Molecular structures and description of the different generations of quaternary ammonium (QAC).
\r\n\t
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Sepasgozar and Dr. Farzaneh Soflaei",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11926.jpg",keywords:"3D modeling, Facility Management, Risk Management, Life Cycle Management, Energy Efficiency, Cost Management, Sustainable Design, Climate Change, Artificial Intelligence, Virtual Reality, Geographical Information System, Internet of Things",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 18th 2022",dateEndSecondStepPublish:"April 15th 2022",dateEndThirdStepPublish:"June 14th 2022",dateEndFourthStepPublish:"September 2nd 2022",dateEndFifthStepPublish:"November 1st 2022",remainingDaysToSecondStep:"a month",secondStepPassed:!0,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"A registered architect, a visiting researcher at the College of Architecture in Texas, USA with a focus on sustainable architecture, experienced faculty member, LEED Green Associate.",coeditorOneBiosketch:"Dr. Sepasgozar is recognized as one of the world's top 2% researchers by Stanford University in 2020, Associate Editor and Editorial board member for Q1 & Q2 journals, reviewer of 33 leading journals including Cleaner Production, Automation in Construction, Construction Innovation. 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Throughout my career, I have explored different aspects of this multi-dimensional issue in a wide range of buildings and urban scale projects. In my Ph.D. research in architecture, I worked on the impact of courtyards as passive cooling/heating strategies on improving thermal comfort and reducing costs and consumption in low-energy housing design. On the other hand, my Ph.D. dissertation in urban design was carried out on the role of public open spaces as potential venues for democratic participation and interactions, in urban social sustainability. 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The spectrum of clinical manifestations of COVID-19 varies from asymptomatic or somewhat mild-disease (81%) to severe clinical conditions characterized by respiratory failure requiring mechanical ventilation (14%) and to critical systemic presentations with multiple organ dysfunction syndromes or failures (5%) [1, 2].
There is a huge effort to develop vaccines, some are developed and received the accelerated access at the moment, however, there is no specific antiviral treatment recommended or appreved for COVID-19, yet. Current therapeutic strategies are only supportive and oxygen therapy represents the primary treatment intervention for patients with severe pneumonia. The medications that have already been known such as anti-viral, anti-malarial, and anti-inflammatory agents have been taken from the shelves and began to be used as the emergency state action to improve the recovery of the patients and increase the survival. Whilst these treatments can improve patient’s recovery and survival to some extend, these therapeutic strategies do not lead to unequivocal restoration of the lung damage inflicted by this disease [3]. The outcome so far shows that the antibiotics are ineffective; although systemic corticosteroids seem be effective, they also reduce the immune system activity and thus its ability to fight against the infection. It is of crucial importance to save the patients with severe COVID-19 pneumonia, to prevent and even reverse the cytokine storm along with inhibiting the viral replication [4].
The cellular therapies with mesenchymal stem cells (MSCs) are attracting attention as they could offer a new therapeutic approach in this context. These stem cells have broad pharmacological effects, including anti-inflammatory, immunomodulatory, regenerative, pro-angiogenic and even anti-fibrotic properties [5].
Stem cells, in particular MSCs, exert their immunomodulatory, anti-oxidant, and reparative therapeutic effects likely through secreted extracellular vesicles (EVs), and therefore, could be beneficial, alone or in combination with other therapeutic agents, in patients diagnosed with COVID-19 [3, 6]. They are are emerging as new promising treatments, since they could not only attenuate the inflammation but also regenerate the lung damage caused by COVID-19 [7, 8].
In this chapter, we outline the information about this novel virus, and the pathophysiology of the COVID-19 infection, the mechanisms of cytokine storm and lung damage caused by SARS-CoV-2 virus and how mesenchymal stem cells (MSCs) can be utilized to hamper this damage by harnessing their regenerative properties. The potential of these ancestor cells in the enhanced clinical utility in treating the COVID-19 patients along with the opportunuties major roadblocks to progressing these promising curative therapies toward mainstream treatment for COVID-19 have also been evaluated.
Belonging to the β Coronavirus family, SARS-CoV2 is a single-stranded RNA, enveloped virus of 50-200 nm diameter [9]. Spike Glycoprotein (S) is the vital protein consisting of three S1-S2 heterodimers that bind to angiotensin-converting enzyme 2 (ACE2) receptor on type II pneumocyte in the lung tissue [3, 9, 10]. Besides S protein, enetically SARS-CoV-2 is constructed on structural proteins of membrane (M), envelope (E), and nucleocapsid (N) proteins. Spread of the virus is managed by the high affinity of S proteins to ACE2 receptors that are expressed in human organs, principally in lung alveolar epithelial cells and enterocytes of the small intestine [11, 12].
Once the SARS-CoV-2 virus enters into the type II pneumocyte and capillary endothelium by endocytosis, it increases in the cytoplasm. Apoptosis is induced by yhe stress in the pneumocytes. Besides, the viral RNA acts as a pathogen-associated molecular pattern and is recognized by the pattern recognition receptor or toll-like receptors. Subsequent chemokine attraction causes neutrophil migration and activation. Then the destruction of the alveolar-capillary walls occur. This leads to the lost interface between the intra-alveolar space and the stroma. Therefore, fluid leaks through and fills into the alveolar spaces [13, 14].
One of the prominent features of SARS-CoV-2 is its being more inclinable to infect the human lung and higher, 3.20-fold faster, duplication time than SARS-CoV [15].
Modes of transmission occurs through droplet transmission, fecal-oral route, conjunctiva and fomites [13, 14]. Also, the local transmission can be traced back to the patient’s body fluids such as respiratory droplets, saliva, feces, and urine [15]. The virion is stabilized at lower temperatures, i.e., 4 °C has higher survival than 22 °C [16, 17].
Before the clinical symptoms presentation, during the symptomatic stage and even during the recovery period, the patients with COVID-19 can spread the infection, because SARS-CoV-2 virions are shed throughout the clinical course.
When it comes to the residence time of the SARS-CoV-2 virion on surfaces, it has been known that the viable residence time of SARS-CoV-1 in aerosols, copper, cardboard, stainless steel, and plastic are 3 h, 4 h, 24 h, 48 h, and 72 h, respectively [18].
The symptoms and relevant clinical presentations of COVID-19 was deeply elaborated in WHO-China joint report [19]. Cases of 85%, present with pyrexia in but only 45% are febrile on early presentation [20]. Cough is seen in 67.7% of patients and sputum is seen in 33.4%. Cases show respiratory symptoms such as dyspnea (18.6%), sore throat (13.9%), and nasal congestion (4.8%) [20]. General symptoms such as muscle or bone aches (14.8%), chills (11.4%), and headache (13.6%) are also seen [20]. Gastrointestinal symptoms including nausea/vomiting and diarrhea are observed in 5% and 3.7% of the cases, respectively. These clinical presentations of COVID-19 were consistent in similar studies on COVID-19 cases in China [21, 22, 23, 24].
In SARSCoV- 2 infected severe cases, fatal acute respiratory distress syndrome (ARDS), associated with monocyte and macrophage infiltration, diffuse alveolar damage, and cellular fibromyxoid exudates have been confirmed [25, 26] with mortality reported as high as 52.4% [27]. At the 7th–10th days of the manifestations of immune dysregulation, including cytokine release syndrome with elevated cytokine levels (IL-6, IL-8, IL-1, IL2R, IL-10, and TNF-α), lymphopenia (in CD4+ and CD8+ T cells), and decreases in IFN-γ expression in CD4+ T cells [26, 27, 28]. It is suggested that the cytokine storm or response may weaken the adaptive immunity against COVID-19 infection, [29] which is associated with atrophy of the secondary lymphoid tissues [25]. The risk of the success of the anti-inflammatory treatment comes from the secondary infections [30].
In severely damaged the lung tissue the ARDS develops which can further turns to septic shock. These two complications are the major issues in intensive care unit (ICU) care. The mortality from COVID-19 in patients older than 60 years, with smoking history, and comorbid medical conditions including but not limited to hypertension, cardiovascular and cerebrovascular disease, and diabetes also occurs from these complications. Notably, smoking and older age group patients tend to have a higher density of ACE2 receptors [13].
