend around the processus cochleariformis in the tympanic cavity [4, 6]. The ET lies between the tympanic orifice and the isthmus, which has the smallest diameter at the intersection point of the petrous and squamous parts of the temporal bone just behind the sphenoid spine. Brown et al. reported that the ET is subdivided by genu within the membranocartilaginous part into two portions; posterior horizontal ET between the genu and the anterior attachment of the tympanic membrane ridge, whereas the anterior vertical ET lies from the genu to the nasopharyngeal orifice and opens into the nasopharynx. During endoscopic eustachian tube obliteration, the ET is cannulated to treat refractory CSF rhinorrhea by identifying three anatomic parameters: the ET length, isthmus diameter, and genu location. According to a new surgical classification, the cartilaginous portion of the ET is divided into the petrous, lacerum, pterygoid, and nasopharyngeal parts. The bony part attaches to the ET sulcus or sulcus tuba, which is contiguous to the FL medially. The FL is located in the incomplete confluence of the union of the body and the lingular process of the greater sphenoid wing anteriorly, the clivus of the occipital bone medially and the petrous apex posteriorly and covered with the fibrocartilaginous tissue that separates the ET from the ICA [23].
The internal opening of the CC is located near the FL for the passage of the ICA, which is freed at the petrous apex into the cavernous sinus (Figure 2). It is localized medial to the ET, below the greater superficial petrosal nerve (GSPN), a branch of the FN and the trigeminal ganglion [1, 3, 4]. The petrous segment of the ICA within the CC has four anatomic parts, called vertical, posterior genu, horizontal, and anterior genu. During endoscopic endonasal surgery, the junctional part of the ET at the sphenoid spine and FS is crucial landmark to identify and protect the petrous segment of the ICA [13]. The anatomical and surgical relationships between the ET and the petrous segment of the ICA are as follows:
The first curve, posterior genu is located at the level of the bulging basal turn of the cochlea within the bend of the CC. Laterally, the bony part of the ET and the tendon of the tensor tympani muscle; posterolaterally, the promontory and posterosuperiorly, geniculate ganglion are paramount landmarks for the posterior genu of the ICA. The V3 lying anteromedially to the FS and the parapharyngeal segment of the ICA, which passes posteroinferiorly to the sphenoid spine, are critical landmarks. Posterolaterally, the petroclival fissure cartilage is an important landmark to separate the pharyngobasilar fascia from the anterior genu of ICA.
The second turn of the ICA, anterior genu, above the fibrous tissue of the FL is in close proximity to the lacerum segment of the cartilaginous ET laterally and continues as the paraclival ICA in the carotid groove. During the endoscopic approach, the Vidian artery and nerve (VN) are critical landmarks for the second curve of the ICA.
For safe manipulation of the horizontal part of the ICA, the GSPN can be used as surgical landmark. Above the anterolateral margin of the FL the union of the GSPN and the deep petrosal branch of the carotid neural plexus forms the VN which is located anteroinferiorly and lateral to the second turn of the ICA. Malignancies that involve the petrous apex or the carotid artery require the extended endoscopic endonasl approach (EEA). During this procedure, the medial and lateral optico–carotid recesses in the cavernous sinus and the vidian canal (VC) are vital surgical landmarks, which allow to identify the position of the ICA for safe surgical resection near the ICA [13].
At the apex above the CC, a shallow fossa called trigeminal impression (Figure 2) is located for the lodgement of the sensory ganglion of the TN (semilunar ganglion or Gasser’s ganglion) that is covered by a pouch-shaped dura mater called Meckel’s cave [3]. Vascular compression and arachnoid adherence of the TN branches result in trigeminal neuralgia. During endoscopic vascular decompression and Meckel’s cave approaches, the VC, the bone between V2 and the VC and the pneumatization of the sphenoid sinus form a safe route to access and to decompress Gasser’s ganglion with branches, the cranial nerves (III, IV, VI), and the petrous ICA [13, 23].
Behind the trigeminal impression, the roof of the IAM is indicated as a shallow fossa, then it continues with the AE, which is a surgical landmark for the middle fossa approach and located at the junction of the posterior third and the anterior two-thirds of the petrous portion (Figure 2). It is a valuable guide to signify the SSC and the roof of the vestibule up to 93% of the temporal bones [19, 22].
