Comparative quantification between two different bone marrow aspiration systems and bone marrow concentrate, in a bilateral patient model.
\r\n\tThere will be a chapter on secondary causes of sexual dysfunction disorders related to diabetes, cardiovascular disease, and obesity. A chapter on remedial measures to enhance sexual activity and maintain human relationships will be discussed. As there is a growing number of cancer survivors a chapter on cancer-related sexual dysfunction will be welcomed for including it.
",isbn:null,printIsbn:null,pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,isNomenclature:!1,hash:"b988fda30a4e2364ee9d47e417bd0ba9",bookSignature:"Dr. Dhastagir Sultan Sheriff",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11889.jpg",keywords:"Sex, Sexual Response Cycle, Erection, Premature Ejaculation, Libido, Orgasm, Painful Intercourse, Psychological, Female, Lack of Desire, Erectile Disorders, Pain Disorders",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"April 8th 2022",dateEndSecondStepPublish:"May 6th 2022",dateEndThirdStepPublish:"July 5th 2022",dateEndFourthStepPublish:"September 23rd 2022",dateEndFifthStepPublish:"November 22nd 2022",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"3 months",secondStepPassed:!0,areRegistrationsClosed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Dhastagir Sultan Sheriff is a life member of the European Society for Human Reproduction and Early Human Development, Association of Physiologists and Pharmacologists of India, member of the National Academy of Medical Sciences, New Delhi, and resource person for UNESCO for Medical and Bioethics. Dr. Sheriff has authored five books including a textbook on medical biochemistry with additional interest in human sexology. He has done extensive research in andrology, sex education, and counseling.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"167875",title:"Dr.",name:"Dhastagir Sultan",middleName:null,surname:"Sheriff",slug:"dhastagir-sultan-sheriff",fullName:"Dhastagir Sultan Sheriff",profilePictureURL:"https://mts.intechopen.com/storage/users/167875/images/system/167875.jpg",biography:"Dhastagir Sultan Sheriff is a life member of the European Society for Human Reproduction and Early Human Development, Association of Physiologists and Pharmacologists of India, member of the National Academy of Medical Sciences, New Delhi, and resource person for UNESCO for Medical and Bioethics. Dr. Sheriff has authored five books including a textbook on medical biochemistry with additional interest in human sexology. He had editorials written in the British Journal of Sexology, Journal of Royal Society of Medicine, Postgraduate Medicine, and Scientist. He was a former Rotarian, Citizen Ambassador, and was selected for the Ford Foundation Fellowship.",institutionString:"University of Benghazi",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"2",institution:{name:"University of Benghazi",institutionURL:null,country:{name:"Libya"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:null},relatedBooks:[{type:"book",id:"6934",title:"Psycho-Social Aspects of Human Sexuality and Ethics",subtitle:null,isOpenForSubmission:!1,hash:"44731b106aa0d1ab5c64a7394483c7d5",slug:"psycho-social-aspects-of-human-sexuality-and-ethics",bookSignature:"Dhastagir Sultan Sheriff",coverURL:"https://cdn.intechopen.com/books/images_new/6934.jpg",editedByType:"Edited by",editors:[{id:"167875",title:"Dr.",name:"Dhastagir Sultan",surname:"Sheriff",slug:"dhastagir-sultan-sheriff",fullName:"Dhastagir Sultan Sheriff"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"7163",title:"Infertility, Assisted Reproductive Technologies and Hormone Assays",subtitle:null,isOpenForSubmission:!1,hash:"6db6e4ccb7088f17f819121f7eb6424d",slug:"infertility-assisted-reproductive-technologies-and-hormone-assays",bookSignature:"Dhastagir Sultan Sheriff",coverURL:"https://cdn.intechopen.com/books/images_new/7163.jpg",editedByType:"Edited by",editors:[{id:"167875",title:"Dr.",name:"Dhastagir Sultan",surname:"Sheriff",slug:"dhastagir-sultan-sheriff",fullName:"Dhastagir Sultan Sheriff"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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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:"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:"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:"2270",title:"Fourier Transform",subtitle:"Materials Analysis",isOpenForSubmission:!1,hash:"5e094b066da527193e878e160b4772af",slug:"fourier-transform-materials-analysis",bookSignature:"Salih Mohammed Salih",coverURL:"https://cdn.intechopen.com/books/images_new/2270.jpg",editedByType:"Edited by",editors:[{id:"111691",title:"Dr.Ing.",name:"Salih",surname:"Salih",slug:"salih-salih",fullName:"Salih Salih"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"71327",title:"The Rationale of Autologously Prepared Bone Marrow Aspirate Concentrate for use in Regenerative Medicine Applications",doi:"10.5772/intechopen.91310",slug:"the-rationale-of-autologously-prepared-bone-marrow-aspirate-concentrate-for-use-in-regenerative-medi",body:'The objectives of regenerative medicine applications are to support the body to form new functional tissues to replace degenerative or defective ones and to provide therapeutic treatment for conditions where conventional therapies are inadequate. The human body has an endogenous system of regeneration through stem cells, as they are found almost in every type of tissue. Regenerative medicine treatment options using autologous stem cells can be safely executed by well-trained physicians at point of care (POC). This review is not meant to be exhaustive, but our aims are to shed light on the bone marrow progenitor and stem cell mechanisms and highlight present and future applications of autologous bone marrow-derived stem cells in this exciting new regenerative medicine discipline.
In this chapter a definition is provided on embryotic and non-embryotic stem cells, followed by an intensive review of non-embryotic autologous adult stem cells. The use of allogeneic MSCs, the fabrication of engineered constructs by seeding of natural or synthetic scaffolds with cells, released from autologous tissues will not be presented in this chapter, as only relatively few of these cell-based approaches have entered the clinical arena. In particular, we deliberate on the biology and clinical application of mesenchymal stem cells originating from freshly harvested bone marrow. We portray on the techniques of a marrow harvesting procedure using ultrasound and fluoroscopic techniques. Explicit scientific information is provided on the bone marrow aspirate cellular content, their specific biological functions and intercellular interactions, as these, among others, contribute to tissue regeneration following clinical regenerative medicine applications. Furthermore, we underline the differences between bone marrow aspirate and, centrifugated, bone marrow aspirate concentrate injectates, both prepared at point of care from freshly aspirated marrow.
Finally, a condensed literature review addressing a variety of clinical ortho-biological indications, spinal disorders, chronic wounds, and critical limb ischemia is provided. Regenerative medicine technologies, using marrow-derived mesenchymal stem cell-based therapies, as part of the regenerative medicine treatment armamentarium, offer solutions to a number of undeniable clinical conditions that have not been able to adequately result in a solution through the use of medicines or surgeries.
Becker, McCulloch, and Till first conducted experiments that lead to the discovery of stem cells in 1963. After injecting bone marrow cells into irradiated mice, nodules developed in proportion to the number of bone marrow cells injected, and they concluded that each nodule arose from a single marrow cell. At a later stage, they produced evidence that these cells were capable of endless self-renewal, which is as we know now, a fundamental feature of stem cells [1]. A stem cell is a type of cell that is non-specific/specialized in its function; in contrast, for instance, a heart or brain cell is functionally specific.
Generally, we recognize two types of stem cells, embryonic and non-embryonic, with two defining properties. Firstly, they have the capacity of self-renewal, therefore giving rise to more stem cells. Secondly, they are capable of differentiating into different lineages under appropriate conditions.
Embryonic stem cells (ESCs) are obtained from 5- to 12-day-old embryos, and they are pluripotent and have a high plasticity as they can differentiate into ectoderm, mesoderm, and endoderm layers, whereas non-embryonic stem cells (non-ESCs) are multipotent, and it appears that they are able to form multiple cell lineages which form an entire tissue, usually specific to one germ layer, e.g., adult stem cells [2].
The capability for stem cell potency, in combination with the relative ease to prepare bone marrow stem cell injectates, is an invaluable property for regenerative medicine cell-based therapies in general and more specifically to treat, e.g. musculoskeletal disorders (MSK-D), chronic wounds, and critical limb ischemia.
Non-ESCs are undifferentiated, and their proliferation potential compared to embryogenic stem cells is limited, as they have lost their pluripotent capability, placing them lower in the stem cell hierarchy. Nonetheless, it has been suggested that non-ESCs maintain their multipotent differentiation potential. Since they are categorized as allogenic products, they are commercially prepared from several allogenic sources, like amniotic fluid, umbilical cord, and Wharton’s jelly [3]. In this chapter we will only deliberate on non-embryotic, autologous adult bone marrow aspirate (BMA)-derived progenitor/stem cells and other bone marrow (BM) stromal cells, prepared at POC with dedicated and approved centrifuges for BM concentration.
Friedenstein and colleagues reported first on the isolation of bone marrow-derived stem cells from BM stroma and incubated it in plastic culture dishes and identified mesenchymal stem cells as colony-forming unit fibroblasts (CFU-Fs) [4]. The BM stroma is made up of a network of fibroblast-like cells and includes a subpopulation of multipotent cells which are able to generate the mesenchyme, known as the mass of tissue, that develops mainly from the mesoderm of the embryo subpopulation. The cells are referred to as mesenchymal stem cells (MSCs) [5]. The
The bone is an organ composed of cortical and trabecular bone, cartilage, and hematopoietic and connective tissues. The bone tissue has an essential role in the structure and protection of the human body. Spongy, or trabecular bone, is composed of a lattice of fine bone plates filled with hematopoietic marrow, fat-containing marrow, and arterial-venous sinusoidal blood vessels. Furthermore, it consists of bone cells at different developmental stages (including pre-osteoblasts, osteoblasts, and osteocytes), collagen fibrils, and calcium and phosphate deposits [8]. Arterial vessels enter the marrow through foramina nutricia and then divide into several arterioles. Small arterioles and capillaries from these vessels span throughout the bone marrow and supply sinusoids, which are interconnected by inter-sinusoidal capillaries [9]. The BM tissue is soft, similar to the peripheral blood, flexible connective tissue comprising the center and the epiphysis of bones, referred to as the BM cavity. In this place a variety of new blood cells are produced and ultimately released to the peripheral circulation.
We recognize two categories of bone marrow tissue: the red and yellow marrow. Depending on age, the red marrow is replaced by the yellow marrow. In adults, the red bone marrow is a rich source of bone marrow-derived cells and present in most skeletal system bones of the iliac crest, tibia, spine vertebrae, humerus, calcaneus, ribs, and near point of attachment of long bones of legs and arms. In this well-shielded environment, an estimate of 500 billion cells per day can be produced, in particular erythrocytes, granulocytes, and platelets [10]. Regenerative medicine applications have a focus on the use of the red bone marrow as it contains myeloid and lymphoid stem cells and MSCs. In contrast the yellow marrow consists primarily of fat cells with poor vascularity and is deprived of the multipotential MSCs [11].
The BM cavity in the trabecular bone is subdivided into four regions: endosteal, sub-endosteal, central, and perisinusoidal regions [12]. In Figure 1, the four regions, according to the model of Lambertsen and Weis, have been adopted and modified [13]. In general, the bone marrow consists of a hematopoietic component (parenchyma) and a vascular component (stroma). The parenchyma includes hematopoietic progenitor and hematopoietic stem cells (HSCs), which are localized close to the endosteum and around the blood vessels. BM stroma cells, including endothelial cells, are recognized as multipotential non-hematopoietic progenitor cells, capable of differentiating into various tissues of mesenchymal origin, including osteoblasts, chondrocytes, tenocytes, endothelial cells, myocytes, fibroblasts, nerves, and adipocytes, as verified in in vitro and partially in in vivo research [14, 15]. The bone marrow’s microvasculature includes single layers of endothelium arising in sinusoids, where they also contribute in rolling extravasations of leukocytic cells into and out of the BM tissue structures. The function of the vasculature and BM-derived endothelial cells is that they provide a barrier between the BM compartment as a functional and spatial entity from the extra-lymphoid BM section and the peripheral circulation, as described by Kopp et al. [9]. The endothelial cells likewise contribute to tissue regeneration, as endothelial precursor cells are essential in improving vascularization of damaged and degenerative tissue cells by the secretion of pro-angiopoietic factors of invading cells [16].
Bone marrow subdivisions. On the left side, the Aspire introducer (Aspire Bone Marrow Harvesting System™, EmCyte Corporation, Fort Myers, FL, USA) has passed the cortical bone entering the marrow cavity. The harvesting cannula is inserted through the introducer in the marrow cavity. On the right side, a representation of the subdivisions in the bone marrow cavity subdivisions is indicated, showing the endosteal, sub-endosteal, central, and perisinusoidal regions. The endosteal and sub-endosteal regions compose the endosteal niche, harboring the proliferative and quiescent HSC-MSC niches. The marrow tissue is extracted via the side holes of the harvesting cannula (adapted and modified from Lambertsen and Weis [
A
HSC niches are present in various (prenatal) tissues, like the aorta-gonad-mesonephros region and the yolk sac, followed by the placenta, fetal liver, spleen, and bone marrow. Postnatally, the bone marrow is the primary site of HSC presence [22]. The model of the HSC niche was first described by Schofield in 1978 [23], later confirmed by others, to describe the physiologically limited microenvironment in which HSCs, MSCs, and their progenitors reside in the bone cavity where they are enfolded by BM stromal cells [24], covered by the bony structure of the BM cavity. The stem cell niches in bone have been extensively described by Yin and Li, providing insights into the actions of osteoblastic and vascular niches, revealing central roles for numerous signaling and adhesion molecules [25]. A significant portion of these hematopoietic cells is found next to the endosteal bone surface, designating a clear role for osteoblasts in the regulation of HSCs and thus hematopoiesis [26]. Based on flow cytometry research by Kiel et al., HSCs are more likely than other hematopoietic cells to be immediately adjacent to a sinusoid, in the trabecular region of the BM [27]. This location suggests that HSCs and their niche may be directly, or indirectly, regulated by factors present near the bone planes. The HSC niche is comprised of many different niche constituents including osteoclasts, endothelial cells, fibroblasts, adipocytes, and the HSC progenitor cells [28].
The BM is highly vascularized, with large central arteries branching into progressively smaller microvessels like arterioles and transitioning into venous sinusoids near the bone (endosteal) surface. Therefore, it has been suggested that HSCs are maintained in a perivascular niche by endothelial or perivascular cells, as they are frequently located adjacent to the blood vessels [29]. These occurrences resulted in the expression of various perivascular mesenchymal cell makers CD146, stromal cell-derived factor-1 (SDF-1) also referred to as CXCL12, and Nestin-GFP, defining the heterogenous BM stroma cell composition [9], including the MSCs that surround the blood vessels [30]. The more perivascular nature of MSC niches was validated by Shi and Gronthos, demonstrating the expression of α-smooth muscle actin (αSMA) at perivascular sites, with the immunohistochemical localization of specific CD marker cells [31]. Mores studies confirmed the presence of MSCs in BM, expressing a Nestin-GFP transgene, localized and attached around the BM blood vessels and part of the perivascular HSC niche [32]. Kunisaki et al. indicated that most HSCs do not only have a perivascular presence, but they are preferentially located in the BM endosteal regions. The endosteal regions contain a complex network of stromal cells as well that have been implicated in HSC maintenance, including arteriolar and venous endothelial cells, pericytes, and chemokine (C-X-C) ligand 12 (CXCL12) reticular cells. Their study also suggested that quiescent HSCs localize preferentially to small arterioles near the endosteum, suggesting that distinct niches may exist for both quiescent and proliferating HSCs [33]. From all these findings, it can be concluded that pericytes are in fact MSCs, because they can differentiate in osteoblasts, chondrocytes, and adipocytes [34].
Megakaryocytes (MK) are the precursor cells of blood platelets. BM hematopoietic cells are responsible for platelet production. MK may regulate HSCs indirectly as they are closely associated with BM sinusoidal endothelium, extending cytoplasmic protrusions into the sinusoids to produce platelets. A direct regulation of HSC by MK through signaling of transforming growth factor beta 1 was established, with activation of quiescent HSCs and increased proliferation rate. In the event of a sudden response to systemic stress signaling, fibroblast growth factor-1 as part of the MK growth factor pool will start signaling HSCs and will overshadow the TGF-b1 signaling in order to stimulate high volumes of HSC expansion [35].