Asymptomatic or presymptomatic infection takes its naming from the patients which are the most majority of the all cases have no symptoms although they test positive for SARS-Cov-2 by reverse-transcriptase polymerase chain reaction (RT-PCR). The rest of the cases demonstrate the symptoms of fever (98%), cough (76%), dyspnoea (55%) and myalgia or fatigue (44%). Other signs, such as sputum production (28%), headache (8%), haemoptysis (5%) and diarrhoea (3%), may also be present [31]. On the other hand, the severe cases are seen in the clinics and are typically characterised by pneumonia and usually accompanied by the complications of ARDS [31, 32], acute cardiac injury [33], and secondary infections [34].
ARDS is the most significant complication in severe cases of COVID-19, and it affects 20–41% of hospitalized patients [31, 35] besides, heart failure, renal failure, liver damage, shock and multi-organ failure have also been observed as complications.
Clinical manifestation severity has been seen in a stratification which depends on symptomatology [36] (Figure 1). Adult COVID-19 cases may be grouped as follows [37, 38]:
Mild: The cases with any of the various signs and symptoms of COVID-19 (e.g. muscle pain, fever, malaise, headache, cough, sore throat) but the absence of breath shortness, dyspnoea or abnormal chest imaging.
Moderate: The cases with showing signs of lower respiratory illness by clinical assessment or imaging and peripheral oxygen saturation (SpO2) ≥ 94% (room air at sea level).
Severe: The cases characterized by breathing rates ≥30 breaths/min, SpO2 < 94% (room air at sea level); a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) less than 300 mmHg, or lung infiltrates greater than 50%.
Critical: The patients presenting with respiratory failure requiring mechanical ventilation, septic shock and/or multiple organ dysfunctions [36].
Clinical stages and the manifestations of COVID-19 disease.
As the RNA expression is detectable across a wide range of human tissues [39], it is thought that the multi-organ dysfunction is probably linked to the expression pattern of ACE2 gene. The cells, tissues and organs most affected are those with high ACE2 expression, the entry receptor or opening doors for SARS-Cov-2. The research has shown that ACE2 is abundantly expressed in the epithelia of the lung and small intestine in humans, for possible routes of the SARS-Cov-2 [40]. Since the recent data suggest that cell-surface expression on the lungs is below the detection limit [41], it has been proposed that the COVID-19 disease pathology would not be directly correlate with ACE2 cell-surface protein expression [41]. As reported for the heart and kidneys, the said disparity may be linked to the selective, transient expression of ACE2 [42, 43].
Health condition of the patients suddenly detoriates in the later stages of diseases progression. Death comes right after the fast multiple organs’ failure and ARDS. Cytokine storm has been indicated as the causal factor for ARDS and multiple organ failure [44, 45]. WHO has announced the case fatality rate of COVID-19 as ranging from 0.3 to 1%, higher than that of influenza A which is 0.1%. The epidemiological studies reported from the countries implementing COVID-19 mitigation strategies revealed that almost 80% of patients of COVID-19 had no symptoms or mild disease, whereas 14% of the patients had severe symptoms, and 6% of them were in critical condition [46].
The management of viral pneumonia is supportive in absence of specific treatment. The most dominating symptoms are fever and dry cough, therefore the first-line antipyretic agent antitussive medications [47]. Oxygen supplementation at 5 L/min must be administered for patients requiring ARDS treatment and the oxygen saturation target must be ≥92–95% in pregnant cases, ≥90% in other cases [48].
Conventionally, the complications of septic shock and acute kidney injury should be managed with relevant sepsis and renal replacement therapies [49]. In the middle to later course of COVID-19, some of the cases may develop overlapping bacterial and/or fungal infection. In these cases, the empiric antimicrobial treatment should be provided.
The WHO has been recommending the usage of extracorporeal membrane oxygenation in the patients who sustain hypoxia refractory to supplementary oxygen [49]. Or else, the convalescent plasma and IgG are used as rescue therapy in critical cases, without any solid evidence for the benefit of this practice. Most of the cases demonstrate vital health measures to control COVID-19 spreading. If the public health measures are not taken properly, there will be a patient burden that exceeding the volume of ICU beds and mechanical ventilation, as seen in the crisis in Italy. Hence, the objective of the COVID-19 management lies on the maintenance of social distancing to suppress the rapid emergence inflow of new cases. This epidemiological approach is called as flattening of the curve. The public health interest should be for identifying and isolating the infective cases, attain and maintain contact tracing and isolation [50].
The story of the cytokine ‘breeze’ transformation to the ‘storm’ starts with the infection of the cell through receptor−ligand interactions which activates massive numbers of leucocytes, particularly B cells, T cells, natural killer cells, monocytes, dendritic cells and macrophages. The release of inflammatory cytokines from these cells attract and activate more white blood cells. Cytokine breeze starts locally post-primary infection with appearing classical signs of inflammation including, calour (heat), dolour (pain), rubor (redness), tumour (swelling or oedema) and loss of function. At the beginning, the localized response works for eliminating the trigger. The host response involving the increase in blood flow, facilitation of leucocyte extravasation and delivery of plasma proteins to the site of injury, increase in body temperature and pain triggering spreads throughout the body via systemic circulation. These responses along with the host repair processes results in either gradually restored organ function or recovery happening by fibrosis which may lead organ dysfunction [51, 52].
Fibroblasts proliferate and invade the intra-alveolar zone constructing fibroblast foci. This seems as the beginning of the pulmonary fibrosis pathogenesis [53]. Lung sections from two patients with early-phase COVID-19 pneumonia demonstrated the characteristics similar to this initiation step of fibrosis [54]. Fibroblast foci were observed in the airways, besides the edema, type II pneumocyte hyperplasia with infiltration of inflammatory cellular and multinucleated cells. Some reactive epithelial hyperplasia areas are also abundant alveolar macrophages [54]. The SARS-CoV-2 caused progressing injury of the alveolar zone, apears to establish a pro-inflammatory microenvironment triggering this aberrant response with partial replacement of normal tissue by fibrous tissue. Since the severe and critical COVID-19 presentations show strong involvement of inflammatory components and possibly loss of resident stem cell stocks, the research is focused in investigating the role of cellular therapy using immune response-suppressing MSCs for COVID-19 therapy [55].
In COVID-19, the extend of the cytokine release can be the ground for the diversity of the clinical manifestations. The term ‘cytokine breeze’ meaning a mild/nonlethal cytokine release response to infection includes the symptoms of increased local temperature (heat), myalgia, arthralgia, nausea, rash, depression, and other mild flu-like symptoms. The compensatory repair process in the body is launched for the reparation of the organs and tissues affected. The term ‘cytokine strom’ is used to describe the similar sudden and uncontrolled cytokine releases observed in autoimmune, hemophagocytic lymphohistiocytosis, sepsis, cancers, acute immunotherapy responses, and infectious diseases [56, 57].
All these cytokine strom ailments were not only observed in SARS-Cov-2, but also previously reported in SARS-Cov-1 and MERS-Cov cohorts [58, 59]. Hyperinflammation, though, is characteristic for SARS-Cov-2 which is a unique immunological feature of COVID-19. The data reported from recovered and seriously ill patients suggests that there is a significant relationship between severe inflammation and mortality. The main components of the cytokine strom are the critical pro-inflammatory immune elements in the inflammation site [51]. Once the immune system is activated by infection, drug or any stimulus, the cytokines (IFN, IL, chemokines, CSF, TNF, etc.) are released in high levels into the circulation leading to deleterious and diffuse impact on multiple organs.
At the moment, the factors responsible for triggering the inflammatory sequence resulting in cytokine strom are still ambiguous. It is attributed to an imbalance in immune-system regulation resulting from increasing immune cell activation via TLR or other mechanism and decreasing in anti-inflammatory response.