The TT is a thin bony layer covering all of the anterior surface (Figure 2). It forms the roof of the mucosal line including from behind to forward the MA, tympanic cavity and ET which are lined with mucosa. Also, its lateral edge turns downward to subdivide the squamo-tympanic fissure into two parts [1, 3].
On the TT, a bony roof of the geniculate ganglion, there are two foramina, which continue as a small groove adjoining anteromedially; the medial one starts from the hiatus of the facial canal and lodges the GSPN, a branch of the FN and the petrosal branch of the MMA, whereas the lateral one lodges the lesser superficial petrosal nerve, a branch of GPN (Figure 2) [3, 9, 16, 22].
Kaen et al. described the “VELPPHA” area indicating the posterior limit of the transpterygoid EEA. It is composed of the VC (V), the ET (E), the FL (L), the petroclival fissure (P), the pharyngobasilar fascia (PHA), and multiple cartilaginous fibers between them. The posterior opening of the VC, the posterior limit of surgical corridor in the transpterygoid approach, is located above the ET and below the petrous ICA. Behind the posterior margin of the medial pterygoid process, the superomedial border of the ET attaches to the cartilaginous fibers of the FL. The petroclival fissure is situated between the lateral border of the clivus (occipital bone) and the petrous part of the temporal bone and lodges the IPS. The horizontal segment of the petrous ICA turns upward at the medial border of the petrous apex to form the anterior genu of the ICA, and then it continues as the lacerum segment, second vertical segment of the ICA. So, the VC-ET junction is a safe and critical landmark for efficient localization of the lacerum segment of the ICA, as part of the transpterygoid extension of EEA [24].
Tayebi Meybodi et al. described the pterygoclival ligament as a thickened extension of the pharyngobasilar fascia from the pterygoid process to the anteromedial aspect of the lacerum segment of the ICA and reported that the course of the pterygoclival ligament consistently refers to the anteromedial aspect of the lacerum ICA. So, they suggested that the pterygoclival ligament can be used as a safe landmark in case of tumor invasion of the VN, and drilling along the medial aspect of this ligament is more reliable way compared with the VN to avoid the ICA injury during extended EEA. Also, they remarked that this ligament may localize in a venous compartment, which is in contact with the cavernous sinus superiorly and the pterygoid venous plexus posteroinferiorly [25].
The posterior surface, anterior wall of the posterior cranial fossa, is encircled by a venous triangle that is formed by the grooves for SS posteriorly and SPS at the petrous ridge and IPS at the junction of the pars lateralis of the occipital bone and the temporal bone anteroinferiorly. The SS drains into the bulb of the IJV, which exists from the JF together with the cranial nerves (IX-XI) [1, 6, 9].
The IAM is a short canal, about 1 cm long, and has a large orifice, which allows passage of the vestibulocochlear nerve below the FN, the superficial petrosal artery (a branch of the MMA) and the labyrinthine artery (branch of the basilar artery). The bottom (fundus) of the IAM is subdivided into unequal superior and inferior portions by a transverse falciform crest, and into the anterior and posterior portions by a vertical segment, Bill’s bar, respectively (Figure 3) [2, 15]. The localization of the nerves within the IAM is determined by a triangular shaped Bill’s bar as follows; posteriorly the superior and inferior vestibular nerves, anteroinferiorly the cochlear nerve, anterosuperiorly the FN and nervus intermedius pass through the foramina of the fundus (Figure 3) Mortazavi [1, 4, 6].
The aqueductus vestibuli is a bony canal which contains the saccus and ductus endolymphaticus. Its opening is an oblique slit behind the IAM (Figure 3). The endolymphatic sac is located at the lateral part of the posterior surface medial to the posterior SSC [2, 18].
The subarcuate fossa is an indistinct depression (large in new born) located behind the IAM (Figure 3) and transmits a small vein and the subarcuate artery, which is a branch of the meatal segment of the anterior inferior cerebellar artery [4, 5, 9, 14].
The surface landmarks on the posterior surface of the petrous portion: a, petrous ridge; b, arcuate eminence; h, internal acoustic meatus; ı, subarcuate fossa; j, aqueductus vestibuli; k, sigmoid sinus sulcus; m, sulcus of the middle meningeal artery; 12, mastoid foramen.