The role and function of the extracellular matrix (ECM) can be defined as key structural-functional components of cell niches, including soluble factors, cell-cell contacts, and cell-matrix adhesions present in these microenvironments. ECM components include fibrillar proteins, with, among others, collagen fibers, fibronectin, and other filamentous network components. The ECM’s mechanical stability is provided by collagen [36]. Other significant ECM components supporting the BM niches are glycosaminoglycans and mainly hyaluronic acid via its receptor CD44. The surface marker is also expressed by MSCs and HSCs [37]. Intracellular signaling in the ECM occurs through cytokine and growth factor membrane receptors, similar to the MSC niche. These cytokines and receptor activities contribute to cross talk between ECM components and MSC niches, provoking cell differentiation. For instance, Djouad et al. demonstrated that the induction of MSC differentiation towards chondrocytes in articular cartilage was induced and/or influenced by molecules from both the MSC niche and the ECM components of this microenvironment, leading to chondrogenic differentiation of MSCs [38]. Other studies suggested that ECM deposited by microvascular endothelial cells enhances MSC endotheliogenesis [39]. In general, no specific ECM components are identified that maintain MSCs in their immature state, as a niche matrix would do. However, it has become clear that the ECM can regulate MSC differentiation on a solitary basis, indicating potential applications for regenerative medicine applications and tissue engineering.
Exploiting BM preparations at POC seeks to overcome the limitations of ex vivo MSC culturing. Clinicians utilizing regenerative medicine applications have a growing interest in using the concentrated bone marrow products, since it is well acknowledged that BM is a plentiful source of MSCs, progenitors, and other cells residing in the trabecular part of flat and long bones, acquired via appropriately performed BMA procedures [40, 41]. The regenerative medicine market is rapidly growing, with many procedures performed in musculoskeletal, orthopedic, and spinal disorders, wound care management including critical limb ischemia, and tissue engineering [42, 43, 44, 45]. Several groups have mentioned some considerations when performing BM harvesting procedures, addressing a variety of factors that have an impact on patient comfort and the quality of the harvested BM. Emphasis was given to procedural safety issues when using harvesting needle systems, level of experience of the operator, the choice for concentration technology and centrifugation devices, and pain management [46]. Autologous regenerative medicine BM-MSC applications may range from a harvesting a low volume of BM and direct, unprocessed, tissue injection to the use of centrifugation protocols to concentrate and filter the BMA prior to injecting it in patients [47].
Various bone marrow needle harvesting systems are available on the market, each with their own proprietary design characteristics and thus marrow aspiration dynamics when transferring marrow cavity cells through a needle system into collection syringes. In Figure 2, three different needle systems are shown. Potentially, different needle design features might affect the quality and viability of the harvested BM tissue, as well as the cellular yields. Therefore, BM needle system features and harvesting dynamics are important considerations when considering BMA procedures. Physicians have been using a variety of harvesting needles during the last decades, including the traditional Jamshidi™ harvesting needle (Ranfac Corporation, Avon, MA, USA). Based on design differences, not every BMA is born equal, and cellular yields, composition, and viability might vary among harvesting devices. For interpretation purposes, some of the cellular difference between two newly developed BMA needle harvesting systems, the Aspire Bone Marrow Harvesting System™ and the Marrow Cellution Bone Marrow Aspiration Device™ (EmCyte Corporation, Fort Myers, FL, USA, and Ranfac Corporation, Avon, MA, USA, respectively) is shown. A significant difference between the two harvesting systems is that the Marrow Cellution device is developed and used by physicians for BMA aspiration with direct injection only, without filtration or processing prior to patient injection [47]. Therefore, these specimens mimic the marrow cavity cellular content and their specific cell concentrations. This includes a red blood cell (RBC) content and hematocrit which is similar to the peripheral blood values. Conditional negative forces occur with the syringe pull during marrow aspiration; this particular BMA injectate can have high plasma-free hemoglobin (PFH) concentrations, which cannot be removed from the injectate. The Aspire™ harvesting system is designed to penetrate the trabecular bone, maintaining a quiescent tissue environment during deployment and collection, contributing to a reduction in tissue activation, plasma-free hemoglobin content, and clotting. The Aspire™ harvesting system is intended to provide a BMA for centrifugation processing, leading in this occasion to the creation of PurePRP SupraPhysiologic BMC® (EmCyte Corporation, Fort Myers, FL, USA). In Table 1, comparative laboratory data between the abovementioned needle systems in a bilateral patient harvesting model are disclosed.
Bone marrow aspiration devices. (A) Jamshidi™ device (Ranfac Corporation, Avon, MA, USA), with a sharp and open distal tip, allows for more peripheral blood aspiration. (B) Aspire Bone Marrow Harvesting System™ (EmCyte Corporation, Fort Myers, FL, USA) consists of a separate trocar introducer and aspiration needle with a completely closed and blunt tip. The side holes of the aspiration needle are designed to minimize cellular trauma and hemolysis during aspiration. (C) Marrow Cellution Bone Marrow Aspiration Device™ (Ranfac Corporation, Avon, MA, USA) is used as an aspiration device only, to aspirate 10 ml of bone marrow, followed by unfiltered injection.
Laboratory parameters | BMA-MC 10 ml | BMA-A 10 ml | BMC 11 ml |
---|---|---|---|
TNC × 106/mL | 25.8 | 31.8 | 73.7 |
Platelets × 106/mL | 117 | 117 | 576 |
CD34+ (HSCs) × 105/mL | 1.42 | 1.12 | 2.51 |
CFU-F (MSCs) × 103/mL | 446 | 1.13 | 837 |
Hematocrit % | 36.2 | 36.2 | 9.8 |
RBCs × 109/mL | 4.02 | 4.08 | 1.44 |
PFH mg/dl | 913 | 721 | 299 |
Hemolysis % | 4.6 | 3.2 | 1.6 |
Cell viability % | 94.4 | 94.4 | 96.8 |
Comparative quantification between two different bone marrow aspiration systems and bone marrow concentrate, in a bilateral patient model.
BMA-MC, bone marrow aspirate Marrow Cellution System; BMA-A, bone marrow aspirate Aspire System; BMC, bone marrow concentrate; TNC, total nucleated cells; CD34+, hematopoietic stem cell marker/expression in bone marrow; CFU-F, colony-forming unit fibroblast: assay for bone marrow mesenchymal stem cell analysis, MSCs, mesenchymal stem cells. (BMA-MC, Marrow Cellution Device™—Ranfac Corporation, Avon, MA, USA; BMA-A, Aspire Bone Marrow Harvesting System™—EmCyte Corporation, Fort Myers, FL, USA).
In theory, a larger-volume BMA collection syringe should produce a stronger negative pressure and therefore harvest more MSCs. However, Hernigou et al. found that the aspiration of only 10–20% of the full syringe volume resulted in a higher MSC concentration in both 10 and 50 ml syringes, indicating that high-quality harvesting of MSCs requires a significant negative pressure in the marrow cavity to liberate MSCs. They also concluded that the collection of MSCs decreased as the syringe was filled, at a lower negative pressure [40]. Therefore, smaller syringes and thus smaller aspiration volumes result in higher MSC concentrations than with larger aspiration volumes [48]. This translates to the physical equation, “Negative Pressure = Pull Force/Plunger Surface Area,” resulting in the fact that with the same pull force and a smaller diameter syringe plunger, a higher negative pressure is created [49]. Lately, the authors use 10 ml syringes, employing a fast and intermittent pull technique to collect small volumes from different intra-trabecular sites (Figure 3). This is in accordance with a trend towards small-volume HPD aspiration techniques [50]. Another advantage for using 10 ml syringes is that anticoagulation protocols can be better managed. Smaller syringes fill considerably quicker than larger syringes, and smaller syringes can be adequately agitated with the anticoagulant to avoid clotting.
Bone marrow aspiration. After the aspiration needle has been advanced in the marrow cavity, the marrow is extracted using a firm, but a gentle, aspiration pressure is applied to the 10 ml syringe. The aspiration needle is easily rotated to collect marrow from a fresh area.
In order to perform BM-MSC procedures, a certain volume and quality of marrow tissue are required in order to prepare a bone marrow concentrate (BMC). The aspiration volume is contingent on the processing volume of the BMC concentration system that is being used. It is imperative to locate precisely the donor site, as MSCs are located in the marrow cavity subcortical area and around the blood vessels [19]. The precise delivery of local anesthetics and safe trocar placement are accomplished by using image guidance during aspiration procedure. In the following section, we focus on the posterior super iliac spine (PSIS) sites, as it is the most frequently reported anatomical site for BMA.
When the PSIS is targeted, patients are positioned in the prone position, while avoiding lumbar lordosis. Sonographic assessment using a portable ultrasound system with a 5–2 MHz low-frequency curvilinear transducer is positioned in a transverse plane over the hyperechoic bilateral sacral cornua, with the patient lying prone and the monitor screen in the line of sight of the operator. The probe is then translated contralaterally from the physician, keeping the hyperechoic sacrum visualized. Next, the probe is translated proximally, with the hyperechoic ilium coming into view, while maintaining the hyperechoic sacrum, until the most superficial depth of the ilium is reached, known as the PSIS, contralateral to the examiner [51]. After identification of the PSIS, the most superficial depth is confirmed in both transverse and longitudinal orientation (Figure 4). With the probe in the transverse plane at the PSIS, the slope of the iliac wing is noted for correct angulation of the BM trocar, and the most superficial depth of the PSIS is brought under the most medial aspect of the ultrasound probe. Using a sterile marker, a mark and directional line is made in both parallel and perpendicular orientations to form an intersection at the most superficial depth of the PSIS. This mark is maintained on the patient during skin preparation prior to the introduction off the BM trocar, and a superficial wheal of local anesthetic is placed at the point of planned trocar skin entry. Following the local antiseptic measures, sterile ultrasound gel is applied at the marked area, and a sterile probe cover is applied to the 5–2 MHz curvilinear array transducer. Typically, a mixture of local anesthetics is injected around the PSIS cortex and periosteal sleeve, under continued sonographic guidance, making sure to “walk off” the PSIS in four directions (superiorly, medially, laterally, and inferiorly), confirmed by sonographic guidance. The trocar is then introduced, using either a manual force that is perpendicular and slightly lateral to the patient, at 9–12 counterclockwise-clockwise rotations, or a mallet. The next steps of the procedure are subject to the implementation of the instructions for use provided by the manufacturer of the aspiration harvesting system.
Ultrasound imaging of the PSIS. With the probe in the transverse plane, the PSIS is confirmed, and the slope (D) of the iliac wing is noted for correct angulation of the BM trocar (B), and the most superficial depth (C) of the PSIS is brought under the most medial aspect of the ultrasound probe. Note: (A) indicates the skin surface, and (E) marks the depth of the PSIS below the skin, in this patient approximately 1.6 cm (courtesy of J. Rothenberg).
After proper patient positioning, the fluoroscopic equipment is installed to optimize the positioning for fluoroscopic imaging, using ipsilateral or contralateral oblique beam angulations for viewing the targeted PSIS site. The
Fluoroscopy imaging of the PSIS. General prone position of the patient on a fluoroscopic table for BMA. The
As MSCs represent a small population of BM cells [7], it is of critical importance to choose a BMA site that will yield the most MSCs. BM is relatively easy to harvest, largely available, and dispensable. Obviously, it is important that the BMA procedure is performed impeccably to obtain an optimal quality of viable BM tissue [5, 53]. In humans, the most common anatomical location to obtain BM is the iliac crest, but other BMA sites have been utilized (Table 2). Recently, McDaniel and co-workers, using a porcine model, reported that all studied anatomical bone marrow harvesting locations contained MSCs but the iliac crest was the most abundant source of MSCs [10]. These findings were confirmed in a clinical study, where MSCs were found in BM acquired from the metaphysis of the distal femur, the proximal tibia, and iliac crest. A similar MSC immunophenotype and differentiation potential (into the bone, fat, and cartilage) were seen in BMA from all sites. However, in their study the concentration of MSCs in the iliac crest was significantly higher than in samples from the distal femur and proximal tibia. More specifically, the literature indicates high yields of BM-MSCs acquired from the posterior superior iliac spine (PSIS) [50, 54]. Noteworthy, the group of Narbona-Carceles commented on the relative easiness and safety of lower extremity aspiration techniques [55].
Anterior superior iliac spine (ASIS) |
Posterior superior iliac spine (PSIS) |
Proximal tibia |
Distal tibia |
Distal femur |
Proximal humerus |
Vertebral body |
Calcaneus |
Sternum |
Bone marrow aspiration sites in humans.
The literature pronounces BMAs as a heterogenous mix of cells, referring in most instances to MSCs, HSCs, and mononuclear cells. Platelets, megakaryocytes, and RBCs are seldomly mentioned, let be subject to BM research [24].
The major function of the bone marrow is to generate blood cells. In particular in adults, marrow-derived HSCs are the principle cells of origin of all mature hematopoietic cell phenotypes. HSCs are adult stem cells with extensive self-renewal capabilities and are able to differentiate into specialized blood cells with key roles in some biological activities: control homeostasis balance, immune functions, and response to microorganisms and inflammation. Most HSCs are in a quiescent state within the BM niches. They respond to the signals after the balance of blood cells, or HSC pool, is disturbed from either intrinsic or extrinsic stimuli and signaling processes [56].
Hematopoiesis—the process by which mature blood cells are formed—has been studied intensely for over a century. The vast majority of hematopoiesis occurs in the bone marrow where it must balance enormous production needs. More than 500 billion blood cells are produced every day, with precise regulation of the number of each blood cell type released in the circulation [57]. Hematopoiesis is considered as a pyramidal/hierarchical process with cells of greatest maturation potential or primitiveness sitting at the top of the hierarchy and cells that have undergone terminal differentiation at the bottom. Terminally differentiated blood cells are classified into one of the two major lineages: those derived from myeloid lineages and from lymphoid progenitors. Myeloid cells include erythrocytes, platelets, and cells responsible for cellular immunity, such as macrophages and granulocytes (Figure 6). Cells derived from lymphoid progenitors are major participants in coordinating humeral and cellular immunity. Experimental data suggested that HSCs differentiate into hematopoietic progenitor cells that are capable of exponential proliferation as well as continuing the process of differentiation. Alternatively, HSCs are capable of self-replicating activities, which may give rise to one or two identical daughter cells. As a result, HSC activity must be tightly regulated to meet physiologic demands but also to protect HSCs from oncogenic, physical, and chemical damage to occur. The site or physical location that regulates self-renewal, proliferation, and differentiation of HSCs has been discussed in the HSC niche paragraph.
Hematopoietic stem cell hierarchy. Self-renewing HSCs give rise to common myeloid progenitors and common lymphoid progenitors, producing different types of progenitor cells and ultimately fully differentiated cells. The myeloid progenitors produce granulocyte-macrophage progenitors giving rise to differentiated leukocytic cells and mast cells. The megakaryocyte/erythrocyte progenitors give rise to megakaryocytes, platelets, and erythrocytes. The lymphoid progenitors differentiate ultimately in lymphocytic cell variances.
Emerging evidence suggests that BM-derived endothelial cells and HSCs, including their progenitor cells, contribute to tissue vascularization. HSCs deliver specific angiogenetic factors, facilitating the incorporation of endothelial progenitor cells into newly sprouting vessels. Several clinical studies have shown that BM-derived cells contribute to neo-angiogenesis during wound healing [44], critical limb ischemia [45], and postmyocardial infarction [58]. This should contribute to the clinical discussion of the value of BM-derived HSC and vascular progenitor as they are able to contribute to tissue restoration by accelerating tissue vascularization and regeneration [15, 59].