Altough the local and systemic cytokine responses of host to theinfection are essential parts of the host’s initial response to infection, a cytokine strom, due to the harmful effects on the host, almost always is a pathological process [31]. Normally, to keep the pathogen under control, the cytokines released from natural killer (NK) cells and macrophages, activated T cells, and humoral immunity work to resolve the inflammation, along with the antibody-dependent cell-mediated cytotoxicity [60]. When looked in some more detail, epithelial cells produce local cytokines like IFN-𝛼/𝛽 and IL-1𝛽 which can protect neighbouring cells by stimulating IFN-stimulated gene expression. This also activates the immune competent cells such as NK cells. In turn, the lytic potential of NK cells increased and IFN-𝛾 secretion is potentiated [61]. IFN-𝛾 actives the resident macrophages which amplifies TLR-mediated stimulation, specifically induce the high NK cells release [62]. On one side, the IL-12 acts to increase NK IFN-𝛾 secretion, on the other side, increased levels of IL-6 also may limit the immune response by its effects on the cytotoxic activity of NK cells via the down-regulation of intracellular perforin and granzyme B levels [63]. The disease does not regress but progress further, the activities of the T cells and humoral responses causes additional cytokine responses. This process, like pouring petrol on fire, results in greater or sustained antigen release and added TLR ligands from viral-induced cytotoxicity [64]. Concurrently, an insufficient negative feedback mechanism by IL-10 and IL-4 would be expected to increase the severity of cytokine responses toward a cytokine storm. The exacerbated fire of the lethal cytokine storm reveals widespread alveolar damage characterized by hyaline membrane formation and infiltration of interstitial lymphocytes [65, 66]. In COVID-19 disease, a cytokine storm is demonstrated frequently in patients with severe-to-critical symptoms; concurrently the lymphocytes and NK cell counts are sharply reduced with elevations in levels of D-dimer, C-reactive protein (CRP), ferritin, and procalcitonin which are the inflammation biomarkers [67].
As the reported evidence regarding the immunological response to SARS-CoV-2 is quite limited, we are able to compile and interpret the relevant information from the published information. After the host is invaded by the virus, host innate immune system through pattern recognition receptors (PRRs) including C-type lectin-like receptors, Toll-like receptor (TLR), NOD-like receptor (NLR), and RIG-I like receptor (RLR), is the first to pick out [68]. The inflammatory factors’ expression, dendritic cells’ maturation, and type I interferons (IFNs) synthesis are promoted by the virus for basically two main purposes: limiting the spread of the virus, and phagocytosis of the viral antigens [68]. Whilst the escape of the virus from the immune responses is facilitated by the N protein of the virus [69], a strong troop of the adaptive immune response joins the combat against the virus, with its elements of T lymphocytes including CD4+ and CD8+ T cells. CD4+ T cells stimulate B cells to produce virus-specific antibodies, and CD8+ T cells directly kill virus-infected cells. T helper cells produce proinflammatory cytokines to enhance the antiinflammatory process. Paradoxically, SARS-CoV-2 induces the apoptosis of the T cells, hence inhibit their function. The major role of humoral immunity over its complements such as C3a and C5a and antibodies cannot be overlooked in the fight against the virus [70, 71]. Here comes another paradox where the immune system overreaction of the generates a large amount of free radicals locally causing severe damages to the lungs and other organs, even multi-organ failure and even death [62, 72].
In severe cases, it has been reported that SARS-CoV-2 affects heart, kidney, liver, GI-system, resulting in multiple organ dysfunction and in some cases even death [73]. One study supports that the novel virus also could potentially infect the enterocytes through a ACE2 enzyme; as ACE2 is highly expressed on enterocytes may help to explain why diarrhea occurs with acute infection as well as the fecal shedding observed [74]. Since the ACE-2 receptors are also expressed on other tissues like kidney, liver, heart and digestive system organs; thus, explaining the rapid progression towards systemic inflammatory conditions as observed in critically ill patients [75]. Hence, it is worth to consider that the infection spreading in broader scale would have impact the inflammatory cascade sources in a number of tissues in several organs, besides the lung.
Based on evidence from laboratory, animal, and clinical studies, the WHO recommends the drugs for treatment of COVID-19 includes Remdesivir, Lopinavir/Ritonavir, Lopinavir/Ritonavir with interferon beta-1a, chloroquine, and hydroxychloroquine [76].
Remdesivir is a monophosphoramide prodrug that causes premature termination of viral RNA replication. It was developed against Ebola, MERS-CoV, and SARS-CoV, before the COVID-19 pandemic shook the globe. Potent interference of remdesivir with the NSP12 polymeras3e of SARS-CoV-2 was shown in vitro despite intact ExoN proofreading activity [73, 77]. It is suggested that when the baricitinib which is an inflammatory drug used in combination with anti-viral drugs like Remidesivir, increases the potential of the drug to reduce viral infection [78, 79].
The Lopinavir/Ritonavir drugis a protease inhibitors combination. It is usually used to treat HIV infection; from the laboratory experiments, it is evident that these drugs could be used to treat the COVID-19 infections [80]. The lopinavir and ritonavir are used as a regimen single-agent or combination with either ribavirin or interferon-α [81]. It is also reported that the interferon beta-1a, which is used to treat multiple sclerosis, can also be used as a remedial approach for COVID-19 disease [73].
A randomised controlled trial (ChiCTR 2000029308) aimed to evaluate the efficiency and safety of lopinavir and ritonavir in severe COVID-19 patients, comparing lopinavir-ritonavir (n: 99) to standard care (n: 100). There was a significant difference in the time to clinical improvement between the two groups on day 14, whereas this difference was not statistically significant on day 28. The decrease of 5.8% in mortality at 28 days and the length of stay in the ICU reduced as five days in the lopinavir-ritonavir treatment [82].
Spike protein from virus binds to ACE2 or CD147 on the host cell, mediating viral invasion and dissemination of virus among other cells [55, 83]. In addition to ACE2, it has recently been demonstrated that S protein of novel virus also binds to CD147. Meplazumab which is an anti-CD147 humanized antibody, co-immunoprecipitation, ELISA, and immuno-electron microscope were handled to demonstrate the new CD147 path of viral invasion. This importanty evidence has been providing a key target for the development and administration of specific anti-SARS-CoV-2 medicines [84].
ACE Inhibitor and Angiotensin Receptor-1 Blocker are also medications used for the curative purposes of COVID-19. As already mentioned, SARS-CoV-2 enters the type II pneumocytes via the ACE2 receptor. Functionally ACE2 receptor has a mutual physiological action to ACE1, it converts the angiotensin II back into angiotensin I. Thus, patients taking receptor blocker will have an increased plasma angiotensin II. On the contrary, patients taking inhibitor will have low angiotensin II levels [85, 86]. Its effect in the alveolar tissue is still unknown. Discontinuation of ACEi or ARBs is not recommended yet as hypertension is an acute risk of discontinuation and can exacerbate the clinical course and increase mortality of COVID-19 if infected by SARS-CoV-2. Although chloroquine is an anti-malarial medication, it can inhibit pH-dependent stages of replication in viruses, as well as having immunomodulation which is dependent on the suppression of cytokines (IL-6 and TNF-α) production and dissemination. Secondary COVID-19 rates can be minimized with pre- and post-exposure prophylaxis in an individual with document exposure to SARS-CoV-2. Therefore, hydroxychloroquine has been hypothesized to be an adequate chemoprophylaxis candidate to reduce secondary COVID-19 [87].
WHO recommends to continue the use of ibuprofen as antipyretic agent, yet the first-line antipyretic remains to be acetaminophen.
The use of systemic corticosteroids in the management of ARDS secondary to viral pneumonia is debatable. The rationale behind this that the corticosteroids prolong the viral shedding time and maintain a systemic anti-inflammatory condition. This will minimize the precipitation of ARDS, dyspnea, and severe pneumonia.
The systemic corticosteroid usage in the management of ARDS developed due to viral pneumonia is still under discussion. The aim of this medication use is that corticosteroids prolong the viral shedding time and maintain a systemic anti-inflammatory state that will minimize the precipitation of ARDS, dyspnea, and severe pneumonia [76].
Considerable amount of protection is provided by the convalescent plasma collected from donors who have survived an infectious disease by developing antibodies is considered to provide a great degree of protection for recipients affected by the emerging virus [88]. Convalescent plasma is an old tool that has been successfully used to treat numerous infectious diseases, including the 2003 SARS-CoV-1 epidemic, 2009–2010 H1N1 influenza virus pandemic, and 2012 MERS-CoV epidemic [88, 89, 90, 91] for which there is no effective treatment.