The inferior surface articulates with the basilar part of occipital bone medially, and the greater wing of the sphenoid bone anteriorly and forms an irregular external surface of the base of the skull. Below the apex, there is a quadrilateral area that serves as an attachment for the levator veli palatini muscle. The lateral part of this area merges with the posterior margin of the greater wing of sphenoid to form the sulcus tuba in front of the cartilaginous portion of the auditory tube [4, 5, 21]. It presents some anatomical landmarks as follows:
The external opening of the CC, which shows an inverted L-shape course, forms the entrance for the ICA, which is surrounded by a plexus of sympathetic nerves (Figure 4). The anterior margin of the horizontal segment of the CC is separated from the musculotubal canal by a thin layer of bone laterally [1, 5, 18].
The jugular fossa is a deep dome-shaped depression at the lateral wall of the JF and located behind the CC and below the floor of the tympanic cavity. It houses the superior bulb of the IJV and the mastoid canaliculus (Figure 4) for the entry of the Arnold’s nerve, which provides sensory innervation of the EAC and auricle [9, 15]. The jugular spine in the jugular notch of the occipital bone divides the JF into the pars nervosa (anterior) and pars venosa (posterior) [4, 5, 9]. Normally, the jugular bulb is located between the IJV and the horizontal course of the SS. Abnormalities of it (80% below the FN in the mastoid cavity) result in dehiscence of the adjacent structures such as: the mediolateral enlargement of the JB results in the vestibular aqueduct, PSC, and IAC dehiscence, whereas the anteroposterior enlargement of the JB may cause the FN dehiscence. Abnormal high riding JB shows both mediolateral and anteroposterior enlargement and results in dehiscence of the FN [26].
Between the jugular fossa and the CC, the inferior ganglion of the GPN is localized in a triangular depression, whereas the inferior tympanic canaliculus penetrates into wedge-shaped bony ridge and transmits the tympanic branch of the GPN and inferior tympanic artery. At the apex of this triangular depression, there is an external opening of the cochlear aqueduct (Figure 4), which connects the perilymphatic space to the subarachnoid space and transmits the cochlear vein [1, 5, 14].
Behind the CC the vaginal process which is the extension of the sharp lower border of the tympanic plate wraps the root of the styloid process (Figure 4). The lower border of that extension serves an attachment for the deep layer of parotid fascia [1, 3, 5, 6].
The surface landmarks on the inferior surface of the petrous portion: FM, fossa mandibularis; FS, foramen stylomastoideum; FJ, fossa jugularis; ET, eustachian tube; eocc, external opening of carotid canal; ıocc, internal opening of carotid canal; star: inferior tympanic canaliculus; arrowhead: cochlear aqueduct.
Internal structures in the petrous portion contain the ME and inner ear. The ME contains an air-filled tympanic cavity and the ossicular chain which is composed of the malleus, incus, and stapes [14]. The walls of the ME:
Lateral wall contains the tympanic membrane and the scutum pointed infero-medially from the squamous portion. The tympanic membrane has two parts; pars flaccida is located in a fibrocartilaginous ring called the tympanic sulcus and susceptible to perforations and pars tensa is situated in the notch of Rivinus above the lateral process of the malleus. At the medial surface of the membrane a depression called umbo is formed by attachment of the manubrium of the malleus.
Medial wall consists of the cochlear promontory, the FC, the oval and round windows. It is divided into three part by the bony ridges: the ponticulus superiorly and the subiculum inferiorly. The oval window (vestibular window) is located above the ponticulus whereas the round window (cochlear window) is below the subiculum, and the tympanic sinus between them is located medial to the FC. The vestibular window is closed by the base of the stapes. The facial recess lies below the lateral SSC and superolateral to the oval window.
Superior wall, the TT, which forms the roof of the ME.
Inferior wall is a bony roof of the IJV.
Anterior wall includes the anterior epitympanic recess superiorly, below it the tensor tympani muscle lies posteriorly and attaches to the neck of the malleus after turning laterally. The orifice of the ET and below it the CC is located inferiorly.