In recent decades, physicians performing regenerative medicine applications have been more interested in the potential of BM-MSCs than of HSCs. Imaginable reasons for this particular interest in MSCs might be recent published expert opinions: the
An effective BM-MSC injection is reliant on the performance of the marrow aspiration procedure, minimizing cellular trauma, while maximizing cellular yields and simultaneously avoiding peripheral RBC infiltration [62]. BM aspiration procedures, and not diagnostics, are routinely performed to collect bone marrow tissue to be processed using dedicated BM-MSC concentration kits for regenerative medicine applications. Kits may include a harvesting needle system and/or BM concentration device (Figure 7). These at POC MSC isolation techniques are a streamlined method to concentrate marrow cells, including MSCs, HSCs, and progenitor cells. These MSC centrifugation procedures demand less time and attention than laboratory preparation and culturing methodologies which are technically demanding. Double-spin centrifugation protocols create a layered BMC buffy coat stratum, based on different centrifugal forces that accomplish density cellular separation, as a result of the specific cellular gravity of the individual marrow components, as shown in Figure 8. Furthermore, BMA concentration-based technologies provide an economic and clinical/patient advantage when compared to the culturing technologies.
Bone marrow preparation essential components. In bone marrow concentration and preparation kits, the foremost components are a bone marrow harvesting needle and a concentration device (courtesy of EmCyte Corporation, Fort Myers, FL, USA).
Bone marrow gravity separation following centrifugation. (A) Bone marrow aspirate in concentration device before centrifugation. In (B), the bone marrow aspirate is concentrated, with a view on the buffy coat stratum (gray layer on top of the RBC layer), referenced by the two black lines. Following a two-step centrifugation protocol, the centrifugal forces achieve density marrow cell separation due to the specific gravities of the individual marrow components.
Traditionally, BM-MSCs have been separated from other BM cells following strict laboratory cell processing protocols. These cell processing techniques are lengthy procedures, as they cannot be performed at POC, as a same-day procedure. In many parts of the world, clinicians are allowed to use autologous, fresh, and non-cultured BMA and BMC products that are prepared at POC. In the USA, regenerative medicine biological procedures demand the use of the so-called 510-K FDA-approved devices. The use of MSCs following laboratory expansion techniques is facing considerable legislative barriers. Furthermore, the literature has cited potential risks associated with laboratory MSC cell processing techniques, like tumorigenicity [63], genetic instability [64], and immunogenicity [65]. Others raise concern regarding the efficacy and function of cultured MSCs by in vitro culture conditions during the cell passages for cell expansion. Karp and Teo reported on the loss of specific MSC surface receptors functions, negatively affecting chemotaxis [66]. Others have informed on impaired homing abilities and disappearing CXCR4 receptors following cell culturing, when compared to non-cultured BMA, in which high CXCR4 concentrations were measured [67]. Last but not least, laboratory MSC cell culturing methods for regenerative medicine practices require the availability of a specialized and dedicated facility, using strict regulatory protocols which will increase costs [68].
In order to better understand the specifics of MSC cell concentrations, counts, and quality, it’s important to understand the differences between laboratory techniques analyzing HSCs and MSCs, as they differ with regard to the specificity and relevance of the different BM cell types, possibly effecting regenerative medicine therapy outcomes.
The International Society of Cellular Therapy (ISCT) has developed criteria in order to outline human MSCs for both laboratory-based scientific investigations and for preclinical studies [69]. MSCs are defined as those cells that are able to adhere to plastic and express a number of cell surface markers while undergoing multilineage differentiation [70].
It has been difficult to determine what type of cells is neighboring both MSCs and HSCs and contributes to the regulation of the functional continuation of stem cell, as immunostaining methods are complex procedures to perform. Flow cytometry is a laboratory technique used to detect and measure characteristics of cell/particle populations by measuring their physical and chemical properties. A specific protocol for the identification of dissimilar cell surface molecules is called cluster of differentiation (CD) where monoclonal antibodies (markers) are used to establish positive and negative staining for certain cell types. Specifically for MSC and HSC, explicit CD markers are established to validate BM cellular content, as it is widely accepted that MSC cultures are a heterogenous source of cells with varying self-renewal and differentiation properties [71]. This indicates that there is no single unique indicator for identification. Hence, a panel of both positive and negative protein markers is used to identify the cell surface markers that are expressed by MSC populations, like CD29, VD44, CD51, CD73, CD90, CD105, CD166, and Stro1. While they must be negative for hematopoietic stem cell markers like CD14, CD34, and CD45 [72], some of these markers are included in the minimum ISCT criteria.
In the initial BM monolayer, several hematopoietic oriented cells (macrophages, endothelial cells, and lymphocytes) adhere to plastic [7]. Nevertheless, in culturing conditions only fibroblast-like spindle-shaped cells proliferate and form ultimately CFU-F colonies. These cells are representative of the more highly proliferative cells in MSCs [73]. The CFU-F assay is a different method used to determine the MSC presence in a vial of BM tissue. Unlike a complete blood count test, which is a quantitative blood cell analysis, the CFU-F assay is a laboratory assay in particular for stem and progenitor cell determination (Figure 9) [74]. The CFU-F assay is a qualitative indicator of the proliferative and differentiation capability of individual MSC cells within a BMA or BMC sample. The cells are seeded and cultured in a growth medium where they have to adhere to plastic, at 37°C. After 14 days the cultures are evaluated, and CFU-Fs are counted, whereby a minimum of 50 cells per CFU need to be defined.
MSC culturing. A picture of a flask cultured with stained human MSCs. The zoomed-in area is a light micrograph showing the morphology of a MSC colony in a patient treated with BMC. After culturing for 14 days, the MSC count in this example was 1065/mL (picture courtesy of BioSciences Research Associates, Cambridge, MA, USA).
MSCs are multipotent stem cells which can be obtained from various adult tissues, like the BM stroma, adipose tissue, synovium, periosteum, and trabecular bone. Typical features are their ability for self-renewal, defined as sustaining biological pathways and mechanisms to preserve the undifferentiated stem state, and the regulation of lineage-specific differentiation [39]. Although the number of MSCs represents only a small fraction of non-hematopoietic, multipotent cells of the bone marrow (0.001–0.01%), understanding these unique cells has taken great strides forward. Generally, MSCs have developed a great attractiveness for regenerative medicine autologous therapeutic applications and tissue engineering opportunities, because of their multipotentiality and relative ease of isolation from numerous tissues, like BM [75]. MSCs can be also identified as specialized populations of mural cells or pericytes, sharing a niche with HSCs. Under appropriate conditions and an optimal microenvironment, MSCs can differentiate into various mesodermal lineages like osteoblasts, chondrocytes, endothelial cells, adipose tissue, and smooth muscle cells (Figure 10) [76]. These MSC proficiencies have led to the use of MSC as a potential strategy for treating various diseases, since they encourage biological processes, for example angiogenesis, cell proliferation, and cell differentiation [77]. Furthermore, they synthesize cytokines and trophic mediators which participate in tissue repair processes, immune modulation, and the regulation of inflammatory processes [78]. Caplan also suggested that the modulation of inflammation is instigated by the suppression of inflammatory T-cell proliferation and inhibition of monocyte and myeloid cell maturation [79]. Based on the above characteristics, it can be assumed that MSCs are capable to institute a regenerative microenvironment at the site of release and improve the various cell recruitment, cell-signaling, and differentiation of endogenous stem cells, with the potential to instigate tissue repair in a variety of disease states.
MSC differentiation potential. MSC differentiation potential into endodermal, ectodermal, and mesodermal lineages. The mesodermal lineage differentiation has been recognized as the most attractive differentiation lineages for regenerative medicine applications, executed at point of care, as these produce osteoblasts, chondrocytes, tenocytes, adipose tissues, and smooth muscle cells.
In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contact, secretion of cytokines, and/or by a combination of both mechanisms. MSCs have been shown to exert profound anti-inflammatory and immunomodulatory effects on almost all the cells of the innate and adaptive immune systems via a variety of mechanisms, notably cytokine and chemokine secretion [80]. The immunosuppressive capabilities of MSCs are only exploited when they are exposed to sufficiently high concentrations of pro-inflammatory cytokines, like interferon-gamma (IFN-γ), tissue necrosis factor α, (TNF- α), and interleukins α or ß (IL-1α, IL ß). In order for MSCs to become “immunosuppressants,” they need to be triggered by these inflammatory cytokines, and the inflammatory environment is then a crucial factor for MSCs to exert their immunomodulatory effects. These are wielded by blocking apoptosis of native and activated neutrophils, aside from decreasing neutrophils from binding to vascular endothelial cells and the mobilization of neutrophils to the area of damage [81]. Furthermore, MSCs constrain the complement-mediated effects of peripheral blood mononuclear cell proliferation [82], and they limit mast cell degranulation and the secretion of pro-inflammatory cytokines, while at the same time MSCs migrate towards CXCL12 and other chemotactic factors [83]. In Figure 11 the MSC cell-dependent trophic support mechanisms are shown. Data from Jiang and others suggested that MSCs can block the differentiation of CD34+ cells from BM or blood monocytes into mature dendritic cells by direct contact as well as by secreted paracrine factors [84]. Under their influence, M1 (pro-inflammatory) macrophages are transformed into M2-type cells with an anti-inflammatory phenotype, and the interleukin-10 secreted by them inhibits T-cell proliferation. This immunosuppressive effect related to T-cell proliferation and decrease in cytokine production by MSCs was, among others, confirmed by Sato et al. [85]. However, the mechanisms by which MSCs are mobilized and recruited to damaged sites are not known. In addition, how they survive and differentiate into distinct cell types is still not clear. Once MSCs have been applied to the microenvironment of injured or degenerated tissues, many factors stimulate the release of many growth factors by MSCs; a detailed growth and trophic factor overview is shown in Table 3. These growth factors stimulate the development of fibroblasts, endothelial cells, and tissue progenitor cells [86]. It is credible to state that the use of MSCs and their potential in immunomodulation in regenerative medicine applications hold great promise [87].
MSC trophic mechanisms. After bone marrow cell injections, MSCs produce a variety of trophic factors impacting healing cascades by reducing cell apoptosis, fibrosis, and inflammation. Furthermore, by acting on cell proliferation cascades, they contribute to differentiation and mobilization of cells. MSC paracrine trophic factors are potentially important in maintaining endothelial integrity and promoting angiogenesis and the secretion of various growth factors and reparative cytokines.
Growth factor/cytokine | Activity in MSC regenerative repair |
---|---|
Epidermal growth factor | Wound healing |
Tissue regeneration | |
Fibroblast growth factor | Tissue repair |
Intrinsic stem cell survival | |
Tissue regeneration | |
Neurogenesis | |
Hepatocyte growth factor | Vasculogenesis |
Intrinsic neural cell regeneration | |
Insulin-like growth factor | Wound healing |
Neurogenesis | |
Keratinocyte growth factor | Wound healing |
Platelet-derived growth factor | Tissue repair |
Transforming growth factor beta | Wound healing |
Vascular endothelial growth factor | Angiogenesis |
Wound healing | |
Angiopoietin-1 | Angiogenesis |
Tissue repair | |
Erythropoietin | Angiogenesis |
Interleukin-8 | Wound healing |
Stem cell-derived factor-1 | Neuroprotective effect |
Wound healing |
Growth and trophic factors contributing to MSC tissue regenerative processes.
In order for MSCs to differentiate into several cell lineages, the action of specific growth factors and chemical mediators are needed in these processes [88, 89]. Once MSCs are mobilized, or after BM tissue injections, they produce a number of trophic factors that impact healing responses. At a local tissue level, they act by reducing cell apoptosis, fibrosis, inflammation, and activation of cascades that lead to cell proliferation and differentiation, mobilization of cells, and an onset of angiogenesis via paracrine and autocrine pathways [90]. Crucial agents involved in these processes include a variety of growth factors. The MSC trophic effects are associated with the secretion of reparative cytokines and growth factors [91], which contribute finally to tissue repair of inflamed and degenerated tissues, retaining positive MSC paracrine effects [92]. Many of the MSC growth factors are generated on the principle of the cell regulating protein nuclear factor-κB activation, after an initial exposure to pro-inflammatory stimuli such as IFN-γ, TNF-α, and IL-1β or even hypoxia [93]. These factors most likely coexist in prepared MSC-containing BM vials and delivered at tissue injury sites. In this situation, MSC growth factors and other cell mediators may have the potential to exert their specific activities via molecular interplays and subsequently promote optimal MSC-associated therapeutic tissue healing, in particular in a highly concentrated environment [94]. The endothelial monolayer barrier function of tissue capillary beds is often disturbed under degenerative and inflamed conditions, allowing for the blood to release proteins and white blood cells, while MSCs produce and release growth factors that affect endothelial cell and subsequently promote the development of tissue progenitor cells and fibroblasts and support tissue regeneration and repair [95]. Some clinicians combine platelet-rich plasma concentrations [96] with BM products in order to have a more biologically active graft, projected to optimize regenerative medicine treatment outcomes. However, it is important to comprehend the detailed mechanisms underlying the inflammation-modulated production of growth factors by MSCs, as this will provide a better perspective for the clinical application of MSCs or their paracrine factors in tissue regeneration.
MSC paracrine trophic factors are potentially important in maintaining endothelial integrity and promoting angiogenesis through their ability to regulate endothelial cell proliferation and ECM production [97]. Furthermore, endothelial cell permeability is reduced, and MSCs inhibit interactions between leukocytes and endothelial cells [98]. Apart from MSC trophic factors, fibroblasts have fundamental functions in maintaining tissue integrity and promote tissue healing through their secretion of cytokines that support ECM building. These endothelial and angiogenetic capabilities have been demonstrated in clinical studies addressing chronic wound healing [99] and recovery from postmyocardial infarction [100].
An enduring problem in the field of cell-based regenerative medicine therapies is the factual delivery of the harvested and prepared cells to the site of injury, a process termed “homing” [101]. One of the major characteristics of MSCs after administration is that they are able to migrate to sites of inflammation and tissue damage, which are typically associated with cytokine outburst [102]. Homing mechanism to degenerated and injured tissue sites are influenced by factors like age, cell viability, the number of available cells (dosage), and the delivery method. Unlike the well-characterized phenomenon of leukocyte homing by de novo, or exogenously delivered (BM) MSCs, is still unclear. Evidently, an increase in leukocyte migration, with induced rolling response to inflamed tissue sites, has been noted by engineered MSCs [103]. For successful cell-based regenerative therapies, it is critically important for MSCs to control cell adhesion in the ECM of the treated tissue. This will occur through the expression of fibronectin and specific integrin and selectin protein adhesion molecules, which are binding to collagen and fibrin ECM components [102], initiating tissue healing and regeneration through cell adhesion, cell growth, migration, and differentiation [104]. The migration ability of MSCs is further controlled by a wide range of growth factors, acting under the receptor tyrosine kinase signaling principle [105], once more illustrating the importance and presence of platelets and their growth factors in the collected BM vial. Furthermore, the administration of MSCs via various delivery routes (intravenous, intraperitoneal, intra-arterial, in situ injections) seems to have an effect on MSC homing [66].
When applying regenerative medicine MSC applications, physicians have a choice to use either a BMA as a regenerative injectate, without any processing steps, or they can harvest a particular BMA volume necessary to produce a BMC, with dedicated devices and centrifuges. Additionally, the differences between a BMA injectate only and a BMA concentrate are discussed.
In the freshly aspirated BMA samples, the heterogenous cellular content is pervasively distributed in the vial, as long as clotting is prevented.
Prior to a BMA procedure, it is recommended that bone marrow harvesting devices, concentration devices, and all of the processing accessories that will be in contact with BM are subject to a thorough heparin rinsing. Furthermore, several instructions for use advice to leave a volume of anticoagulant in the aspiration syringes and processing device as well, as BM tissue has the potential for rapid clotting. Before a BMC concentration device is loaded for processing, the aspiration syringes volumes are transferred into one consolidating collection syringe and subsequently filtered through a 200u heparin rinsed filter to eliminate particles, fibrin strands, and fat tissue.