Based on the clinical effectiveness of convalescent plasma, such as signs of improvement approximately 1 week after convalescent plasma transfusion, effectively neutralizing SARS-CoV-2, leading to impeded inflammatory responses and improved symptom conditions without severe adverse events the FDA has granted clinical permission for applying convalescent plasma to the treatment of critically ill COVID-19 patients [92]. Antibiotics with immunomodulatory actions are used in therapy with antiviral drugs and to avoid secondary infections, such as bacterial and fungal infections in patients. Besides their antimicrobial function, antibiotics such as Azithromycin show immunomodulatory properties, which can reduce inflammatory macrophage polarization and inhibit NF-κB signaling pathways, minimizing the hyperinflammation damage. Since the beginning, antibiotics have been used with good results in mortality reduction and shortening of intubation time in COVID-19 disease [93, 94].
The expressive number of deaths and confirmed cases of SARS-CoV-2 call for an urgent demand of effective and available drugs for COVID-19 treatment. Currently, multiple avenues for therapies are being explored.
Human mesenchymal stromal cells are also recognised as mesenchymal stem cells and medicinal signaling cells (MSCs). The reason of why the MSCs are named as ‘mesenchymal’ is their residence in the mesodermal niche, and their multipotency. They are also termed as mesenchymal stromal cells, if they fulfill the minimum criteria of adherence, expression of CD105, CD73, and CD90, absence of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR cell surface markers as well as gives rise to descendant lineages including myocytes, adipocytes, chondrocytes, and osteocytes [95, 96] as characterized by International Society of Stromal Therapy (ISCT) in 2005 [97].
Since initial isolation from the bone marrow (BM), MSCs have been found in numerous adult and fetal-derived organs/tissues such as adipose tissue, dental pulp, umbilical cord, and placenta [98]. For translational research, MSCs are categorized into different generations according to their preparation strategy as minimally manipulated, culture-expanded, lineage induced, or genetically modified [99].
Another recommendation of naming is “medicinal signaling cells”, as inspired by the fact that these cells possess the properties of homing and secrete bioactive factors that possess the immunomodulatory and regenerative potential. These features give these cells the ability to act as drugs in situ and they have shown site-specific therapeutic outcomes. The infused exogenous MSCs were shown to signal resident stem cells of the patient to repair the damage via their bioactive factors instead of undergoing their differentiation [100]. For both research and clinical purposes, large amounts of MSCs can be isolated from the embryo, fetus as well as adult stem cell sources, including bone marrow, umbilical cord blood, adipose tissue, menstrual blood, Wharton’s jelly, amniotic fluid and human deciduous teeth [101, 102].
Our age is the time of MSCs, thanks to the self-renewal and differentiation properties, have already demonstrated a promising role in treating numerous life-threatening diseases as part of the modern research and regenerative medicine [103]. Depending on their origin, stem cells can be divided into three categories of embryonic, fetal and adult stem cells [104]. Indeed, fetal and embryonic stem cells have a higher potential than adult stem cells. The adults stem cells are more used in the research and development because they have the availability and less ethical issues. Bone marrow, fat tissue, human dental pulp and umbilical cord blood are amongs the numerous sources of adult stem cells, which are of crucial importance in regenerative medicine. The technology, moreover, allow these stem cells to be isolated and protected in stem cell banks under low temperature for many years without losing their potential. Particularly, the umbilical cord blood and bone marrow are the reservoirs of both hematopoietic stem cells and MSCs. It has been known that the MSCs are the most explored and exploited categories of stem cells in treating various disorders [102].
MSCs have the differentiation capacity toward trilineage paraxial mesodermal derivates such as bone, cartilage, and fat. Besides, immunomodulatory properties of MSCs allow for expansion of therapeutic use of them in regenerative medicine in inflammatory diseases, in addition to the allogeneic allogeneic use [105, 106, 107, 108]. Interestingly, the first published evidence in the allogeneic MSCs use in inflammatory disease is a pediatric case with acute refractory graft-vs-host-disease (GVHD) in 2004 in which MSCs derived form bone marrow were given. In this study, the patient transplanted MSCs survived in well condition 1 year after MSC treatment, while other 24 patients having severe GVHD showed the median survival rate as 2 months [109]. After this pioneering evidence, MSC immunomodulation has shown to be broad-based, best detailed for CD4 lymphocytes but also for dendritic cells and natural killer cells [110, 111]. As evident by the increasing MSC trials focusing on immune/inflammatory diseases in recent years which are accounted for almost one-third of the trials, the clinical importance of the immunomodulatory properties is compromised [112, 113, 114]. General charateristics of the MSCs is their being fibroblast shaped cells which are plastic adherent; fulfilling the criteria of stem cells and MSC as well as stromal cell types [109, 115]. The immunomodulatory activities are suggested to include:
inhibition of the proliferation and function of dendritic cells, T and B cells, as well as NK cells.
polarization of monocyte to anti-inflammatory macrophages called M2 cells.
production of IL-10 and decreased production of TNF-α and IL-12 [116, 117].
In addition, MSCs have powerful antifibrotic effects which may alleviate lung fibrosis [118, 119].
Lately, there have been increasing reports revealing that the MSCs induce therapeutic characteristics by releasing bioactive substances known as secretomes in a paracrine path [120]. The soluable proteins such as chemokines, growth factors, cytokines, and extracellular vesicles (EVs) including microvesicles and exosomes present in the MSC-secretomes [121]. MSCs, like all other stem cells, when the culture medium or secretome are injected into the patients, show paracrine signalling to take in these molecules to the cells in vicinity [122]. The exosomes contain bioactive molecules, including microRNAs (miRNA), transfer RNAs (tRNA), long noncoding RNAs (lncRNA), growth factors, proteins, and lipids. Of note, the lipid content of the exosomes is an added value by facilitating the infusion of the exosomes attracted to the plasma membrane of the neighboring cells [123]. Once the molecules content of the secretome is internalized, the neighboring cells modulate various downstream pathways, including immunomodulation, suppression of apoptosis, prevention of fibrosis, and remodelling or repair of the damaged tissues [120, 124].
Several studies with drugs targeting GM-CSF, IL-6, IL-1, IL-2 and TNF-α is already in the pipeline which aims to calm down the inflammatory response in COVID-19 patients. MSCs are well-known for their immuno-modulatory properties including the anti-inflammatory cytokines/chemokines secretion, anti-apoptotic effect and their reparative ability for the damaged epithelial cells. Their inherent nature to migrate towards injured lungs and secretion of paracrine factors which protects and repair alveolar cells; make MSCs a potential therapeutic option for COVID-19 treatment. Recently, MSCs have been widely studied from basic research to clinical trials particularly for immune-mediated inflammatory diseases such as systemic lypus erythematous (SLE) and GVHD [125, 126, 127].
The therapy using MSCs usually covers the processes of isolation, culture, subculture, proliferation, and differentiation of exogenously obtained stem cells, which are then transplanted into patients for immune regulation and microenvironment repair. The therapy success determinants are the safety and efficacy for any treatment. Hence the safety and effectiveness of MSCs are important as shown in a number of clinical trials besides fundamental studies.
MSCs have been widely used in the treatment of inflammatory diseases, such as in graft vs. host disease [128] and lupus erythematosus [129]. Some studies have shown that MSCs have definite efficacy in improvement in cardiovascular, kidney, liver, and other diseases [130, 131].
MSCs are able to regulate the immune response by controlling the function and proliferation of various immune cells. They alsocan inhibit monocyte differentiation into dendritic cells (DCs) which results in upregulation of regulatory cytokines and downregulation of inflammatory cytokines [132]. It was suggested that systemic administration of MSC resulted in reduction of H5N1 influenza virus-induced mortality in older patients with severe pulmonary illness [133]. Also, in patients with H7N9 induced ARDS, a significant improvement in survival rate was observed [134]. So far, MSC transplantation in human subjects with diverse disease conditions has not showed any severe adverse events [135]. Therefore, it is plausible that MSC-therapy can be used to treat COVID-19 patients.