Posterior wall consists of the pyramidal eminence, epitympanum, and facial recess. The stapedius muscle passes through the pyramidal eminence and inserts to the head of the stapes [2, 5, 7, 14, 18].
The tympanic cavity is lined with the mucous membrane that extending into the MA posteriorly and the ET anteriorly. This cavity consists of three parts changing according to the level of the tympanic membrane; the epitympanum (superior to the level of the tympanic membrane), mesotympanum (at the level of the tympanic membrane), and hypotympanum (inferior to the level of the tympanic membrane). The hypotympanum has the orifice of the ET. At the lateral part of the epitympanum below the lateral malleal ligament there is the Prussak space which is bounded by the neck of the malleus medially and the pars flaccida and scutum laterally [2, 3, 5, 14].
Inner ear is comprised of the otic capsule (osseous labyrinth), which surrounds the membranous labyrinth and is divided into three parts from anterior to posterior including the cochlea, vestibule, and three SCs [14]. Cochlea is the spiral shaped bony labyrinth of the inner ear that looks like a snail shell making 2¾ turns about the modiolus and consists of the vestibular and the tympanic and the cochlear ducts, which are formed by an inner membranous partition. The vestibular duct (scala vestibuli) locates at the superior part of the cochlear canal and contains perilymph (rich in sodium ions) and is limited by the oval window, and is separated from the cochlear duct by Reissner’s membrane. The cochlear duct (scala media) locates at the middle part of the cochlear canal and contains endolymph (rich in potassium ions) and is separated from the tympanic duct by the basilar membrane, which has the Organ of Corti including the sensory hair cells. The stereocilia of these cells perceives the potential difference between the perilymph and the endolymph and converts that motion to electrical signals and finally hearing occurs. The tympanic duct (scala tympani) locates at the inferior part of the cochlear canal and contains perilymph as the vestibular duct and is limited by the round window [3, 5, 14, 15]. Vestibule contains the utricle and saccule. SSCs containing three semicircular ducts organized like three flower leafs that join the vestibule. They are located perpendicular to each other; the superior corresponds to the AE, the posterior is parallel to the posterior surface of the pyramid, and the lateral is perpendicular the mucosal plane and angled at 30°from the transverse plane [3, 15].
The FN passes through the anterosuperior part of the IAM and enters the fallopian canal (FC). It contains motor, sensory, and parasympathetic fibers and has six segments as follows:
Cisternal segment lies from the brain stem to the IAM. This part runs together with the cisternal part of vestibulocochlear nerve in same pia mater coverage.
Meatal segment is the smallest part of the FC and contains Bill’s bar as an important landmark.
Petrous (labyrinthine) segment forms first genu (geniculate ganglion) above the cochlea at the lateral wall of the ME and gives a branch named as GSPN. Then, it enters the tympanic cavity and forms an angle ranging from 19 to 107° with tympanic segment of the FC [7, 20]. Because of this segment is the narrowest part and lack of arterial anastomoses, it is susceptible to embolic attacks and vascular compression.
Tympanic segment (first part) starts from first genu and turns backwards to lie in a thin-walled bony canal that runs evenly between the lateral SSC superiorly and the oval window inferiorly and medial to the incus. A dehiscence of the bony canal is more common at this segment in average 41–75%.
Pyramidal segment (second part of the tympanic segment) forms second genu at the posterior wall of the ME above the pyramidal process. It forms an angle ranging from 95 to 125° with mastoid segment of the FC [7, 20].
In the mastoid or vertical segment, the FN gives the acoustic branch for the stapedius muscle, the chorda tympani, and sensitive branch for the auricular region. This segment is located 5.50 mm anteromedially to the SS and extends from the level of the LSC to the digastric ridge (~3.8 mm). Then it exits the temporal bone at the SMF and enters the parotid gland [14, 27].