It is our belief that the preparation of a vial of concentrated MSCs is best created by the so-called double-spin protocols, using dedicated and approved disposable concentration devices. BMA centrifugal processing techniques, to produce a viable BM-MSC injectate, are generally accepted methods when executed at POC, because these preparation protocols seek to overcome the limitations of MSC ex vivo cell culturing techniques. In this section we touch on a BMC preparation protocol to produce PurePRP SupraPhysiologic Bone Marrow Concentrate (PureBMC®SP, EmCyte Corporation, Fort Myers, FL, USA). The PureBMC®SP autologous biologic is part of an autologous cellular platform technology, facilitating the preparation of platelet-rich plasma and adipose tissue concentrates. A two-step centrifugation and preparation protocol will concentrate the indispensable BMA cellular content to a BMC. Following a first centrifugation spin, the BMA is sequestered in a BM plasma fraction (BMPF), containing a buffy coat layer and RBCs. The BMPF is aspirated, immediately followed by a separate collection of 2 ml of RBCs, following the instructions for use of the PureBMC® concentration device. Both volumes are then transferred for a second centrifugal spin cycle to the concentration compartment of the same device. During the second spin, a specific centrifugation protocol is accomplished, leaving the bone marrow cells in a concentrated fashion attached at the bottom of the chamber. Excessive BMPF is manually removed, leaving behind a specific BMC volume for resuspension. The amount of this volume depends on the requirements for clinical applications. Therefore, the BMC injectate volume may vary between 3 and 10 ml, with increased cell concentrations according to this final volume varying between 4- and 10-fold the native concentrations.
In a BMA injectate, the concentrations of the cells resemble the concentration of the cells that are present in the bone marrow cavity. However, based on aspiration techniques, the number of MSC might be increased. A BMC is a small volume of fluid containing a high concentration of cells extracted from the bone marrow, such as high yields of MSCs (can be measured as CFU-Fs), HSCs, progenitor cells, total nucleated cells, and platelets, at a significant concentration above BMA baseline values. Furthermore, the heterogenous nature of marrow cells is completed by the presence of increased levels of growth factors [106, 107], cytokines like IL-8, and interleukin-1RA [94]. Additionally, in a BMC injectate following a two-step centrifugation procedure, the RBC and plasma-free hemoglobin (PFH) concentrations are significantly decreased when compared to a BMA injectate.
Throughout the aspiration procedure, RBCs can be damaged as a result of high shear forces [108]. As a consequence, the RBC membrane will start to disintegrate, and hemolysis, with the release of PFH, will occur. Damaged RBCs and free hemoglobin (Hb) lead to the development and release of toxic Hb forms, like free hemin, ferric Hb, and iron [109]. This is of particular concern as PFH and their split products, heme and iron, cannot be cleared, by natural scavenger proteins, when bone marrow injectates are applied in any microenvironment, as these are outside of the blood stream. A graphic representation of the pathophysiological effects and reactions of PFH, leading to various hemolytic-related sequelae and potentially encumbering clinical outcomes, is presented in Figure 12.
Pathophysiological effects and reactions of RBCs in BMC vials. In absence of scavengers and compensatory mechanisms, PFH split products can lead to toxic consequences like inflammation and prooxidant effects, endothelial cell dysfunction, and vasoconstriction. Biological treatment specimen, containing high concentrations of RBCs, will lead to RBC cell membrane disruption (eryptosis,) releasing macrophage migration inhibitory factor (MIF) (courtesy of P. Everts and modified from Schaer et al. [
In Table 1 the effects of concentrating BMA to BMC with regard to some of the most important marrow constituents and factors are shown, as discussed in Section 4.1. The data in the table represent a clinical bilateral BMA model, using two different harvesting systems. For both systems, BMA was aspirated in an identical manner, at three different depth levels collecting in total 10 ml of marrow. Furthermore, to compare the cellular differences between a BMC injectate and a BMA injectate (BMA-MC), we collected an additional 40 ml of BMA with the Aspire system, after the first 10 ml. This allowed for a total processing volume of 60 ml to produce BMC. Laboratory analysis resolved that both BMA devices were almost similar with regard to cell viability and numbers. Interestingly, with regard to CFU-Fs, the data are in accordance with Hernigou [40], and the first marrow aspiration provides the highest number of CFU-Fs. However, when comparing the BMA-MC cellular composition (a patient treatment specimen) with the BMC treatment specimen, significant differences occur. Centrifugation foremost significantly increases cells who take part in regenerative processes when compared to the BMA-MC product. In contrast the non-regenerative RBCs and PFH concentrations are significantly reduced in the treatment vial, while maintaining a higher cell viability after centrifugation. Our findings, with regard to cellular enrichments comparing a BMA with a BMC, are in agreement with others [41, 110], but not regarding RBC content and PFH. The cell concentrations are not only depending on the centrifugation protocols and the final BMC volume but are contingent of a meticulously executed BMA procedure, maintaining high cell viability, with minimal cell destruction.
Currently, eight BMC harvesting devices are available on the market, producing different formulations of BMC and tissue viabilities and yielding different cellular concentration characteristics [111, 112]. As such, BMC preparations may vary widely regarding HSCs, MSCs, progenitors, platelet growth factors, and RBC content. Given this heterogeneity, the impact of BMC therapies on tissue regeneration may vary greatly. Explicitly, it is important to understand that the BMC non-stem cell cellular components in the treatment vial might have significant roles regarding behavior and function of MSCs. Recently, the cellular variances were confirmed in a systematic review, evaluating BMC studies in musculoskeletal pathologies [53]. Presumably, as postulated by numerous authors, the variances in BMC cellular compositions have a significant effect on the biological activity and regenerative potency of the treatment specimen, and these inconsistencies impact clinical outcomes [111, 113]. Unfortunately, an exact understanding of the underlying signaling relationships is not completely understood [114].
The use of autologous BM-derived MSCs for the treatment of a variety of musculoskeletal ailments is progressing significantly. Literature findings demonstrate positive outcomes after regenerative medicine MSC applications, in particular in joints, tendons, and bone, and hold great promise for future MSK-D applications, especially if more research and larger clinical trials are performed, focusing on cell validation processes and elucidating on potential dose responses.
In this section we will present several clinical studies in which autologous, heterogenous BMC was used as a regenerative biologic to treat a variety of musculoskeletal disorders [42, 115]. Studies reporting on similar pathologies using BM-derived/cultured MSCs are not mentioned as it has been reported that these technologies have different biomedical properties and extraction methods [116] and potentially possess new challenges and indications, when compared to at POC prepared fresh autologous BMCs. In clinical settings, BMC has been exploited as an ortho-biological treatment option for a range of indications, like symptomatic focal femoral head osteonecrosis, OA of the knee and hip, focal chondral defects, as well as another MSK-D. The rationale to use autologous BMC in osteoarthritic (OA) joints and other indications is its potential in facilitating anti-inflammatory and anabolic effects after injection. Moreover, the heterogenous BMC cellular assortment is known for its angiogenetic properties, therefore contributing to chondrocyte metabolism and inducing homing of (progenitor) stem cells to the treated areas [117]. Rodriguez-Fotan and co-workers used a two-step BMC preparation protocol in patients with early onset of OA in the knee or hip (Kellgren-Lawrence grades I–II, Tönnis grades I–II, respectively), with BMA aspirated from the anterior iliac crest. After a single BMC injection, 63% of treated patients had improved clinical symptoms at 6-month postinjection. They concluded that the intra-articular BMC injections are safe procedures and no adverse events were reported [118]. In a prospective single-blind, placebo-controlled trial, 25 patients with bilateral knee OA and a median age of 60 years were randomized to receive BMC into one knee and saline placebo into the contralateral knee, thereby utilizing each patient as his/her own control. Safety data, effect of pain relief, knee function (Osteoarthritis Research Society International) measures, and the visual analog scale (VAS) for pain were observed until 6 months after the injections [119]. However, no differences between the two groups were observed. Of interest in this study was that the final injectate composition consisted of a mix of BMC and BMPF suspension. However, the eventual consequences of diluting BMC with BMPF on outcomes were not discussed. In another case series by Kim et al., a more invasive treatment approach was used. BMC was mixed with adipose tissue as a multi-tissue preparation for knee OA injections, in patients with a mean age of 60.7 years. At 9-month follow-up, all patients showed clinical improvement, with satisfactory results in 70.7% of patients [120]. Remarkably, the authors found that patients with inferior treatment results had a greater severity of OA prior treatment, as they were marked at Kellgren-Lawrence grade IV, suggesting that advanced OA may be more restrained to BMC therapy. The side effects encountered in this study, joint inflammation and pain, were in accordance with data from Rodriguez-Fotan [118]. Recently, a similar (retrospective) study was executed by Mautner and associates. Patients were prospectively treated either with bone marrow aspirate concentrate (BMAC) or micro-fragmented adipose tissue (MFAT) injections, for symptomatic knee OA [121]. The follow-up responses consisted of 76 patients (with 106 knees). The Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire, Emory Quality of Life (EQOL) questionnaire, and VAS for pain were compared with baseline scores for all patients, and outcomes between BMAC and MFAT groups were evaluated. Data demonstrated a significant improvement in joint function and VAS pain scores after both MFAT and BMAC injections. No significant difference between the two autologous biological groups was demonstrating that BM- or adipose tissue-derived ortho-biological injections resulted in similar functional improvements.
Lately, Darrow et al. reported on patients treated for shoulder osteoarthritis or rotator cuff tears (
Philippe Hernigou, world renowned for treating femoral head osteonecrosis, advocates the use of autologous BMC cell therapies [123]. He described a substantial repair and stabilization of necrotic femoral heads with percutaneous injection of autologous BMC, in combination with surgical core decompression. In a later paper, he reviews three decades of BMC therapies in hip osteonecrosis, emphasizing the quality of the BMC and cellular competence and addressing the effects of BM cell concentrates on the microenvironmental changes within osteonecrotic bone [124]. Other groups reported on prospective randomized clinical trials for femoral head osteonecrosis, comparing surgical decompression alone versus decompression augmented by autologous BMC preparations. The biologics were implanted during the surgical decompression procedure. In one study, patients were evaluated using the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index questionnaire, VAS pain index, and MRI. The mean WOMAC and VAS scores in all patients improved significantly (
Awad and associates recently published a meta-analysis on knee cartilage repair [127]. They conducted a systematic review using the PRISMA guidelines and the
In a retrospective study, Stein et al. used BMC for primary Achilles tendon repairs, following traumatic injuries [128]. The BMC was adjunct to augment the surgical correction. Although the study lacked a control group, at a mean follow-up of 30 months, there were no re-ruptures reported. In a small patient study, centrifuged BMC specimen were injected in patients, refractory to conservative therapies, with clinical and radiological evidence of chronic patellar tendinopathy. Long-term follow-up showed statistically significant improvement in the majority of its reported scores [129]. A series of patients, diagnosed with clinical lateral epicondylitis, were treated with a single-spin BMC protocol. A significant improvement was noted when pre-BMC scores were compared with postinjection scores, at 12-weekpost-intervention. The authors suggested that BMC injections in patients who have failed non-operative treatment, before a surgical intervention, should be considered, and in their belief BMC injections can be developed as second-line conservative treatment in chronic tendinopathy, potentially reversing the degenerative process [130].
Degenerative disk disease (DDD) affects the disks that separate the spine bones. Age-related changes can lead to arthritis, disk herniation, or spinal stenosis. Pressure on the spinal cord and nerves may cause pain. DDD is associated with significant morbidity. Conservative treatment options, physical therapy, self-care, medication, and spinal injections are used to manage the symptoms. However, these measures are often not significantly responsive. Surgery has been an option if the pain is chronic. Nowadays, autologous regenerative applications have been made available to patients as an alternative treatment option.
Pettine et al. studied the use of intra-discal BMC injections in patients with DDD [131]. The authors injected 26 symptomatic patients for lumbar diskogenic back pain and disability and evaluated their postinjection outcomes using disability scores, pain scores, and MRIs. At 2-year follow-up, patients experienced significant improvements in disability and pain scores. This group was the first to report on MSC dose-dependent outcome responses. Patients receiving greater concentrations of autologous BM-MSC (expressed as CFU-Fs > 2000/ml) experienced a faster and greater reduction in pain scores. Later, these findings were strengthened with a follow-up study at 36 months, showing similar outcome results [43]. At 5-year follow-up, absolute and percentage reductions in pain and disability scores were sustained, with no adverse events reported through the 5-year follow-up period [132]. The American Society of Interventional Pain Physicians published recently guidelines addressing responsible and safe use of autologous biologics in the management of lower back pain [133]. Their extensive analysis revealed that there is level III evidence for the use of PRP and BM-MSCs. The guidelines also state that, following diagnostic evidence, regenerative therapies should be provided to patients as an independent therapy. If appropriate and indicated, regenerative therapies can be combined with conventional medicinal therapy or in conjunction with physical and behavioral therapy.
Hart et al. informed on a prospective, randomized, and blinded study in patients with lumbar disease the use of BMC mixed with allograft spongiosa chips during surgical posterolateral fusion (PLF) procedures. Patients underwent instrumented lumbar spine PLF procedures [134]. Fusion status and the degree of mineralization were evaluated by two radiologists blinded to patient group affiliation. X-ray examination, in control patients at 12-month follow-up, showed that the bone graft mass fused in none of the cases and, at 24-months, in four cases (10%). In the BMC treatment group, 6 cases (15%) achieved fusion at 12 months and 14 cases (35%) at 24 months. Computed tomography scans showed that 40% of control patients and 80% of BMC-treated patients had evidence of at least a unilateral continuous bridging of the bones between neighboring vertebrae at 24 months, significantly favoring the mixture of spongiosa bone with autologous BMC (
Cell-based therapies are an attractive approach for the treatment of recalcitrant chronic wounds. BM-MSCs have been studied as a therapeutic strategy in chronic hard-to-heal wounds [135]. The orchestrated process of wound healing entails cellular and hormonal physiological processes in inflammation, proliferation, collagen matrix formation, and epithelialization which are regulated by various platelet-derived growth factors, such as TGF-b, VEGF, PDGF, granulocyte-macrophage colony-stimulating factor, the interleukin family, EGF, FGF, and TNF-a [44, 105]. In chronic, poor-healing wounds, the activity and effectivity of growth factors and cytokines are often reduced due to a chronically inflamed wound. Under these conditions the neo-angiogenetic wound healing potential is reduced, resulting in poor or no full wound epithelialization. The rationale for using BMC in these patients is the potential to modulate the immune response and secreting paracrine factors which promote (neo) angiogenesis, thereby providing biological ingredients for wound tissue repair that can jumpstart full wound closure [76, 136]. Optimal wound bed preparation is of the essence in wound healing strategies and encompasses tissue debridement with proper management of the bacterial load. Based on BM-MSC characteristics and their biological activity, MSCs are capable of interacting with resident wound cells to transform resident cells to functional matrix building cells, as described by Balaji et al. [137]. This finding might be of particular importance for dermal rebuilding processes, to stimulate (transplanted) keratinocyte-mediated wound epithelialization.
Patients with significant, below the knee, vascular diseases and who are, first of all, not eligible for revascularization surgery or endovascular treatments due to several comorbidities or have high operative risk and had multiple failures of revascularization or high rate of re-stenosis, might be suitable candidates for biological cell-based therapy with BM-MSCs. Patients diagnosed with critical limb ischemia (CLI) might also suffer from chronic non-healing wounds, and the estimated amputation and mortality rates are high [138]. The application of regenerative medicine therapies, in particular the use of BM-MSCs protocols, has merged as a treatment option in patients with CLI. In these patients, the justification to use BMC is to promote the regeneration of impaired endothelium and stimulate neo-angiogenesis in ischemic areas [139]. Several varieties of BM-MSC therapies have been studied in CLI patients, ranging from BM-derived mononuclear cells, CD34+ BM cells, to mesenchymal stromal cells. The outcomes of cell-based trials have been encouraging and demonstrated a significant decrease in the rate of amputation [140]. It can be concluded that BM-MSC applications have the potential to modify the natural history of intractable CLI, while high-quality randomized trials are needed [45].