MSCs are evaluated as one of the most promising candidates for SARS-CoV-2 infection treatment. Since the key target for the treatment of SARS-CoV-2 infection resides in the cytokine storm management in lungs, MSCs are well-suited considering their main mechanism of action is through their immunomodulatory and anti-inflammatory properties [129].
MSCs have immunomodulatory effects and they:
prevent uncontrolled cytokine or inflammatory factors production,
inhibit excessive immune responses, and.
reduce immune damage to tissues and organs.
Having the immunemodulatory properties, MSCs not only take part in suppressing immune injury, but also replace and repair damaged tissue and inhibit lung fibrosis. Treating COVID-19 with MSCs has presented considerably good results [136]. Stem cell therapy can suppress the storm of cytokine release, promote endogenous repair by improving the microenvironment, slow the progression of acute lung inflammation down and relieve the symptoms of respiratory distress [137]. The reports suggested that the potentially COVID-19 can be successfully treated with MSCs therapy by the MSC regulation mechanism of the immune system. Studies revealed that when the MSCs are exposed to an inflammatory microenvironment, they can regulate immune cells and inflammatory factors, such as cytokines, leading the alterations in the specific or nonspecific immune responses in vivo. The said modulation is shown to be related to exosomes or the cytokines secreted by MSCs, such as transforming growth factor (TGF)-b, prostaglandin (PG)E-2, and interleukin (IL)-10 [138, 139].
The regulation of the T and B lymphocytes’ functions is of special interest as it has been done in several ways. One of these is the T cell proliferation, which is controlled by inflammatory stimulation. A study on cell cycle analysis revealed that T cell subsets can be blocked at the G0/G1 phase. Another way of modulation is that the MSCs can control T cell function via cytokines, by releasing TGF-β, inhibiting the immune activity of Th17 cells, inducing their altering to form T regulatory cell Treg cells, or secreting hepatocyte growth factor to regulate the Th17/Treg cell balance [140]. The modulation of the B cells’ proliferation, differentiation, and antibody secretion by the MSCs is also important, since MSCs can affect the G0/G1 phase transition of B cells and regulate the antibody secretion ability of B cells through various transcription pathways [141]. MSCs help to regulatory B cells to multiply; these B cells express IL-10. MSCs activate T cells to release interferons, as well. Suppression of activated B cells regulates the immune function of B cells, and MSCs can also affect innate immune cells, including macrophages and dendritic cells, to realize immune regulation. Under inflammatory conditions, MSCs regulate macrophage function, as well [142]. Once the proinflammatory macrophages (M1) secrete the inflammatory agents, activated MSCs can up-regulate the cyclooxygenase (COX)-2 signal and increase PGE2 secretion. This thereby promotes the transformation of macrophages from activated proinflammatory type to selectively activated anti-inflammatory type (M2).
The MSCs releasing the anti-inflammatory factor TSG-6 and the CD44 macrophages act collectively to destroy the interaction between CD44 and toll-like receptor-2, inhibit the nuclear factor-jB signal downstream, and reduce the inflammatory response [143]. On the other hand, the MSCs can secrete HGF under endotoxin stimulation to induce differentiation into regulatory dendritic cells and alleviate acute lung injury [144].
As explored by research, COVID-19 patients’ blood have large numbers of inflammatory factors including interferon-c, interferon-inducible protein-10, and monocyte chemoattractantprotein-1. Additionally, when the patients staying in ICU is compared with the patients in the inpatient clinics, the concentration of granulocyte colony-stimulating factor (G-CSF), MCP-1, tumor necrosis factor (TNF)-a, and other inflammatory factors were shown to be dramaticaly higher in the ICU patients, hence there is a positive correlation between the severity of the cytokine storm and the clinical manifestations of COVID-19 [145]. As discussed previously (see section 2.2.2) COVID-19 have a variety of clinical manifestations changing from a mild disease to a severe disease. This change in severity results both from complications of the viral infection and the cytokine storm. The cytokine storm damaging effects are well-known. Cytokine storm in patients with severe COVID-19 can lead to the release of nitricoxide, which affects the normal systolic and diastolic function of blood vessels, thereby causing hypotension and multi-organ hypoxia [146]. Severe patients have IL-6 levels ten-times higher than those in non-severe patients. In addition, the IL-6 levesl are closely related to the serum SARS-CoV-2 virus load and vital signs of patients. Some study reports have now shown that tozumab (anti-IL-6 receptor) use can prevent worsening of the disease [147]. The MSCs of umblical cord origin, can also inhibit monocyte activation and IL-6 production to inhibit the development of cytokine storm, these result in the improved patient’s prognosis. It has been reported that the microenvironment having high IL-6 levels, lead the MSCs to produce cytokines and exosomes enriched with mirR-455-3p, thus calming cytokine storm down and treating acute inflammatory injury. However, the effect of MSCs on cytokine storm in patients with COVID-19 still needs further confirmation [148].
MSCs may suppress ARDS exacerbation and pulmonary fibrosis. Studies have revealed that, once infused or transplanted intravenously, MSCs can reside in the lungs and help improving the microenvironment of the lungs, protecting alveolar epithelial cells, promoting neovascularization, and preventing pulmonary fibrosis [149, 150]. So it seems that one of the most important outcome of the MSC treatment is its reparative action. The reperative function of the MSCs is managed over a variety of the cytokines, particularly keratinocyte growth factor (KGF) [151]. KGF functions through promoting alveolar fluid clearance and alleviating the acute lung injury induced by endotoxin by up-regulating ACE-2 [152]. Another up-regulation managed by KGF is that the activity of sodium potassium ATP enzyme in alveolar cells, resulting in the improvement in alveolar fluid transport, and this play a therapeutic role in ARDS and lung injury [153].
MSCs may have bacteriostatic role. There was a controversy in whether the virus could cause MSCs to lose their function when the MSCs are invaded by bacteria. Although conducted in limited number of patient size, the clinical trial reported from Beijing, showed that the COVID-19 virus could not infect umbilical cord MSCs that were infused intravenously [136]. MSCs can exert their anti-COVID-19 virus effect through direct and indirect mechanisms, according to the recent research. Direct function of the MSCs can be lined up as the direct anti-viral effect by secreting antibacterial peptides and proteins, indoleamine 2,3-dioxygenase, IL-17, and other molecules. MSCs can activate a large number of anti-virus genes independent of interferon, such as the IFITM gene, which can encode protein structures that prevent viruses from invading cells [154]. When it comes to the indirect function of the MSCs combating against COVID-19, they also exhibit an indirect antiviral effect through regulating the coordination of pro-inflammatory and anti-inflammatory actors of the patient’s immune system and inducing the macrophages’ functions [155, 156, 157].
The in vitro sepsis model, ARDS model, and alveolar epithelial fibrosis model use in the research activities demonstrated the immunoregulation and antibacterial and antiviral values of MSCs [156, 157]. Studies show that MSCs secrete at least four AMPs including, antibacterial peptide LL-37, human defensin 2, hepcidin, and lipocalin-2. The function of these AMPs includes killing cells, inhibiting the synthesis of essential proteins, DNA, and RNA of infected cells, interacting with certain targets in infected cells, and playing an active regulatory role in the infection and inflammatory progress of COVID-19 patients [158].
The therapeutic properties of the MSCs against SARS-CoV-2 infection include:
Apoptosis induction via activated T-cells alleviating excessive immune responses.
Regeneration and maintenance of the homeostasis in specific lung injuries.
Release of cytokines to inhibit neutrophil intravasation and enhance macrophage differentiation which helps attenuate inflammation and also promotes the release of extracellular vesicles which deliver microRNA, mRNA proteins and metabolites into host cells post lung injury which promotes repair regeneration and lung function restoration. Therefore MSCs should be considered as a potential treatment for critically ill patients with SARS-CoV-2 infection [159, 160].