According to the classical description, the FC has four segments: labyrinthine, tympanic, pyramidal, and mastoid, but the meatal segment is important from an anatomical and surgical perspective. The stylomastoid artery, a branch of the posterior auricular or the occipital arteries, supplies the inferior parts of the FC up to the second genu and anastomoses directly with the petrosal branch of the MMA, which supplies the geniculate ganglion. The FC pathologies are composed of agenesis, aplasia, narrowing, and osteopetrosis of the canal, which result in complete or incomplete facial paralysis. Bell’s palsy depending on the activation of a dormant herpes virus, is responsible for 50% of peripheral FN palsies. The FC dehiscence can be congenital or secondary to the surgical intervention or pathology of adjacent structures and results in cerebrospinal fluid (CSF) otorrhea. Several surgical approaches, including the translabyrinthine, transcochlear and retrosigmoid, are used to treat the FC pathologies [27].
Ossification of the petrous portion begins from the 14 centers that fuse to form otic capsule and is completed at birth. The petrous portion develops from the cartilaginous differentiation of the mesenchyme by endochondral ossification at the 16th week of gestation. The cementum layer in teeth roots and petrous portion of the temporal bone contain the optimal endogenous DNA substrate which can provide information to specify the geographic location for genomic analyses [28]. Damgaard et al. reported that the prevalence of the endogenous DNA contents in nonpetrous bones and teeth is ranged from 0.3 to 20.7%, while the levels for petrous bones ranges between 37.4 and 85.4% [29]. Due to the high density and resistance to harsher climatic conditions of the petrous bone, the otic capsule of the petrous bone preserves DNA substrate extremely well and has much higher endogenous DNA level than the teeth by 5.2-fold on average. So, it is currently acknowledged as the optimal substrates for ancient genomic research [28, 29].
Kawase’s triangle: Borghei-Razavi et al. evaluated the safety of this posteromedial middle fossa triangle for removal of the tumors locating or spreading into the cerebellopontine angle and petroclival area. Kawase’s triangle was identified between the GSPN laterally, the geniculate ganglion at the AE posteriorly, and ganglion gasserian at the trigeminal impression anteriorly. During anterior petrosectomy for accessing the posterior cranial fossa via middle fossa, the GSPN forms the lateral border of the surgical approach (Figure 5) [30].
The surgical triangles on the anterior surface: Kawase’s triangle: Post-med (posteromedial triangle) and Glasscock’s triangle: post-lat (posterolateral triangle). FS, foramen spinosum; GG, geniculate ganglion; TI, trigeminal impression.
Glasscock’s triangle, or the posterolateral middle fossa triangle, is identified between the TN (V3), the geniculate ganglion at the AE and FS (Figure 5). The margins of this triangle are formed by a line between where the GSPN crosses under V3 and the FS medially, a line between the FS and geniculate ganglion laterally, and GSPN describing the base [3, 5, 16].
Rhomboid area (Kawase triangle+postmeatal area) is situated between the GPN, petrous ridge, AE, and the posterior border of the V3. A large tumor located in the midline skull base or spreading into the infratemporal and petroclival region even the cavernous sinus can be removed by extended EEA through V2-V3 corridor to avoid complications including ICA injury, IPS bleeding, TN injury and CSF leak [31].
Trautmann’s triangle is bounded by the SPS superiorly, SS posteriorly, and solid angle which is formed by three SCs anteriorly (Figure 6). In this triangle, the retro-labyrinthine tract from the MA, the endolymphatic sac, and the vestibular aqueduct are located [5, 9].
The surgical triangles on the posterior surface: Trautmann’s triangle margins are formed between the superior petrosal sinus superiorly, the sigmoid sinus posteriorly, and the semicircular canals antero-inferiorly. Star: Citelli’s angle (sinodural angle) is formed between the dural plates of the middle fossa superiorly, the posterior fossa anteriorly and the sigmoid sinus posteriorly.
Donaldson’s line is a surgical line that is parallel to the LSC whereas it is vertical to the posterior SSC and divide it into superior and inferior portions. Below this line medial to the labyrinth the endolymphatic sac is situated. Citelli’s angle (sinodural angle); is bounded by the middle fossa dura plate (SPS) superiorly, posterior fossa dura plate (bony plate covering the MA) anteriorly and the SS posteriorly (Figure 6). During mastoidectomy the air cells in this triangle should be removed [1, 5, 6].
In clinical applications, for fully understanding of the tridimensional architecture of the petrous portion, a reference lines and angles can be defined on the anterior and posterior surfaces from a superior view.