Regenerative medicine technologies offer solutions to a number of compelling clinical problems that have not been able to adequately result in a solution through the use of drugs, surgery, or permanent replacement devices. Reviewing the last decades regarding autologous biological therapies, BM-MSCs have gained great interest. The purpose of this chapter was to review specific characteristics of bone marrow tissue and its cellular content, in particular the mesenchymal stem cells. Considerations when performing aspiration techniques and bone marrow concentrate preparations were presented, including explicit roles of hematopoietic and mesenchymal stem cells and other cytokines. Among autologous tissue-based cellular therapies, bone marrow mesenchymal cell therapies have been the most frequently employed and reported on, despite the fact that effects of coadjuvants, dosing, repetitive procedures, etc. are not yet established. Cultured MSC therapeutic interventions require strict procedures and biological license agreements, making them less attractive for same-day regenerative therapies. Using at POC BM-MSC concentrates overcomes these lengthy regulatory processes without the need for mandates. Clinical translation of BM-MSC-based therapies remains a work in progress, as proper standardization has not yet been recognized [53]. However, in the clinical setting, effective and safe autologous BMA harvesting and preparation of BMC have been reported [42]. More, better, organized randomized clinical trials that are warranted with accurate follow-up data revealing the efficacy of BM-MSC therapy, including laboratory validation of the used products, should be a future goal. Furthermore, proper deliberations should manage the enormous variability aspects, like aspiration techniques, imaging options and procedures, BMC preparation protocols, effect of patient age, as well as tissue disease state. Therapy failures should also be highlighted in order to understand how they impact the therapy outcomes. Ultimately, the adoption of an accepted standard of overall regenerative biological preparations, including critical and ambivalent nuances, is crucial for future regenerative medicine practices.
Author P. Everts is also the chief scientific officer of the EmCyte Corporation.
A number of studies have been carried out in the area of interlanguage pragmatics. Most of them focus on the production/realization of speech acts (e.g., requests, thanks, compliments, advice, complaints, apologies, etc.) in general and on the performance of learners of English in particular ([1], p. 261–270; [2]). The present study focuses on the realization of apologies by a group of Canadian learners of French as a second language, through a comparative analysis of apology strategies in French L1 and French L2. Data for the study were collected from two groups of respondents: a group of Canadian native English speakers and a group of learners of French L2. The chapter is structured as follows: Section 2 presents the theoretical background, in which the communicative act of apologizing is defined, and a brief literature review is presented. The methodology is outlined in Section 3, and the findings are presented and discussed in Section 4. The chapter concludes with remarks and perspectives for future research.
An apology is an expressive speech act by which a speaker intends to remedy an offense for which she/he takes responsibility and intends to restore equilibrium between him/her and the addressee, that is the apology recipient ([3], p. 155). From this point of view, an apology is “remedial work” [4]. There are two opposing views on apologies based on Brown and Levinson’s [5] theory of politeness, which uses the central concept of the face of Goffman [4]. The first view considers apologizing as a face-threatening act for the speaker ([5], p. 68): it is perceived as a self-demeaning act because the speaker, by apologizing, directly or indirectly recognizes his or her fault or responsibility for the offense. On the other hand, apologizing is a face-enhancing act for the speaker, since it serves to portray the speaker as someone who recognizes his or her mistakes and humbly says something to “set things right” ([6], p. 373) or to restore a strained relationship with the offended person. Apologies could also be considered as attempts to restore the hearer’s face and social harmony. These contradictory perceptions certainly have an impact on the choices of apology strategies.
Several studies have examined similarities and differences in apologizing across languages and cultures ([7], p. 558–562 for an overview of studies on apologies). Apologies have also been explored from an interlanguage pragmatics perspective. The focus here is on how learners apologize in L2. These include studies such as Cordella’s [8] work on Spanish speakers apologizing in English, Trosborg’s [6] work on apologies by Danish learners of English, Abe’s [9] thesis on apologies in English L2 by Japanese learners, a study of apologies in English by Jordanian Arabic learners [10]. Studies of apologies in L2 French include Warga and Schölmberger’s [11] study of apologies by Austrian learners of French in a study abroad situation and Esmonds’ [12] work on apologies by American learners.
These few studies on apologies in L2 French add to a growing body of research on the production of other speech acts in French L2. These include Kraft and Geluykens’ [13] analysis of complaint strategies in L2 French by German learners of French; Schaeffer’s [14] analysis of complaints in L2 French by English-speaking learners; Mulo Farenkia’s [15] study of compliments in L2 French by English-speaking Canadian learners of French; Bae’s [16] thesis on request strategies in L2 French by Korean learners of French; Warga’s work [17] on requests in L2 French by Austrian learners of French, and Mulo Farenkia’s [18] study of the realizations of offer refusals by Canadian learners of French as a second language. The present study adds to this growing body of research aimed at understanding “how non-native-speaking (…) learners of a language acquire pragmatic competence in their target language” ([1], p. 261), and if they can communicate effectively in an “L2-speaking environment where the learner’s target linguistic behavior is, ultimately, that of the [native speaker]” ([1], p. 261). The present study thus addresses the following questions:
How do L2 French learners apologize in their target language?
Which (realization) forms do they use to apologize in L2 French?
Are there qualitative and quantitative differences or similarities between apology strategies used by L1 French speakers and L2 French learners?
The next section centers on the description of the methodology used to answer these questions. It presents the participants, the data collection instrument, the scenarios as well as aspects of data analysis.
The research is based on material collected for a larger project on French interlanguage pragmatics in the Canadian context. The examples used here were provided by two groups of respondents. The first group consisted of 16 native speakers of French (
Situation 1: Vous arrivez chez votre ami(e) et, en enlevant votre manteau, vous renversez son vase qui se brise en plusieurs morceaux. Qu’est-ce que vous lui dites ? [While taking off your jacket you accidently break your friend’s vase. What do you say to him/her?]
Situation 2. En entrant dans un restaurant, vous heurtez accidentellement le serveur/la serveuse et cela la pousse à renverser le plateau qu’il/qu’elle transportait. Qu’est-ce que vous lui dites ? [While entering a restaurant you inadvertently bump into a waiter/waitress, and she/he spills the content of the plates she/he was carrying to another client. What to you tell him/her?]
Situation 3. Vous avez accepté d’aider votre enseignant(e) dans le cadre de son projet de recherche. Vous avez une rencontre avec lui/elle à cet effet à 9: 00 le lendemain. Vous serez en retard au rendez-vous. Vous l’appelez. Qu’est-ce que vous lui dites ? [You were supposed to meet with your professor to help him/her in a research project. You will arrive late for the appointment. You call him/her. What do you tell him/her?]
In situation 1, the participant has broken a friend’s vase. In situation 2, the respondent accidentally bumped into a waiter/waitress and caused her/him to throw the entire content of the tray he/she was carrying. In situation 3, the participant is late for an appointment with his/her professor. The first situation is symmetrical, that is the offender and the offended are equal in social status and the relationship is a close one (it involves two friends), while in the second situation the relationship is distant, that is the interactants do not know each other. The third situation is an asymmetrical one: the offended person has a higher power position (professor), and the offended and the offender know each other as acquaintances. The respondents were asked to imagine themselves in each of the situations and to write down what they would say in order to apologize in the three situations. The 32 informants provided 95 answers for the three questionnaire tasks: 48 responses by the FL1 group and 47 responses by the FL2 group.
The apologies collected were analyzed based on the schemes used in previous studies in which apologies are examined with respect to the degree of the directness of utterances, the number of moves involved in the same utterance, use of additional speech acts or supportive acts, and mitigating or intensifying devices, etc. ([6], p. 373–409; [20], p. 143–168). The first step was to segment the apology utterances produced by the participants and to classify each occurrence or token as a strategy belonging to one of the following three pragmatic categories: direct apologies, indirect apologies, and supportive acts.
Direct apologies and indirect apologies are realized using many different strategies and forms. For example, direct apologies are realized through strategies such as expressions of regret, requests for forgiveness, or offers of apologies, and each of these strategies may be realized using different linguistic structures (cf. Section 4.2). Indirect apologies can be realized in the form of explanations, taking responsibilities, offering repairs, or promise of forbearance and these strategies can be framed in many different ways as well (cf. Section 4.3). Also, the data show that the apology acts occurred either in single moves, that is alone as in (1), or in association with other moves, that is as complex apologies. Complex apologies may result from the combination of two or more apologies or may be made up of apologies that are accompanied by supportive moves (cf. Section 4.1) as in (2) and (3). In (2), the speaker combines three moves to apologize, namely a preparatory act (
1.
‘I am so sorry.’
2.
‘Oh my God, I am so sorry. I can help you to clean the mess.’
3.
‘Allo, this is X. I am so sorry. I did not set my alarm clock. I could be there in 30 minutes. Do I still need to come? Once again, sorry.’
In the next section, we will present the results of the analysis of the data.
Table 1 shows the overall distribution of strategies used by both the FL1 and the FL2 participants. It appears that the FL1 group used more strategies than the FL2 population (FL1: 116 vs. FL2: 106). The FL1 speakers mostly preferred direct apologies: this strategy accounted for 50% of the examples provided in the FL1 data set. The FL2 respondents most commonly used indirect apologies: this strategy accounted for 56.6% (60 examples) of their responses. While supportive acts had the lowest frequency in both data sets, Table 1 shows that the FL1 informants used more supportive acts than the FL2 learners did.
FL1 | FL2 | |
---|---|---|
Direct apologies | 58 (50%) | 44 (41.5%) |
Indirect apologies | 43 (37%) | 60 (56.6%) |
Supportive acts | 15 (13%) | 2 (1.9%) |
Total | 116 (100%) | 106 (100%) |
The overall distribution of strategies used by the FL1 and the FL2 participants.
We also compared the use of the three strategies mentioned above across the three situations. Table 2 shows the situational distribution of these strategies in both data sets. In the FL1 corpus, direct apologies were much more employed with friends (36%), while the FL2 respondents used direct apologies much more towards waiters/waitresses (36%). Similarities emerged between the two groups regarding the use of indirect apologies. For instance, the majority of all indirect apologies occurred in the professor situation, with 48.8% in the FL1 examples and 45% in the FL2 data set. The supportive moves essentially appeared in the professor’s situation in both data sets.
Direct apologies | Indirect apologies | Supportive acts | ||||
---|---|---|---|---|---|---|
FL1 | FL2 | FL1 | FL2 | FL1 | FL2 | |
S1 – Friend | 21 (36%) | 14 (32%) | 13 (30.2%) | 20 (33.3%) | 1 (6.7%) | 0 |
S2 – Waiter | 19 (32.7%) | 16 (36%) | 9 (21%) | 13 (21.7%) | 0 | 0 |
S3 – Professor | 18 (31%) | 14 (32%) | 21 (48.8%) | 27 (45%) | 14 (93.4%) | 2 (100%) |
Total | 58 (100%) | 44 (100%) | 43 (100%) | 60 (100%) | 15 (100%) | 2 (100%) |
Situational distribution of apology strategies in French L1 and French L2.
As already indicated above, apologies were realized by the participants of both groups either as unsupported head acts or single moves (simple apology utterances) or as combinations of multiple moves (complex apology utterances). Table 3 shows the distribution of these two realization patterns in FL1 and FL2.
FL1 | FL2 | |||||
---|---|---|---|---|---|---|
S1 | S2 | S3 | S1 | S2 | S3 | |
Simple apology utterances | 3 | 3 | 0 | 1 | 4 | 0 |
Complex apology utterances | 13 | 12 | 16 | 15 | 12 | 16 |
Total | 16 | 15 | 16 | 16 | 16 | 16 |
Distribution of single and complex apology utterances.
Table 3 shows that both the FL1 and the FL2 respondents mostly used complex apology utterances. Of the 47 examples provided by the FL1 speakers, there were 6 (12.8%) simple apology utterances and 41 (87.2%) complex apology utterances. Of the 48 apology utterances provided by the FL2 learners, there were 5 (10.4%) simple apology utterances and 43 (89.6%) complex apology utterances. While simple apology utterances were distributed equally in S1 (Friend) and S2 (Waiter) and did not occur in S3 (Professor) in the FL1 examples, the vast majority (4 of 5, i.e., 80%) of simple apology utterances used by FL2 participants were found in S2 (Waiter). No simple apology utterance was used in S3 (professor), the more formal situation.
This section presents results regarding the way in which the respondents of both groups realized their apologies, that is the various linguistic structures employed to apologize. In the first section (Section 4.3.1) the devices used to introduce apologies are discussed. The realization forms of direct apology strategies are presented in Section 4.3.2. Realization forms of indirect apology strategies are discussed in Section 4.3.3. The use of supportive moves is examined in Section 4.3.4.
The participants of both groups used specific interjections to introduce their apologies. These devices function as “emotional exclamations” ([21], p. 190) to express the speaker’s negative feelings about the offense (e.g., surprise, shame, regret, etc.), before issuing the apology. Overall, there were 31 tokens of interjections in both data sets. As can be seen in Table 4, the FL1 used five times more interjections than the FL2 informants. With respect to situational distribution, the findings show that the participants of both groups did not use interjections in apologies directed to professors. Also interesting are the types of interjections used.
FL1 | FL2 | Total | |
---|---|---|---|
S1 – Friend | 15 (57.7%) | 2 (40%) | 17 (54.8%) |
S2 – Waiter | 11 (42.3%) | 3 (60%) | 14 (45.2%) |
S3 – Professor | 0 | 0 | 0 |
Total | 26 (100%) | 5 (100%) | 31 (100%) |
Distribution of interjections to introduce apologies in FL1 and FL2.
The interjections found in the examples provided by the FL2 were “
4.
5.
6.
Three realization types of direct apologies were found in both data sets. These are “expression of regret”, “offer of apology”, and “request for forgiveness”. Their frequencies are summarized in Table 5.
FL1 | FL2 | |
---|---|---|
Expression of regret | 42 (72.4%) | 37 (84%) |
Offer of apology | 13 (22.4%) | 7 (16%) |
Request for forgiveness | 3 (5.2%) | 0 |
Total | 58 (100%) | 44 (100%) |
Distribution of direct apologies used by FL1 and FL2 respondents.
First, Table 5 shows that the participants of both groups mostly used expressions of regrets when apologizing directly, albeit with different frequencies; This type accounted for 72.4% of all direct apologies produced by the FL1 participants and 84% of direct apologies used by the FL2 group. The FL1 speakers expressed regret are by means of constructions like
With respect to their situational distribution, the results show that the FL1 group mostly used expressions of regret in the professor situation and their offers of apology were equally distributed in situations 1 (friend) and 2 (waiter). By contrast, expressions of regret used by the FL2 participants appeared much more in the waiter situation, while offers of apology were equally distributed across the three situations in FL2 examples (Table 6.).
S1 – Friend | S2 – Waiter | S3 - Professor | ||
---|---|---|---|---|
FL1 | Expression of regret | 14 | 11 | 17 |
Offer of apology | 6 | 6 | 1 | |
Request for forgiveness | 1 | 2 | 0 | |
Total | 21 | 19 | 18 | |
FL2 | Expression of regret | 12 | 14 | 11 |
Offer of apology | 2 | 2 | 3 | |
Request for forgiveness | 0 | 0 | 0 | |
Total | 14 | 16 | 14 |
Situational distribution of direct apologies in FL1 and FL2.
We also analyzed the way in which the respondents of both groups used adverbs to reinforce direct apologies. Table 7 presents the frequencies of intensified direct apologies.
FL1 | FL2 | |
---|---|---|
S1 – Friend | 13 (34.2%) | 8 (34.8%) |
S2 – Waiter | 11 (30%) | 10 (43.5%) |
S3 – Professor | 14 (36.8%) | 5 (21.7%) |
Total | 38 (100%) | 23 (100%) |
Frequency of intensified direct apologies in FL1 and FL2.