It has been observed that most of the clinical trials for COVID-19 treatment have used allogeneic stem cell source. The curative effect of MSCs in the treatment of COVID-19 has been shown by the two recent clinical trials. In one of them, human umbilical cord derived MSCs were used in three consecutive intravenous infusions administered to patients with COVID-19; it was reported from this trial that subject demonstrated the neutrophil levels decreased significantly, lymphocytes increased, CD4+ T and CD8+ T cells returned to normal level, and vital signs were improved, after the second intravenous infusion [161]. The other trial recruited seven patients with COVID-19 (two mild cases, four severe cases, and one critical case) to receive one intravenous MSC transplantation each. According to the published results, The patient’s regulatory dendritic cell population increased, the level of the pro-inflammatory factor TNFa decreased, and the level of antiinflammatory factor IL-10 increased, after 2–4 days after MSC transplantation [136]. This was a pilot study Clinical grade MSCs were injected intravenously (1 × 106 cells/kg body weight) and the patients were followed-up for 14 days. From clinical point of view, a significant reduction in clinical symptoms and pneumonia infiltration was observed in chest CT of critically ill COVID-19 patient within 2–4 days of MSC-therapy. An increase in peripheral lymphocyte levels, decrease in C-reactive protein (CRP), drastic disappearance of activated cytokine-secreting immune cells (CXCR3+CD4+T-cells, CXCR3+CD8 + T-cells and CXCR3 + NK-cells) and restoration of regulatory DC cell population to normal levels was observed after day 6 of MSC transplantation. From cytokines point of view, the level of anti-inflammatory cytokine IL-10 was increased and the levels of serum pro-inflammatory cytokine TNF-α was significantly decreased. These were considered as the indicators of the efficient regulation of cytokine storm in COVID-19 patients on MSC transplantation. On the other hand, the absence of ACE-2 receptor and TMPRSS2 on the transfused MSCs affirmed that they cannot get infected with SARS-Cov-2, suggesting the beneficial effects of the MSC-therapy in COVID-19 infection. The authors suggested that this clinical trial showed that transplantation of MSCs can improve the prognosis of patients with COVID-19 [145]. In a case report of one critically all COVI-19 case who is 65-year-old woman with underlying with type-II diabetes and hypertension, it was reported that after receiving MSC-based treatments her health improved and she left the ICU. The authors of this case report proposed that the possible effects of hUCMSCs might be anti-inflammation and tissue repair to COVID-19 patient. They also suggested that MSCs could down regulate proinflammatory cytokines and chemokines and increase IL-10 and VEGF which could promote the lung repair [161]. The patient didn’t respond to any anti-viral drug and the disease progressed to multiple organ injury. During this critical stage when the patient is ventilated, hUC-MSC was infused in 3 consecutive administrations in 50 × 106 cells/dose. After second MSC administration, ventilator was removed as the vital signs had improved with gradual decrease in serum albumin and CRP levels. CT images showed no infiltration patches of pneumonia by the end of MSC infusions. These results suggest that hUC-MSC can be beneficial for patient who showed resistantance to anti-viral drugs. The therapeutic potential of MSCs in viral infections and immunomodulation capabilities to alleviate the cytokine storm, are being tested in clinical studies that have been initiated to further evaluate their efficiency for COVID-19 treatment.
The evidence pf the published results of the clinical trials in which the MSC transplantation is used for curative purposes, shows the beneficial effect of MSCs on the treatment of severe patients. However, more clinical data are still needed to confirm its effectiveness [162].
Several anti-viral drugs such as remdesivir, favipiravir, ribavirin functioning as RNA dependent RNA polymerase inhibitors, lopinavir, ritonavir which are protease inhibitors and drugs suc as hydroxychloroquine targeting endocytic pathway are being evaluated for COVID-19 but standard therapeutics yet not available. To fight against the cytokine storm, immune-therapy targeting TNFα, IL-1, IL-2, and IL-6 and are evaluated. One of the promising immune-modulators is the MSCs administered as add-on therapy can surmount the severity of COVID-19 infections. Recent studies have shown that MSC-therapy significantly dampens the cytokine storm in critically ill COVID-19 patients [163].
The published results of MSC add-on therapy for ARDS, with focused clinical outcome measures’ analysis on safety, efficacy, and related immunologic and pulmonary responses [164]. The clinical studies have demonstrated that MSC therapy is safe and has the potential to mitigate inflammatory and physiologic damage for a variety of conditions involving the central nervous, [165] cardiac, [166] renal, [167] gastrointestinal, [168] and respiratory [169, 170] systems. The data in the literature suggests similar results for MSC therapy for treating ARDS in COVID-19.
As expected, safety is the most important matter for all new therapies, especially in patients at high risk for death from the condition being treated and was carefully evaluated for MSC-treated patients in the clinical trials published. According to the literature review, out of the 200 ARDS patients were treated with intravenously or intratracheally administered MSCs or placebo, 30patients died in the active treatment group. None of these 30 deaths were found to be related to MSC therapy. Also, no other SAEs attributed to the MSC therapy. Some transient adverse effects reported, but all of them resolved on its own in short term. This safety profile is consistent with the experience of other human clinical trials involving MSC therapy [165, 171].
The clinical trials of cell-based therapy using MSCs and their safety has been reported in several clinical trials related to GVHD and SLE [127, 172, 173, 174]. The approach of MSC transplantation has been used to treat H7N9-induced ARDS patients and the outcome showed significant reduction in mortality rates [134]. Similarly, the study of MSC-based treatment for SARS-CoV-2 suggested that MSCs lack SARS-CoV-2 infection-vital receptors (ACE2- and TMPRSS2-); so MSCs are SARS-CoV-2 infection-free. Also, the these cells’ infusion in SARSCoV- 2-infected patients improved the outcomes because of their extraordinary immunosuppressant potential [136].
The potential efficacy of MSC therapy for ARDS in COVID- 19-infected patients is reported from a phase 1 trial. There were 9 patients enrolled. In-hospital mortality was reported as 33.3% (3/9), including two with septic shock and one with ventilator-induced severe pneumomediastinum and subcutaneous emphysema. No serious prespecified cell infusion-associated or treatment-related adverse events was identified in any patient. The circulating inflammatory (CD14CD33/CD11b+CD16+/CD16+MPO+/CD11b+MPO+/CD14CD33+) and MSC markers (CD26+CD45-/CD29+CD45-/CD34+CD45-/CD44+CD45-/CD73+CD45−/CD90+CD45-/CD105+CD45-/CD26+CD45-) were reported as progressively reduced and the immune cell markers such as Helper-T-cell/Cytotoxity-T-cell/Regulatory-T-cell were notably increased after cell infusion. As a result, this phase I clinical trial showed that a single-dose intravenous infusion of hUC-MSCs was safe with favourable outcome in nine ARDS patients [175]. According to the available evidence, SARS-CoV-2 affects not only the lung, but also the heart and kidney with reported cardiomyopathy and kidney injury [171, 176]. It has been reported that the improved resolution of multiple organ failure or increased organ failure-free days with MSC treatment, which further supports their consideration for clinical use.
The safety and efficacy profile of MSCs is well-constituted based on the results from several completed clinical studies conducted on the therapeutic potential of these therapies in lung diseases such as ARDS [134, 177] as well as bronchopulmonary dysplasia cardiovascular diseases), diabetes [178, 179] and also spine injuries [180]. Although it has been still in experimental phase, the stem cell types investigated for possible cure of SARS-CoV-2 infections include human induced pluripotent stem cells. Recently, it has been reported that when iPSCs were exposed to SARS-CoV-2, it was presented a deleterious effect on the cells in vitro where the pluripotency of iPSCs was lost leading to fibroblast-like phenotype [181, 182]. Therefore, evidence-based selection of stem cell type for the treatment of COVID-19 is critical for safety and efficacy.
Wraping up, it seems the MSC-therapy, when applied as add-on treatment, suppresses the over activated immune system through its immuno-modulatory properties and promotes the tissue repair of alveolar cells in lung microenvironment of SARS-CoV-2 infected patients. Clearly, the data of the recent studies are encouraging, however they have major limitations such as the small-sized patient recruitment. Hence, the need for larger randomized control trials to establish the effectiveness and safety of MSC-therapy in SARS-Cov-2 infection is obvious.