Peris-Celda et al. reported that the EAM and the IAM are located in the same coronal plane on the anterior surface forming surgical triangle [9]. Tawfik-Helika et al. separated the pyramid into four compartments and described two segmentation method to provide better understanding of the distributions of these compartments. They identified four compartments based on their connections: mucosal, cutaneous, neural, and vascular [3, 21].
The mucosal compartment consists of an air filled and mucosa lined cavities from anterior to posterior: the ET, ME, and the MA (Figure 7). The mucosal line in an oblique anteromedial direction extends along these structures and is used for segmental description of this pyramid, and all major anatomical landmarks can be identified relative to this axis for surgical approaches [3, 9, 21].
(A) The margins of the anterior surface of the left petrous portion from a superior view are shown posteriorly by a (thick black) line along the PR, petrous ridge; anteriorly by a (dashed black) line lying from the preauricular burrhole to PA, petrous apex and passing through the FS, foramen spinosum; and laterally by a (dashed white) line along the petro-squamous suture. OC, optic canal; ACP, anterior clinoid process; FL, foramen lacerum; SOF, superior orbital fissure; FR, foramen rotundum; FO, foramen ovale; MMA, middle meningeal artery; IOCC, internal opening of carotid canal; GSPN, greater petrosal nerve; AE, arcuate eminence; TT, tegmen tympani; JF, jugular foramen; IAM, internal acoustic meatus; SSS, sulcus sigmoid sinus. (B) The segmentation of the left petrous pyramid into four compartments including mucosal, cutaneous, neural, and vascular is shown on the left petrous portion.
Extending the mucosal line posteriorly, the MA is separated into medial and lateral parts, whereas anteriorly, the bony portion of the ET is localized at the junction of the petrous and squamous parts and the cartilaginous part opens into the pharynx anteriorly. Medially the line passing through the sulcus of the GSPN and laterally a straight line lying between the foramen ovale and FS are parallel to this line (Figure 7) [3, 9, 21].
The cutaneous compartment is composed of the EAM, which is covered by the skin and separated from the ME by the tympanic membrane medially.
The neural compartment is composed of the otic capsule, which is located medial to ME and the mucosal line. In this bony container, the cochlea, vestibule, and SCs are located from anterior to posterior around the fundus of the IAM (Figure 7).
The vascular compartment is composed of the ICA. The axis passing through the horizontal part of the CC is parallel and medial to the mucosal line (Figure 7) [3]. Moreover, Tawfik-Helika et al. described X and V segmentation methods to advance and enhance education of the compartments.
The X method divides the petrous pyramid into four spaces by using two reference lines intersecting with each other at the ME; the mucosal line and the EAM-IAM line form the X letter (Figures 8 and 9). These four spaces around the ME and the contents in it are as follows:
The anteromedial space—the cochlea and the petrous apex including the ICA
The anterolateral space—the roof of the TMJ
The posterolateral space—the lateral part of the MA
The posteromedial space—the posterior labyrinth and the medial part of the MA
Schematic representations of the segmentation of the left petrous portion by using X and V methods.
Schematic representation of the external and internal landmarks on the left petrous portion. V, trigeminal nerve and branches (V1, V2, V3); TI, trigeminal impression; IOCC, internal opening of carotid canal; ET, Eustachian tube; GG, geniculate ganglion; ME, middle ear; MA; mastoid antrum; EAM, external acoustic meatus; TMJ, temporomandibular joint; SCCs, semicircular canals; IAM, internal acoustic meatus; VII, facial nerve; VIII, vestibulocochlear nerve; IX, glossopharyngeal nerve; X, vagus nerve; XI, accessory nerve.
The V method arranges five segments around the mucosal line (Figures 8 and 9) These five segments and the contents in it are as follows:
The petrous apex segment—the ICA medial to the ET
The otic capsule segment—the IAM, cochlea, vestibule and SCs
The mastoid segment—the angle around the MA
The EAM segment—the lateral part of the ME
The TMJ segment—the roof of the TMJ lateral to the ET [3].
Detailed description of the temporal anatomy pointing to relationships between internal and external landmarks and a holistic approach including X an V segmentation methods that break down the petrous pyramid into spaces and compartments can provide an easy way to understand and to use surgical applications. The compartmental approach can be helpful in the fields of education and radiological applications as well as surgery.
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