Overall, the FL1 speakers used more intensifiers than the FL2 participants. Of the 61 intensified direct apologies identified in our corpus, there were 38 (62.3%) tokens used by the FL1 group and 23 tokens (37.7%) produced by the FL2 informants. As shown in Table 7, while intensified direct apologies in the FL1 data set mostly appeared in the professor situation, the FL2 population mostly boosted their direct apologies in the waiter situation.
The choices of adverbial intensifiers in the direct apologies made by the participants of both groups were different to some extent. The results show that of the 38 intensifiers used by the FL1 participants, there were 23 tokens of
Overall, the FL1 speakers produced 43 indirect apologies, while the FL2 learners provided 60 indirect apologies. The results further show that the attested realization types of indirect apologies, that is the speech acts used to apologize, were distributed differently in both data sets (Table 8).
FL1 | FL2 | |
---|---|---|
Taking responsibility | 4 (9.4%) | 10 (16.7%) |
Explanation / Justification | 12 (27.9%) | 9 (15%) |
Offer of repair | 21 (48.8%) | 38 (63.3%) |
Promise of forbearance | 0 | 2 (3.3%) |
Concern for addressee | 6 (13.9%) | 1 (1.7%) |
Total | 43 (100%) | 60 (100%) |
Realization types of indirect apologies in FL1 and FL2.
As seen in Table 8, the FL1 group used four different speech acts (
Let us now turn to the individual speech acts used as indirect apologies, focusing on their realizations and pragmatic functions.
We will begin with
7.
“I am so sorry, I got up late, I will arrive later than expected.”
8.
“I am sorry, but I am going to be late because I overslept.”
9.
“I am very sorry. It was an accident. I offer to help pay in order to replace or repair it.”
10.
“I am very sorry but when I was taking off my coat, I knocked over your vase.”
11.
“I am so sorry, my alarm clock did not ring. I apologize for the late-coming/delay.”
12.
“Good morning sir/ma’am, I am very sorry, I will be late for our meeting. My alarm clock did not ring today, so I got up late.”
13.
14.
“I am sorry. Can we repair it?”
It is worth mentioning that two different types of offers to repair the damage caused appeared in the data. The first type occurred predominantly in situations 1 (friend) and 2 (waiter), where the offender offered to help clean up the mess caused by the incident, using constructions like
15.
“I am so sorry to be late for our appointment. I forgot to set my alarm clock. I will be on time next time.”
16.
“Sorry. Is there anything I can do to make you happy? It will not happen again.”
Finally,
17.
“I am so sorry, this generally does not happen to me but I forgot to set my alarm clock so I overslept this morning. I hope that this did not inconvenience you that much. If you are still available, I can come right now and we can still have our meeting.”
The expression of concern in the FL1 corpus took many different forms. In the friend situation, the speakers showed concern by asking if the vase meant a lot to the addressee as in (18). In situation 2, the speaker wanted to know if the waiter was okay as in (19). In situation 3, a speaker wanted to know more about the professor’s feelings regarding the late coming. More precisely, the speaker wanted to know whether the meeting would be postponed or if the professor still wanted to meet the student as in (20).
18.
“Oh shit, excuse me. Oh shit,
19.
“Oh my God, I am so sorry.
20.
“Good morning, I am really sorry, I forgot to set my alarm clock. Can I come now or another time to participate in the project anyway? I am so sorry.”
Supportive acts are different kinds of speech acts, which may come before or after direct and indirect apologies. Supportive acts alone cannot be used to apologize. Rather, they are external modification devices used to soften or reinforce apologies. The speech acts used to support the apologies focused on many different face-wants of the speaker or the addressee. In the data used, 17 supportive moves were found and the FL1 participants used more supportive acts than the FL2 group (FL1: 15 tokens vs. FL2: 2 tokens). The two supportive acts attested in the FL2 data set were greetings and they appeared in the professor situation, where they served to initiate contact with the interlocutor prior to the apology proper (21).
21.
“Good morning sir/ma’am, I am very sorry, I will be late for our meeting. My alarm clock did not ring today, so I got up late.”
Of the 15 supportive acts found in the FL2 data set, there was one example in situation 1 and 14 examples in situation 3. The supportive act attested in the friend situation was a question of whether the broken vase could be repaired. This question served to introduce the forthcoming offer to take care of the repair as in (22).
22.
“Oh shit, excuse me. Oh shit, did it mean a lot to you? Are there any means to repair it? I can take care of that.”
The 14 supportive acts used by the FL1 respondents in the professor situation include greetings and self-introductions, which were used to introduce the apologetic utterances as in (23). Other supportive acts attested were comments by which the speaker attempted to protect his/her positive face by indicating the offense was not a result of a bad habit: it was an incident that would not repeat itself as in (24).
23.
24.
“I am so sorry, my alarm clock did not ring, it is really not in my habit (to be this late).”
The purpose of this study was to compare strategies used by a group of Canadian university students to apologize in L2 French with those employed by L1 French speakers and to contribute to research in L2 French pragmatics in the Canadian context. Differences and similarities emerged between both groups in the use of various apology strategies.
With respect to the overall use of strategies, the results reveal that the FL1 participants produced more strategies than the FL2 group (FL1: 116 tokens vs. FL2: 106 instances). It was also found that most of the strategies used by the FL1 speakers were direct apologies, while the FL2 learners mostly used indirect apologies. Both groups used emotional exclamations or interjections to introduce and boost their apologies, albeit with a different distribution across the three situations and different types of interjections. Regarding the use of direct apologies, the FL1 speakers were found to use three different types, namely “expression of regret”, “offer of apology”, and “request for forgiveness”, while the FL2 informants made use of only two types, namely, namely “expression of regret” and “offer of apology”. This result seems to suggest that the teaching of apologetic behavior to such a group of FL2 group should endeavor to also draw their attention to requests for forgiveness like
Differences and similarities were documented in the area of indirect apologies. The FL1 group used four different speech acts, namely
On the level of external modification, it was found that the FL1 speaking participants produced more supportive acts than the FL2 speakers (FL1: 15 tokens vs. FL2: 2 tokens).
Overall, the study reveals that while many aspects of apologetic behavior of the L2 French learners approximate that of the FL1 French speakers, some linguistic realization of apology strategies by the L2 French group remained problematic. The grammatical and lexical errors found in their apologies, which are partly due to the influence of the source language (English), represent an aspect that should be taken into account in the teaching and learning of apologetic behavior in L2 French. Also, learners’ attention should be directed to the possibility of using different types of supportive moves to mitigate or intensify aspects of their apologies.
This study has a number of limitations. First, the small-scale nature of the study, based on a corpus of only 16 L2 French learners, does not yield results that could be generalized to a larger group of L2 French learners. This means that a larger-scale investigation is required to establish the strategies L2 French learners choose and the problems they are confronted with when apologizing in the target language. Second, the study focused on apologies in only three situations. It is important to include more situations highlighting various levels of social distance and power distance as well as many different types of offenses in order to have a better picture of apologetic behavior in L2 French. Third: since the study carried out here is based on written data, it would be necessary to employ other types of data (e.g., role-play data) in forthcoming studies in order to establish how negotiations of complaints-apologies exchanges are enacted by L2 French learners. Fourth, in order to understand the motivations behind the use of certain strategies in the target languages, it would also be necessary to tap into the perceptions and cultural representations or cultural schemas underlying apologies in Anglo-Canadian contexts and the way in which they influence the production of apologies in L2 French. It would be also important to look at the way in which apologies are realized in Canadian English (L1), in order to indicate whether some of the “uncommon” apology strategies and realizations found in the FL2 data represent traces of the impact of L1 (English) or pragmatic transfers in the apologies of the FL2 learners in question.
This article is part of my research project “
"Open access contributes to scientific excellence and integrity. It opens up research results to wider analysis. It allows research results to be reused for new discoveries. And it enables the multi-disciplinary research that is needed to solve global 21st century problems. Open access connects science with society. It allows the public to engage with research. To go behind the headlines. And look at the scientific evidence. And it enables policy makers to draw on innovative solutions to societal challenges".
\n\nCarlos Moedas, the European Commissioner for Research Science and Innovation at the STM Annual Frankfurt Conference, October 2016.
",metaTitle:"About Open Access",metaDescription:"Open access contributes to scientific excellence and integrity. It opens up research results to wider analysis. It allows research results to be reused for new discoveries. And it enables the multi-disciplinary research that is needed to solve global 21st century problems. Open access connects science with society. It allows the public to engage with research. To go behind the headlines. And look at the scientific evidence. And it enables policy makers to draw on innovative solutions to societal challenges.\n\nCarlos Moedas, the European Commissioner for Research Science and Innovation at the STM Annual Frankfurt Conference, October 2016.",metaKeywords:null,canonicalURL:"about-open-access",contentRaw:'[{"type":"htmlEditorComponent","content":"The Open Access publishing movement started in the early 2000s when academic leaders from around the world participated in the formation of the Budapest Initiative. They developed recommendations for an Open Access publishing process, “which has worked for the past decade to provide the public with unrestricted, free access to scholarly research—much of which is publicly funded. Making the research publicly available to everyone—free of charge and without most copyright and licensing restrictions—will accelerate scientific research efforts and allow authors to reach a larger number of readers” (reference: http://www.budapestopenaccessinitiative.org)
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The Open Access publishing movement started in the early 2000s when academic leaders from around the world participated in the formation of the Budapest Initiative. They developed recommendations for an Open Access publishing process, “which has worked for the past decade to provide the public with unrestricted, free access to scholarly research—much of which is publicly funded. Making the research publicly available to everyone—free of charge and without most copyright and licensing restrictions—will accelerate scientific research efforts and allow authors to reach a larger number of readers” (reference: http://www.budapestopenaccessinitiative.org)
\n\nIntechOpen’s co-founders, both scientists themselves, created the company while undertaking research in robotics at Vienna University. Their goal was to spread research freely “for scientists, by scientists’ to the rest of the world via the Open Access publishing model. The company soon became a signatory of the Budapest Initiative, which currently has more than 1000 supporting organizations worldwide, ranging from universities to funders.
\n\nAt IntechOpen today, we are still as committed to working with organizations and people who care about scientific discovery, to putting the academic needs of the scientific community first, and to providing an Open Access environment where scientists can maximize their contribution to scientific advancement. By opening up access to the world’s scientific research articles and book chapters, we aim to facilitate greater opportunity for collaboration, scientific discovery and progress. We subscribe wholeheartedly to the Open Access definition:
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\n\nAs a firm believer in the wider dissemination of knowledge, IntechOpen supports the Open Access Initiative Protocol for Metadata Harvesting (OAI-PMH Version 2.0). Read more
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\n\nPeer Review Policies
\n\nAll scientific works are Peer Reviewed prior to publishing. Read more
\n\nOA Publishing Fees
\n\nThe Open Access publishing model employed by IntechOpen eliminates subscription charges and pay-per-view fees, enabling readers to access research at no cost. In order to sustain operations and keep our publications freely accessible we levy an Open Access Publishing Fee for manuscripts, which helps us cover the costs of editorial work and the production of books. Read more
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Poggi"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"8524",title:"Lactation in Farm Animals",subtitle:"Biology, Physiological Basis, Nutritional Requirements, and Modelization",isOpenForSubmission:!1,hash:"2aa2a9a0ec13040bbf0455e34625504e",slug:"lactation-in-farm-animals-biology-physiological-basis-nutritional-requirements-and-modelization",bookSignature:"Naceur M'Hamdi",coverURL:"https://cdn.intechopen.com/books/images_new/8524.jpg",editedByType:"Edited by",editors:[{id:"73376",title:"Dr.",name:"Naceur",middleName:null,surname:"M'Hamdi",slug:"naceur-m'hamdi",fullName:"Naceur M'Hamdi"}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}],booksByTopicTotal:37,seriesByTopicCollection:[{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:11,numberOfPublishedChapters:112,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],seriesByTopicTotal:1,mostCitedChapters:[{id:"41563",doi:"10.5772/53504",title:"Fish Cytokines and Immune Response",slug:"fish-cytokines-and-immune-response",totalDownloads:5578,totalCrossrefCites:23,totalDimensionsCites:65,abstract:null,book:{id:"3193",slug:"new-advances-and-contributions-to-fish-biology",title:"New Advances and Contributions to Fish Biology",fullTitle:"New Advances and Contributions to Fish Biology"},signatures:"Sebastián Reyes-Cerpa, Kevin Maisey, Felipe Reyes-López, Daniela Toro-Ascuy, Ana María Sandino and Mónica Imarai",authors:[{id:"92841",title:"Dr.",name:"Mónica",middleName:null,surname:"Imarai",slug:"monica-imarai",fullName:"Mónica Imarai"},{id:"153780",title:"Dr.",name:"Sebastian",middleName:null,surname:"Reyes-Cerpa",slug:"sebastian-reyes-cerpa",fullName:"Sebastian Reyes-Cerpa"},{id:"157025",title:"Dr.",name:"Kevin",middleName:null,surname:"Maisey",slug:"kevin-maisey",fullName:"Kevin Maisey"},{id:"157026",title:"Dr.",name:"Felipe",middleName:"Esteban",surname:"Reyes-López",slug:"felipe-reyes-lopez",fullName:"Felipe Reyes-López"},{id:"157027",title:"MSc.",name:"Daniela",middleName:null,surname:"Toro-Ascuy",slug:"daniela-toro-ascuy",fullName:"Daniela Toro-Ascuy"},{id:"157028",title:"Dr.",name:"Ana",middleName:null,surname:"Sandino",slug:"ana-sandino",fullName:"Ana Sandino"}]},{id:"39623",doi:"10.5772/50192",title:"Use of Yeast Probiotics in Ruminants: Effects and Mechanisms of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet",slug:"use-of-yeast-probiotics-in-ruminants-effects-and-mechanisms-of-action-on-rumen-ph-fibre-degradation-",totalDownloads:7936,totalCrossrefCites:17,totalDimensionsCites:39,abstract:null,book:{id:"2991",slug:"probiotic-in-animals",title:"Probiotic in Animals",fullTitle:"Probiotic in Animals"},signatures:"Frédérique Chaucheyras-Durand, Eric Chevaux, Cécile Martin and Evelyne Forano",authors:[{id:"151065",title:"Dr.",name:"Frederique",middleName:null,surname:"Chaucheyras-Durand",slug:"frederique-chaucheyras-durand",fullName:"Frederique Chaucheyras-Durand"},{id:"151068",title:"Mr.",name:"Eric",middleName:null,surname:"Chevaux",slug:"eric-chevaux",fullName:"Eric Chevaux"},{id:"151069",title:"Dr.",name:"Evelyne",middleName:null,surname:"Forano",slug:"evelyne-forano",fullName:"Evelyne Forano"},{id:"160177",title:"Dr.",name:"Cécile",middleName:null,surname:"Martin",slug:"cecile-martin",fullName:"Cécile Martin"}]},{id:"28679",doi:"10.5772/32100",title:"Values of Blood Variables in Calves",slug:"values-of-blood-variables-in-calves",totalDownloads:9619,totalCrossrefCites:16,totalDimensionsCites:36,abstract:null,book:{id:"1667",slug:"a-bird-s-eye-view-of-veterinary-medicine",title:"A Bird's-Eye View of Veterinary Medicine",fullTitle:"A Bird's-Eye View of Veterinary Medicine"},signatures:"Martina Klinkon and Jožica Ježek",authors:[{id:"90171",title:"Prof.",name:"Martina",middleName:null,surname:"Klinkon",slug:"martina-klinkon",fullName:"Martina Klinkon"}]},{id:"16107",doi:"10.5772/16563",title:"Effect of Cryopreservation on Sperm Quality and Fertility",slug:"effect-of-cryopreservation-on-sperm-quality-and-fertility",totalDownloads:15484,totalCrossrefCites:10,totalDimensionsCites:35,abstract:null,book:{id:"185",slug:"artificial-insemination-in-farm-animals",title:"Artificial Insemination in Farm Animals",fullTitle:"Artificial Insemination in Farm Animals"},signatures:"Alemayehu Lemma",authors:[{id:"25594",title:"Dr.",name:"Alemayehu",middleName:null,surname:"Lemma",slug:"alemayehu-lemma",fullName:"Alemayehu Lemma"}]},{id:"57645",doi:"10.5772/intechopen.71780",title:"Antibiotics in Chilean Aquaculture: A Review",slug:"antibiotics-in-chilean-aquaculture-a-review",totalDownloads:1951,totalCrossrefCites:17,totalDimensionsCites:29,abstract:"Aquaculture in Chile has been practiced since the 1920s; however, it was not until the 1990s that aquaculture became an important sector here. Important species in Chilean aquaculture include salmonids, algae, mollusks, and turbot. Salmonids are the dominant species in Chilean aquaculture for both harvest volume and export value, their production reaching greater than 800-thousand tons in 2015. However, this growth has been accompanied by an increase in disease presence, requiring greater drug use to control. This increase in drug use is an environmental and public health concern for the authorities, the salmon industry itself, and the destination markets. In this chapter, we review the literature on drug use, antibiotic resistance, regulatory framework, and alternatives, with focus on Chile.",book:{id:"6179",slug:"antibiotic-use-in-animals",title:"Antibiotic Use in Animals",fullTitle:"Antibiotic Use in Animals"},signatures:"Ivonne Lozano, Nelson F. Díaz, Susana Muñoz and Carlos Riquelme",authors:[{id:"208847",title:"Dr.",name:"Ivonne",middleName:null,surname:"Lozano",slug:"ivonne-lozano",fullName:"Ivonne Lozano"},{id:"208895",title:"Dr.",name:"Nelson F.",middleName:null,surname:"Díaz",slug:"nelson-f.