The immense knowledge available with reference to the mechanism of action of MSCs and their effective potencies at a specific disease stage makes MSCs as an promising and effective therapeutic candidate.
It has been demonstrated that MSCs have broad immunomodulatory, anti-inflammatory capacity [183, 184], as well as regenerative properties [185]. MSCs can induce the repair of damaged tissue, and eventually prevent long-term lung damage resulting from COVID- 19. The stabilization of the endothelial fluid leakage and maintenance of the alveolar-capillary barrier function are also characteristics demonstrated by MSCs; obviously, these features are irrevocable to decrease lung permeability and attenuating the development of interstitial lung oedema [186]. These are the main grounds for the MSC based cellular therapy as potentially effective treatment for COVID-19 infection.
Severe cases of COVID-19 infection is characteristic with high levels of cytokines in the plasma, particularly IL-6 which is a biomarker of inflammation and immune response. From this perspective, clinical trials using the medications such as Sarilumab and Tocilizumab, the antibodies anti-IL6 receptors, has been testing such therapeutic strategy in hospitalized COVID-19 infected patients.
Azithromycin is an antibiotic with immunomodulatory effects and invasion inhibitory activity. That is why this drug has been also administrated for the therapy of chronic inflammatory conditions, such as bronchiolitis and rosacea. Although the exact mechanisms of this anti-inflammatory effect are still not fully known, some studies presented a reduction of IL-6 levels after azithromycin treatment [187, 188]. What is more, another study has demonstrated that azithromycin increases rhinovirus-induced interferons and interferon-stimulated mRNA and protein expression as well as decreases rhinovirus replication and release, resulting in induced anti-viral responses in epithelial cells of the human brochiols [189].
After administered systemically, the majority of MSCs reside in the vascular bed of lungs through the interactions with the capillary endothelial cells. When labelled MSCs are traced, it was seen that most are cleared within 24–48 h, and there can be persistence in injured or inflamed lungs for a longer period [190]. It has been suggested that the apoptosis and subsequent efferocytosis and phagocytosis by resident inflammatory and immune cells could be amongst the clearance process [191]. MSCs can secrete various soluble mediators including anti-inflammatory cytokines [192], antimicrobial peptides [193], angiogenic growth factors, as well as extracellular vesicles [194] in their vicinity.
There are evidence for cell–cell transmission of mitochondria from MSCs to respiratory epithelial and immune cells [195]. This reveals the release of anti-inflammatory mediators is specific for the inflammatory lung environment and is mediated through differential activation of damage- and pathogen-associated molecular pathogen receptors expressed on MSC surfaces [196, 197]. Amongst these receptors, Toll-like receptors are crucial; since these are activated by viral RNA in COVID-19 and viral unmethylated CpG-DNA (e.g. TLR9). This leads to modulate the pathways of cell signalling resulting in MSC activation [198]. MSCs derived angiopoietin-1 and keratinocyte growth factor (KGF) contribute to the reparation or restoration of alveolar–capillary barriers disrupted as part of ARDS pathogenesis [199]. On the other hand, the specific inhibitory microRNAs in extracellular vesicles are also described as mediating the protective effects of MSCs in pre-clinical models of infectious or non-infectious acute lung injuries [200].
Currently, there are 82 clinical trials investigating the therapeutic potential of mesenchymal stem cells in COVID-19 patients that are registered on clinicaltrials.gov website; out of all these, 70 trials have (83%) the MSCs as therapeutic agent being tested. The allogeneic bone-marrow or umbilical cord-derived MSCs transplanted intravenously on three different occasions is involved in 21 studies (63%). Most of these trials are either recruiting patients or have not yet started the enrolment. MSCs have been investigated and reported in ARDS both in pre-clinical [201] and clinical settings [127]. Now that, a number of promising trials are currently underway, which could revolutionize the regenerative or MSC-based cellular treatment prospects for severe COVID- 19 patients.
Regardless of how urgent the development of MSC-based therapies for COVID-19 is, it is critically important that the manufacturing of MSCs is in compliance with good manufacturing practices (GMP) and follows strict regulations prior to being approved for the use in humans.
The current findings clearly show that there is a huge unmet need for globally coordinated approach to support to conduct multicentre clinical trials aiming to demonstrate safety and effectiveness of various types of stem cells to treat health complications of novel virus. Also, there is a need in biomedical research and development to establish the most effective stem cell types that are ideally suited for the treatment of the complications.
The development of the stem cell advanced medicinal products will also require: (a) GMP compliant technologies to enable massive stem cell production, and (b) testing platforms that mimic human pathophysiology as much as possible, such as 3D bio-printed organoids, organon- chip, to allow targeted screening and rapid testing of stem cells safety and efficacy. EVs appears as an attractive alternative to cell–based therapy, recently. EVs have several advantages compared to the whole cell therapy including lower risk of oncogenic effects, lower susceptibility to harm by hostile disease tissues and for longer-term storage. The long-term storage is fundamental to make the treatment accessible globally and it surrounds the requirement to have expensive GMP cell manufacturing facilities. The production of EVs must follow the same strict guidelines that apply to stem cells and any EV-based therapy needs to be approved by the health authorities after being tested in clinical trials to demonstrate and confirm the safety and efficacy.
In most clinical trials investigating the MSC treatments of SARS-CoV-2 infection so far, MSCs are delivered via the intravenous route by infusion. The direct target of the intravenous route is not the lungs, that is why the inhalation route delivering the cells directly to lungs could be theoretically more effective. However, the inhalation route has the risk of not able to manage the uniform delivery of cells to lungs [202]. The evidence is being more and more visible to suggest that the curative potential of MSCs is attributed mostly to their secreted EVs via paracrine effects [203].
As evident from several clinically available inhaled medications for chronic lung disease, the inhalation route of delivering therapeutics to the lungs is a more direct route with lower the number of adverse effects, compared to the intravenous route. However, it must be appropriately managed for inhaled administration of a treatment in COVID-19 patients in the hospital setting. Many studies have showed the feasibility of delivering stem cells via spray for direct pulmonary delivery with high viability [204]. Inhalation route of stem cell administration is an opportunity for efficient delivery of stem cells directly to the lungs, yet it needs further research and proof of concept.
Once the globe has suddenly got into the pandemic of COVID-19, all the scientific community has been making every effort to understand the etiology, pathophysiology, societal and clinical aspects of the SARS-CoV-2 viral infection all over the world. As of time this chapter is compiled, there are several vaccines developed in several countries. However, despite all the efforts, there is yet no specific and validated treatment for the infection. Instead, the medicinal products already available are being used in all clinical presentations, including antivirals, antibiotics, antimallarians, ans the agents aiming to take the disease under control. Here, taking the available published evidence in place, we elaborated the structure and pathophysiological aspects which are treatment targets to fight against the pandemic of our age. In this context the mesenchymal stem cells apear as advanced medicinal product of the cellular treatment option. Having the available knowledge refering the mechanism of action of MSCs and their safe and effective potencies at a specific disease stage makes MSCs as an ideal therapeutic candidate. Although still the data to be obtained from future the large scale randomised controlled clinical trials conduct remains an under-explored research area in the field, we suggest that under the light of the available evidence today, MSCs can be used as add-on therapy with promising effectiveness and safety to control, and even treat the COVID-19 infection with regenerative, anti-inflammatory, anti-fibrotic, immunomodulatory and reparative characteristics.
The authors declare that they have no conflict of interest.