-diaz",fullName:"Nelson F. Díaz"},{id:"208897",title:"Dr.",name:"Carlos",middleName:null,surname:"Riquelme",slug:"carlos-riquelme",fullName:"Carlos Riquelme"},{id:"208898",title:"MSc.",name:"Susana",middleName:null,surname:"Muñoz",slug:"susana-munoz",fullName:"Susana Muñoz"}]}],mostDownloadedChaptersLast30Days:[{id:"56612",title:"Reproduction in Goats",slug:"reproduction-in-goats",totalDownloads:2924,totalCrossrefCites:3,totalDimensionsCites:4,abstract:"Reproductive activity of the goat begins when the females reach puberty, which happens at 5 months of age. The ovarian or estrous cycle is the period between two consecutive estrus. It is also the time that lasts the development of the follicle in the ovary, until rupture occurs and ovulation takes place, which coincides with the appearance of estrus. This chapter will describe the physiological and endocrinological bases of estrus in the goat. Likewise, factors affecting the presence of estrus and ovulation will be described. At another point, synchronization of estrus and ovulation, factors affecting the presence of estrus and external symptoms of estrus, will be described. To achieve synchronization of estrus or induction of ovulation within or outside the breeding season, it may be necessary to manage light hours, male effect, and/or use of hormones. The importance of artificial insemination is described, as well as the current situation of this technique worldwide. Currently, the techniques of artificial insemination in goats have been limited worldwide, due to the lack of resources of producers and trained technicians. The techniques of artificial insemination with estrous synchronization programs and ovulation with current research results will be described.",book:{id:"5987",slug:"goat-science",title:"Goat Science",fullTitle:"Goat Science"},signatures:"Fernando Sánchez Dávila, Alejandro Sergio del Bosque González\nand Hugo Bernal Barragán",authors:[{id:"201830",title:"Dr.",name:"Fernando",middleName:"Sanchez",surname:"Davila",slug:"fernando-davila",fullName:"Fernando Davila"},{id:"206127",title:"Dr.",name:"Alejandro Sergio",middleName:null,surname:"Del Bosque-Gonzalez",slug:"alejandro-sergio-del-bosque-gonzalez",fullName:"Alejandro Sergio Del Bosque-Gonzalez"},{id:"206128",title:"Dr.",name:"Hugo",middleName:null,surname:"Bernal-Barragán",slug:"hugo-bernal-barragan",fullName:"Hugo Bernal-Barragán"}]},{id:"58095",title:"The Innovative Techniques in Animal Husbandry",slug:"the-innovative-techniques-in-animal-husbandry",totalDownloads:3817,totalCrossrefCites:4,totalDimensionsCites:8,abstract:"Technology is developing rapidly. In this development, the transfer of computer systems and software to the application has made an important contribution. Technologic instruments made farmers can work more comfortable and increased animal production efficiency and profitability. Therefore, technologic developments are the main research area for animal productivity and sustainability. Many technologic equipment and tools made animal husbandry easier and comfortable. Especially management decisions and applications are effected highly ratio with this rapid development. In animal husbandry management decisions that need to be done daily are configured according to the correctness of the decisions to be made. At this point, smart systems give many opportunities to farmers. Milking, feeding, environmental control, reproductive performance constitute everyday jobs most affected by correct management decisions. Human errors in this works and decisions made big effect on last product quality and profitability are not able to be risked. This chapter deal with valuable information on the latest challenges and key innovations affecting the animal husbandry. Also, innovative approaches and applications for animal husbandry are tried to be summarized with detail latest research results.",book:{id:"6384",slug:"animal-husbandry-and-nutrition",title:"Animal Husbandry and Nutrition",fullTitle:"Animal Husbandry and Nutrition"},signatures:"Serap Göncü and Cahit Güngör",authors:[{id:"215579",title:"Prof.",name:"Serap",middleName:null,surname:"Goncu",slug:"serap-goncu",fullName:"Serap Goncu"},{id:"218971",title:"Dr.",name:"Cahit",middleName:null,surname:"Güngör",slug:"cahit-gungor",fullName:"Cahit Güngör"}]},{id:"58486",title:"Quality of Chicken Meat",slug:"quality-of-chicken-meat",totalDownloads:3351,totalCrossrefCites:19,totalDimensionsCites:28,abstract:"Chicken meat is considered as an easily available source of high-quality protein and other nutrients that are necessary for proper body functioning. In order to meet the consumers’ growing demands for high-quality protein, the poultry industry focused on selection of fast-growing broilers, which reach a body mass of about 2.5 kg within 6-week-intensive fattening. Relatively low sales prices of chicken meat, in comparison to other types of meat, speak in favor of the increased chicken meat consumption. In addition, chicken meat is known by its nutritional quality, as it contains significant amount of high-quality and easily digestible protein and a low portion of saturated fat. Therefore, chicken meat is recommended for consumption by all age groups. The technological parameters of chicken meat quality are related to various factors (keeping conditions, feeding treatment, feed composition, transport, stress before slaughter, etc.). Composition of chicken meat can be influenced through modification of chicken feed composition (addition of different types of oils, vitamins, microelements and amino acids), to produce meat enriched with functional ingredients (n-3 PUFA, carnosine, selenium and vitamin E). By this way, chicken meat becomes a foodstuff with added value, which, in addition to high-quality nutritional composition, also contains ingredients that are beneficial to human health.",book:{id:"6384",slug:"animal-husbandry-and-nutrition",title:"Animal Husbandry and Nutrition",fullTitle:"Animal Husbandry and Nutrition"},signatures:"Gordana Kralik, Zlata Kralik, Manuela Grčević and Danica Hanžek",authors:[{id:"207236",title:"Dr.",name:"Gordana",middleName:null,surname:"Kralik",slug:"gordana-kralik",fullName:"Gordana Kralik"},{id:"227281",title:"Prof.",name:"Zlata",middleName:null,surname:"Kralik",slug:"zlata-kralik",fullName:"Zlata Kralik"},{id:"227283",title:"Dr.",name:"Manuela",middleName:null,surname:"Grčević",slug:"manuela-grcevic",fullName:"Manuela Grčević"},{id:"227284",title:"BSc.",name:"Danica",middleName:null,surname:"Hanžek",slug:"danica-hanzek",fullName:"Danica Hanžek"}]},{id:"56453",title:"Goat System Productions: Advantages and Disadvantages to the Animal, Environment and Farmer",slug:"goat-system-productions-advantages-and-disadvantages-to-the-animal-environment-and-farmer",totalDownloads:4379,totalCrossrefCites:5,totalDimensionsCites:20,abstract:"Goats have always been considered very useful animals. Goats success is related to its excellent adaptability to the difficult mountain conditions, extreme weather and low value feed acceptance, versatile habits and high production considering their size. These are some reasons because goats are among the first animals to be domesticated. In terms of evolution, goats could be separated by their dispersion area in three large groups: the European, the Asian, and the African. Global goat populations, mainly in Africa and in Asia, have increased for centuries but very strongly in the past decades, well above the world population growth. They are also used for forest grazing, an integrated and alternative production system, very useful to control weed growth reducing fire risk. Despite some exceptions, no large‐scale effort to professionalize this industry has been made so far. There are consumers for goat dairy products and there is enough global production, but misses a professional network between both. Regarding goat meat, the world leadership also stays in Africa and Asia, namely in China, and there is a new phenomenon, the spreading of goat meat tradition through Europe due to migrants from Africa and other places with strong goat meat consumption.",book:{id:"5987",slug:"goat-science",title:"Goat Science",fullTitle:"Goat Science"},signatures:"António Monteiro, José Manuel Costa and Maria João Lima",authors:[{id:"190314",title:"Prof.",name:"António",middleName:"Cardoso",surname:"Monteiro",slug:"antonio-monteiro",fullName:"António Monteiro"},{id:"203680",title:"Prof.",name:"Maria João",middleName:null,surname:"Lima",slug:"maria-joao-lima",fullName:"Maria João Lima"},{id:"203683",title:"MSc.",name:"José Manuel",middleName:null,surname:"Costa",slug:"jose-manuel-costa",fullName:"José Manuel Costa"}]},{id:"70760",title:"Induction and Synchronization of Estrus",slug:"induction-and-synchronization-of-estrus",totalDownloads:1750,totalCrossrefCites:1,totalDimensionsCites:2,abstract:"Estrus cycle is a rhythmic change that occur in the reproductive system of females starting from one estrus phase to another. The normal duration of estrus cycle is 21 days in cow, sow, and mare, 17 days in ewe, and 20 days in doe. The species which exhibit a single estrus cycle are known as monstrous and species which come into estrus twice or more are termed polyestrous animals. Among them some species have estrus cycles in a particular season and defined as seasonal polyestrous. It includes goats, sheep, and horses. On the other hand, cattle undergo estrus throughout the year. The estrus inducers can grossly be divided into two parts, that is, non-hormonal and hormonal. Non-hormonal treatments include plant-derived heat inducers, mineral supplementation, uterine and ovarian massage, and use of Lugol’s iodine. The hormones that are used in estrus induction are estrogen, progesterone, GnRH, prostaglandin, insulin, and anti-prolactin-based treatment. Synchronization can shorten the breeding period to less than 5 days, instead of females being bred over a 21-day period, depending on the treatment regimen. The combination of GnRH with the prostaglandin F2α (PGF2α)- and progesterone-based synchronization program has shown a novel direction in the estrus synchronization of cattle with the follicular development manipulation.",book:{id:"8545",slug:"animal-reproduction-in-veterinary-medicine",title:"Animal Reproduction in Veterinary Medicine",fullTitle:"Animal Reproduction in Veterinary Medicine"},signatures:"Prasanna Pal and Mohammad Rayees Dar",authors:[{id:"299126",title:"Dr.",name:"Mohammad Rayees",middleName:null,surname:"Dar",slug:"mohammad-rayees-dar",fullName:"Mohammad Rayees Dar"},{id:"311663",title:"Dr.",name:"Prasanna",middleName:null,surname:"Pal",slug:"prasanna-pal",fullName:"Prasanna Pal"}]}],onlineFirstChaptersFilter:{topicId:"25",limit:6,offset:0},onlineFirstChaptersCollection:[{id:"82991",title:"Diseases of the Canine Prostate Gland",slug:"diseases-of-the-canine-prostate-gland",totalDownloads:0,totalDimensionsCites:0,doi:"10.5772/intechopen.105835",abstract:"In dogs, the most frequent diseases of the prostate gland are benign prostate gland hyperplasia (BPH), acute and chronic prostatitis, squamous metaplasia, and prostate tumors. New diagnostic tools comprise diagnostic markers in the blood and urine, as well as advanced imaging methods. The therapy can be initialized with the 5α-reductase-inhibitor finasteride or an anti-androgenic compound, and prolonged with a long-acting gonadotropin-releasing-hormone (GnRH)-agonist such as deslorelin. In case of prostatitis, effective antibiotics must be applied for weeks. Antibiotics must be able to penetrate into the prostate tissue; fluoroquinolones, clindamycin, and erythromycin are good choices and are in addition effective against mycoplasms. The chronical prostatitis cannot be differentiated from a neoplasia by sonography; a biopsy, histological, and bacteriological examination are required. Tumors of the prostate gland are seldom and mostly occur in castrated but in intact dogs. For the final diagnosis, a biopsy must be taken. Partial and total resection of the prostate gland by use of laser technique is possible but coincedes with many side effects and the prognosis is still futile. Immunotherapy combined with NSAIDs, targeted noninvasive thermotherapy, BRAF gene inhibitors, or prostate artery chemoembolization are promising methods.",book:{id:"11580",title:"Recent Advances in Canine Medicine",coverURL:"https://cdn.intechopen.com/books/images_new/11580.jpg"},signatures:"Sabine Schäfer-Somi"},{id:"82956",title:"Potential Substitutes of Antibiotics for Swine and Poultry Production",slug:"potential-substitutes-of-antibiotics-for-swine-and-poultry-production",totalDownloads:2,totalDimensionsCites:0,doi:"10.5772/intechopen.106081",abstract:"Early of the last century, it was detected that antibiotics added to the animal feeds at low doses and for a long time can improve technical performances such as average daily gain and gain-to-feed ratio. Since then, the antibiotics have been used worldwide as feed additives for many decades. At the end of the twentieth century, the consequences of the uses of antibiotics in animal feeds as growth promoters were informed. Since then, many research studies have been done to find other solutions to replace partly or fully to antibiotic as growth promoters (AGPs). Many achievements in finding alternatives to AGPs in which probiotics and direct-fed microorganism, prebiotics, organic acids and their salts, feed enzymes, bacteriophages, herbs, spices, and other plant extractives (phytogenics), mineral and essential oils are included.",book:{id:"11578",title:"Antibiotics and Probiotics in Animal Food - Impact and Regulation",coverURL:"https://cdn.intechopen.com/books/images_new/11578.jpg"},signatures:"Ho Trung Thong, Le Nu Anh Thu and Ho Viet Duc"},{id:"82905",title:"A Review of Application Strategies and Efficacy of Probiotics in Pet Food",slug:"a-review-of-application-strategies-and-efficacy-of-probiotics-in-pet-food",totalDownloads:15,totalDimensionsCites:0,doi:"10.5772/intechopen.105829",abstract:"In companion animal nutrition, probiotics (direct-fed microbials) are marketed as functional ingredients that add value to pet foods due to the impact they have on gastrointestinal and immune health of dogs and cats. The nature of the beneficial effect each probiotic strain exerts depends on its metabolic properties and perhaps most importantly, the arrival of a sufficient number of viable cells to the large bowel of the host. Pet food manufacturing processes are designed to improve food safety and prolong shelf-life, which is counterproductive to the survival of direct-fed microbials. Therefore, a prerequisite for the effective formulation of pet foods with probiotics is an understanding of the conditions each beneficial bacterial strain needs to survive. The aims of this chapter are: (1) To summarize the inherent characteristics of probiotic strains used in commercial pet foods, and (2) To review recently published literature on the applications of probiotics to pet foods and their associated challenges to viability.",book:{id:"11578",title:"Antibiotics and Probiotics in Animal Food - Impact and Regulation",coverURL:"https://cdn.intechopen.com/books/images_new/11578.jpg"},signatures:"Heather Acuff and Charles G. Aldrich"},{id:"82773",title:"Canine Transmissible Venereal Tumor: An Infectious Neoplasia in Dogs",slug:"canine-transmissible-venereal-tumor-an-infectious-neoplasia-in-dogs",totalDownloads:15,totalDimensionsCites:0,doi:"10.5772/intechopen.106150",abstract:"Canine transmissible venereal tumor is the oldest cancer in dogs and is transplanted via viable cancer cells. This cancer has a specific host, easy transmission, noticeable gross lesions, a predictable growth pattern, an immunologic relative host response, unique molecular characteristics, and is responsive to chemotherapeutic treatment. These points make researchers and practitioners interested in this cancer. Genital cases are noticeable and therefore easier to diagnose and treat than extragenital cases. By contrasting the anatomical features of the two types of cases, we highlight the uniqueness of canine transmissible venereal tumors and discuss the diagnosis, treatment, and prevention of this ancient cancer.",book:{id:"11580",title:"Recent Advances in Canine Medicine",coverURL:"https://cdn.intechopen.com/books/images_new/11580.jpg"},signatures:"Chanokchon Setthawongsin, Somporn Techangamsuwan and Anudep Rungsipipat"},{id:"82797",title:"Anatomical Guide to the Paranasal Sinuses of Domestic Animals",slug:"anatomical-guide-to-the-paranasal-sinuses-of-domestic-animals",totalDownloads:8,totalDimensionsCites:0,doi:"10.5772/intechopen.106157",abstract:"Paranasal sinuses are paired cavities within the skull, which develop by evagination into the spongy bone between the external and internal plates of the cranial and facial bones. Thus, each sinus is lined by respiratory epithelium and has direct or indirect communication to the nasal cavity. The purpose of this chapter is to present an anatomical reference guide of the paranasal sinuses in domestic animals, including large and small ruminants (cattle, buffalo, sheep, and goats), camels, canines (dog) and equines (horse and donkey), appropriate for use by anatomists, radiologists, clinicians, and veterinary students. Topographic descriptions and the relationships between the various air cavities and paranasal sinuses have been visualized using computed tomography and cadaver sections images. The anatomical features (including head bones, muscles, and soft tissues) have been compared using both dissected heads and skulls and computed tomography images. This chapter will therefore be useful as a normal reference guide for clinical applications.",book:{id:"10665",title:"Updates on Veterinary Anatomy and Physiology",coverURL:"https://cdn.intechopen.com/books/images_new/10665.jpg"},signatures:"Mohamed A.M. Alsafy, Samir A.A. 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In this chapter, we review the studies on reproduction of marine mammals using δ13C and δ15N values analyzed in several tissues and describe the typical changes reported to date in those values and Hg concentrations in offspring and milk during lactation. Next, we present data on ontogenetic changes in δ15N and δ13C profiles and Hg concentration, especially focusing on the lactation period, in muscle samples of hunted bowhead whale, and stranded common minke whale (mysticetes), Dall’s porpoise (odontocete), and the harbor seal (phocid). 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She has been a faculty member at the University of California, Riverside in the School of Education since 2016. Her research focuses on translational studies to explore the reward system in ASD, as well as how anxiety contributes to social challenges in ASD. She also investigates how behavioral interventions affect neural activity, behavior, and school performance in children with ASD. She is also involved in the diagnosis of children with ASD and is a licensed clinical psychologist in California. 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Ben-Soussan leads international studies on training and neuroplasticity from neurophysiological and psychobiological perspectives. As a neuroscientist and bio-psychologist, she has published numerous articles on neuroplasticity, movement and meditation. She acts as an editor and reviewer in several renowned journals and coordinates international conferences integrating theoretical, methodological and practical approaches on various topics, such as silence, logics and neuro-education. She lives in Assisi, Italy.",institutionString:"Research Institute for Neuroscience, Education and Didactics, Patrizio Paoletti Foundation",institution:null},editorTwo:null,editorThree:null}]},overviewPageOFChapters:{paginationCount:20,paginationItems:[{id:"82526",title:"Deep Multiagent Reinforcement Learning Methods Addressing the Scalability Challenge",doi:"10.5772/intechopen.105627",signatures:"Theocharis Kravaris and George A. 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He is currently a principal researcher in data analytics and optimisation at TECNALIA (Spain), a visiting fellow at the Basque Center for Applied Mathematics (BCAM) and a part-time lecturer at the University of the Basque Country (UPV/EHU). His research interests gravitate on the use of descriptive, prescriptive and predictive algorithms for data mining and optimization in a diverse range of application fields such as Energy, Transport, Telecommunications, Health and Industry, among others. In these fields he has published more than 240 articles, co-supervised 8 Ph.D. theses, edited 6 books, coauthored 7 patents and participated/led more than 40 research projects. 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He is currently a full professor in\nthe Department of Automation and Applied Informatics at the\nsame university. Dr. Voloşencu is the author of ten books, seven\nbook chapters, and more than 160 papers published in journals\nand conference proceedings. He has also edited twelve books and\nhas twenty-seven patents to his name. He is a manager of research grants, editor in\nchief and member of international journal editorial boards, a former plenary speaker, a member of scientific committees, and chair at international conferences. His\nresearch is in the fields of control systems, control of electric drives, fuzzy control\nsystems, neural network applications, fault detection and diagnosis, sensor network\napplications, monitoring of distributed parameter systems, and power ultrasound\napplications. 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He has an excellent track record in the herpesvirus field, and his group is engaged in clinical research in the field of Epstein-Barr virus diseases. He is the editor of the online Encyclopedia of Environment and he coordinates the Universal Health Coverage education program for the BioHealth Computing Schools of the European Institute of Science.",institutionString:null,institution:{name:"Grenoble Alpes University",country:{name:"France"}}},{id:"131400",title:"Prof.",name:"Alfonso J.",middleName:null,surname:"Rodriguez-Morales",slug:"alfonso-j.-rodriguez-morales",fullName:"Alfonso J. Rodriguez-Morales",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/131400/images/system/131400.png",biography:"Dr. Rodriguez-Morales is an expert in tropical and emerging diseases, particularly zoonotic and vector-borne diseases (especially arboviral diseases). He is the president of the Travel Medicine Committee of the Pan-American Infectious Diseases Association (API), as well as the president of the Colombian Association of Infectious Diseases (ACIN). He is a member of the Committee on Tropical Medicine, Zoonoses, and Travel Medicine of ACIN. He is a vice-president of the Latin American Society for Travel Medicine (SLAMVI) and a Member of the Council of the International Society for Infectious Diseases (ISID). Since 2014, he has been recognized as a Senior Researcher, at the Ministry of Science of Colombia. He is a professor at the Faculty of Medicine of the Fundacion Universitaria Autonoma de las Americas, in Pereira, Risaralda, Colombia. He is an External Professor, Master in Research on Tropical Medicine and International Health, Universitat de Barcelona, Spain. He is also a professor at the Master in Clinical Epidemiology and Biostatistics, Universidad Científica del Sur, Lima, Peru. In 2021 he has been awarded the “Raul Isturiz Award” Medal of the API. Also, in 2021, he was awarded with the “Jose Felix Patiño” Asclepius Staff Medal of the Colombian Medical College, due to his scientific contributions to COVID-19 during the pandemic. He is currently the Editor in Chief of the journal Travel Medicine and Infectious Diseases. His Scopus H index is 47 (Google Scholar H index, 68).",institutionString:"Institución Universitaria Visión de las Américas, Colombia",institution:null},{id:"332819",title:"Dr.",name:"Chukwudi Michael",middleName:"Michael",surname:"Egbuche",slug:"chukwudi-michael-egbuche",fullName:"Chukwudi Michael Egbuche",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/332819/images/14624_n.jpg",biography:"I an Dr. Chukwudi Michael Egbuche. I am a Senior Lecturer in the Department of Parasitology and Entomology, Nnamdi Azikiwe University, Awka.",institutionString:null,institution:{name:"Nnamdi Azikiwe University",country:{name:"Nigeria"}}},{id:"284232",title:"Mr.",name:"Nikunj",middleName:"U",surname:"Tandel",slug:"nikunj-tandel",fullName:"Nikunj Tandel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/284232/images/8275_n.jpg",biography:'Mr. Nikunj Tandel has completed his Master\'s degree in Biotechnology from VIT University, India in the year of 2012. He is having 8 years of research experience especially in the field of malaria epidemiology, immunology, and nanoparticle-based drug delivery system against the infectious diseases, autoimmune disorders and cancer. He has worked for the NIH funded-International Center of Excellence in Malaria Research project "Center for the study of complex malaria in India (CSCMi)" in collaboration with New York University. The preliminary objectives of the study are to understand and develop the evidence-based tools and interventions for the control and prevention of malaria in different sites of the INDIA. Alongside, with the help of next-generation genomics study, the team has studied the antimalarial drug resistance in India. Further, he has extended his research in the development of Humanized mice for the study of liver-stage malaria and identification of molecular marker(s) for the Artemisinin resistance. At present, his research focuses on understanding the role of B cells in the activation of CD8+ T cells in malaria. Received the CSIR-SRF (Senior Research Fellow) award-2018, FIMSA (Federation of Immunological Societies of Asia-Oceania) Travel Bursary award to attend the IUIS-IIS-FIMSA Immunology course-2019',institutionString:"Nirma University",institution:{name:"Nirma University",country:{name:"India"}}},{id:"334383",title:"Ph.D.",name:"Simone",middleName:"Ulrich",surname:"Ulrich Picoli",slug:"simone-ulrich-picoli",fullName:"Simone Ulrich Picoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334383/images/15919_n.jpg",biography:"Graduated in Pharmacy from Universidade Luterana do Brasil (1999), Master in Agricultural and Environmental Microbiology from Federal University of Rio Grande do Sul (2002), Specialization in Clinical Microbiology from Universidade de São Paulo, USP (2007) and PhD in Sciences in Gastroenterology and Hepatology (2012). She is currently an Adjunct Professor at Feevale University in Medicine and Biomedicine courses and a permanent professor of the Academic Master\\'s Degree in Virology. She has experience in the field of Microbiology, with an emphasis on Bacteriology, working mainly on the following topics: bacteriophages, bacterial resistance, clinical microbiology and food microbiology.",institutionString:null,institution:{name:"Universidade Feevale",country:{name:"Brazil"}}},{id:"229220",title:"Dr.",name:"Amjad",middleName:"Islam",surname:"Aqib",slug:"amjad-aqib",fullName:"Amjad Aqib",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229220/images/system/229220.png",biography:"Dr. Amjad Islam Aqib obtained a DVM and MSc (Hons) from University of Agriculture Faisalabad (UAF), Pakistan, and a PhD from the University of Veterinary and Animal Sciences Lahore, Pakistan. 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His research interests include epidemiological patterns and molecular analysis of antimicrobial resistance and modulation and vaccine development against animal pathogens of public health concern.",institutionString:"Cholistan University of Veterinary and Animal Sciences",institution:{name:"University of Agriculture Faisalabad",country:{name:"Pakistan"}}},{id:"333753",title:"Dr.",name:"Rais",middleName:null,surname:"Ahmed",slug:"rais-ahmed",fullName:"Rais Ahmed",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/333753/images/20168_n.jpg",biography:null,institutionString:null,institution:{name:"University of Agriculture Faisalabad",country:{name:"Pakistan"}}},{id:"62900",title:"Prof.",name:"Fethi",middleName:null,surname:"Derbel",slug:"fethi-derbel",fullName:"Fethi Derbel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62900/images/system/62900.jpeg",biography:"Professor Fethi Derbel was born in 1960 in Tunisia. He received his medical degree from the Sousse Faculty of Medicine at Sousse, University of Sousse, Tunisia. He completed his surgical residency in General Surgery at the University Hospital Farhat Hached of Sousse and was a member of the Unit of Liver Transplantation in the University of Rennes, France. He then worked in the Department of Surgery at the Sahloul University Hospital in Sousse. Professor Derbel is presently working at the Clinique les Oliviers, Sousse, Tunisia. His hospital activities are mostly concerned with laparoscopic, colorectal, pancreatic, hepatobiliary, and gastric surgery. He is also very interested in hernia surgery and performs ventral hernia repairs and inguinal hernia repairs. He has been a member of the GREPA and Tunisian Hernia Society (THS). During his residency, he managed patients suffering from diabetic foot, and he was very interested in this pathology. For this reason, he decided to coordinate a book project dealing with the diabetic foot. Professor Derbel has published many articles in journals and collaborates intensively with IntechOpen Access Publisher as an editor.",institutionString:"Clinique les Oliviers",institution:null},{id:"300144",title:"Dr.",name:"Meriem",middleName:null,surname:"Braiki",slug:"meriem-braiki",fullName:"Meriem Braiki",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/300144/images/system/300144.jpg",biography:"Dr. Meriem Braiki is a specialist in pediatric surgeon from Tunisia. She was born in 1985. She received her medical degree from the University of Medicine at Sousse, Tunisia. She achieved her surgical residency training periods in Pediatric Surgery departments at University Hospitals in Monastir, Tunis and France.\r\nShe is currently working at the Pediatric surgery department, Sidi Bouzid Hospital, Tunisia. Her hospital activities are mostly concerned with laparoscopic, parietal, urological and digestive surgery. She has published several articles in diffrent journals.",institutionString:"Sidi Bouzid Regional Hospital",institution:null},{id:"229481",title:"Dr.",name:"Erika M.",middleName:"Martins",surname:"de Carvalho",slug:"erika-m.-de-carvalho",fullName:"Erika M. de Carvalho",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229481/images/6397_n.jpg",biography:null,institutionString:null,institution:{name:"Oswaldo Cruz Foundation",country:{name:"Brazil"}}},{id:"186537",title:"Prof.",name:"Tonay",middleName:null,surname:"Inceboz",slug:"tonay-inceboz",fullName:"Tonay Inceboz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/186537/images/system/186537.jfif",biography:"I was graduated from Ege University of Medical Faculty (Turkey) in 1988 and completed his Med. PhD degree in Medical Parasitology at the same university. I became an Associate Professor in 2008 and Professor in 2014. I am currently working as a Professor at the Department of Medical Parasitology at Dokuz Eylul University, Izmir, Turkey.\n\nI have given many lectures, presentations in different academic meetings. I have more than 60 articles in peer-reviewed journals, 18 book chapters, 1 book editorship.\n\nMy research interests are Echinococcus granulosus, Echinococcus multilocularis (diagnosis, life cycle, in vitro and in vivo cultivation), and Trichomonas vaginalis (diagnosis, PCR, and in vitro cultivation).",institutionString:"Dokuz Eylül University",institution:{name:"Dokuz Eylül University",country:{name:"Turkey"}}},{id:"71812",title:"Prof.",name:"Hanem Fathy",middleName:"Fathy",surname:"Khater",slug:"hanem-fathy-khater",fullName:"Hanem Fathy Khater",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/71812/images/1167_n.jpg",biography:"Prof. Khater is a Professor of Parasitology at Benha University, Egypt. She studied for her doctoral degree, at the Department of Entomology, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, USA. She has completed her Ph.D. degrees in Parasitology in Egypt, from where she got the award for “the best scientific Ph.D. dissertation”. She worked at the School of Biological Sciences, Bristol, England, the UK in controlling insects of medical and veterinary importance as a grant from Newton Mosharafa, the British Council. 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She was a recipient of a Commonwealth fellowship supported by British Council tenable at the Centre for Entomology and Parasitology (CAEP), Keele University, United Kingdom between 2004 and 2005. She was awarded an Honorary Visiting Research Fellow at the same university from 2005 to 2007. \nShe has been an external examiner to the Department of Veterinary Microbiology and Parasitology, University of Ibadan, MSc programme between 2010 and 2012. She is a member of the Nigerian Society of Experimental Biology (NISEB), Parasitology and Public Health Society of Nigeria (PPSN), Science Association of Nigeria (SAN), Zoological Society of Nigeria (ZSN), and is Vice Chairperson of the Organisation of Women in Science (OWSG), LASU chapter. She served as Head of Department of Zoology and Environmental Biology, Lagos State University from 2007 to 2010 and 2014 to 2016. 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His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. 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