ACE2 | angiotensin-converting enzyme 2 |
ARDS | acute respiratory distress syndrome |
CoV | Corona viruses |
COVID-19 | Coronavirus Disease 2019 |
CP | convalescent plasma |
CRP | C-reactive protein |
CRS | cytokine release syndrome |
CT | Chest computerized tomography |
HCQ | Hydroxychloroquine |
HIV | human immunodeficient virus |
Ig | Immunoglobulin |
IL | Interleukin |
MCP-1 | monocyte chemoattractant protein-1 |
MERS-CoV | middle East respiratory syndrome-coronavirus |
MIP-lα | macrophage inflammatory protein-1 alpha |
MOD | multiorgan dysfunction |
TNF-α | tumor necrosis factor alpha |
Microorganisms play both beneficial and harmful roles in our lives. Some of the beneficial roles include production of oxygen via photosynthesis, nitrogen fixation, circulation of carbon by decomposition of dead organic matter, formation of crude oil, and helping animals such as cows digest their food. They are used by humans in making bread, beer, cheese, and antibiotics. Some of the harmful effects are caused by the virulence of pathogenic microorganisms, i.e., infection causing bacteria such as
Textiles have been recognized as a media for the growth of microorganisms such as fungi and bacteria. The growth of these microorganisms on textiles inflicts unwanted effects not only on the textile material, but also on the consumer. These effects can include the generation of unwanted odor, discoloration in the fabric, an increased probability of contamination, and an overall reduction in the fabric mechanical strength [1, 2]. The spread of infections through textile materials can be controlled by the use of antimicrobial textiles that kill pathogens on contact or hinder their ability to reproduce prior to being transferred on to another material or person. Antimicrobial textiles are made by treating textile substrates with antimicrobial agents or by using textile fiber with inherent antimicrobial efficiency. Antimicrobial agents are bound to textiles by different methods depending on the chemistry between the antimicrobial agent and the textile [3]. Consumers’ attitudes toward hygiene and their desire for comfort and well-being have created a rapidly increasing market for antibacterial materials. Therefore, there has been extensive research in recent years in this area. Estimations have shown that there was approximately a production of 30,000 tons of antimicrobial textiles in Western Europe and 100,000 tones worldwide in year 2000. It was also estimated that the production increased by over 15% annually in Western Europe between 2001 and 2005, making antimicrobial textiles a rapidly growing sector of the textile market [1]. While synthetic fibers have been known to be more resistant to microorganisms due to hydrophobicity, natural fibers are more vulnerable to microorganism attack. In addition, soil, sweat, and dust can be nutrient sources for microorganisms [2]. Socks, active-wear, shoe linings, and lingerie account for approximately 85% of the total antimicrobial textile production. In addition, there has recently been a large market for antimicrobial fibers in air filters, outdoor textiles, upholstery, and medical textiles [1].
Other than the antimicrobial ability, there are certain basic requirements to be satisfied by an antimicrobial agent for its successful application on textiles rendering them to be used commercially. The basic requirements of a good antimicrobial agent for textile substrates are summarized below [3, 4]:
Should possess affinity for specific fabric and fiber types.
Be easy to apply on textile substrates.
Be able to inactivate undesirable microbes while simultaneously not affect desired microbes.
Inert to chemicals to which the textile might be exposed during processing.
Durable to repeated laundering, dry cleaning, ironing and prolonged storage including resistance to detergents used to care for the textiles.
Stable during usage without degrading into hazardous secondary products.
Not adversely affect the user or the environment.
Many antibacterial product and chemistry are available in the current market using different technologies. Most antibacterial agents applied on textiles have been used for many years in food preservatives, disinfectants, wound dressings, and pool sanitizers. The attachment of these compounds to textile surfaces or their binding with the fiber can reduce their activity largely and limits the antibacterial agents’ availability. In addition, the antibacterial agent can gradually be lost during the washing and use of the textile material. The most widely used antimicrobial agents for textile applications are based on metal salts (for e.g., silver), quaternary ammonium compounds (QAC), halogenated phenols (for e.g., triclosan), polybiguanide (for e.g., PHMB), chitosan, and N-halamines [5]. The aim of this section is to present the general family of antibacterial textile finishing.
In general, antibacterial agents can either kill the microorganisms (−cidal) or inhibit their growth (−static). Almost all the commercial antimicrobial agents used in textiles (silver, polyhexamethylene biguanide (PHMB), quaternary ammonium compounds, and triclosan) are biocides. They can damage the cell wall or disrupt the cell membrane permeability, and inhibit the activity of enzymes or synthesis of lipids, while all these functions are essential for microorganism’s survival [3].
The antibacterial material can be separated in two categories: antimicrobials with controlled release or ‘leaching’ mechanism and bound or non-leaching type antimicrobials. The mechanism of the leaching type will act upon contact of the cell. On the other hand, the non-leaching types will diffuse a disruptive chemical to the cell. This type is preferred for an environment supporting the diffusion of the chemical, such as water.
The antimicrobial agents that belong to this category do not form strong bonds with the textile substrate. The chemical species responsible for biocidal activity are released slowly from the treated fabric surface, thus killing all the microbes surrounding the agent. An advantage of leaching antimicrobials effect are their superior antimicrobial activity than compounds based on other modes of action on the same fabric under similar environmental conditions [6]. The flip side is that the antimicrobial agent in the textile substrate is depleted eventually and loses its effectiveness. Metal salts (e.g., silver) and halogenated phenols (e.g., triclosans) are examples of antimicrobial agents that utilize the leaching mechanism [7].
The interest of metal and metal oxide particle reside in the high antibacterial activity against microorganism, durability and stability, while having a low mammalian cell toxicity, meaning they are safe for close to the skin application. Even at very low concentrations, many heavy metals are toxic to microorganisms. Metal particles are synthesized from different precursor and reducing agent to obtain different end material, morphology or to lower the impact of on cost or environment. Plethora of synthesis reaction are available from the scientific literature. However, the reaction principle is similar for each technique, using a sol–gel. The precursor is usually a water-soluble salt such as silver nitrate, copper chloride, and zinc acetate. The metal ion is reacted with a reducing agent, such as conventional reducer like sodium borohydride, citric acid, citrate, and ascorbic acid, or with bio-based reducer such as glucose, polysaccharide, cellulosic fiber and plant or microorganism extract. The precursor is mixed with a reducing agent under different conditions such as heat, mixing, sonication to surpass the activation energy of the reaction. Strong reducing agent will require milder reaction condition, while weak reducing agent will require stronger reaction condition. During the reaction, the particle could be stabilized using a capping agent in order to control the shape, size and stability of the final product. In some reaction, the reducing agent will also be used as a capping agent. While metals such as zinc, cobalt and copper have had some applications in past years as antibacterial agents for textiles, silver, having an MIC value of 0.05–0.1 mg/L against
Silver can be applied in other forms: silver ion exchangers, silver salts, and silver metals. Silver zirconium phosphate and silver zeolites are examples of ion exchangers. Silver chloride (AgCl), nanosilver chloride, and AgCl microcomposites (AgCl nanoparticles attached to titanium dioxide as a carrier material) are types of silver salts. Silver metal can be used in the form of filaments and silver metal composites [12]. With concerns regarding bacterial resistance to silver [3], there is efforts to increase the efficiency of metal-based antimicrobial. Other metal based antimicrobial agents found to exhibit good antimicrobial properties are based on copper and zinc compounds, in the form of their sulfides and sulfates [13]. Many studies on metal salts have focused on preparation of nano sized metal particles, which has led to the development of new generation of biocides [5]. Above all, AgNP (Silver Nanoparticles), a nanometric form of silver element without an ionic charge, can be used as a catalyst, an optical sensor and an antibacterial agent [14, 15, 16]. The antibacterial activities of the silver ion and salts are well studied, but research about antibacterial mechanism of AgNP is relatively recent [14]. Different methods have been developed to synthesize and incorporate AgNP in some biomedical applications, and some reports have proven AgNP to be a potent antibacterial agent, that is effective against both Gram-positive and Gram-negative bacteria [17, 18, 19].
All silver-based antimicrobials generate and release different amounts of silver ions, with silver metals releasing the least, silver ion exchangers releasing the most, and silver salts somewhere in between [20]. In the presence of moisture, silver releases ions that bind the bacterial cell’s surface with proteins. On binding, the following action occurs [21].
Denaturing effect of the silver causes DNA to get condensed and lose its replication abilities.
Induces inactivation of bacterial proteins by reacting with thiol group [21, 61].
The form of the silver used impacts its antibacterial effectiveness. For example, a concentration of AgNO3 should be higher than 1 mM to kill silver resistant E.coli. While only 80 nM of AgNP is necessary for the same result [17]. The antibacterial efficacy of silver is directly proportional to the amount of bioactive silver ions released in the presence of moisture, as well as its ability to penetrate bacterial cell membranes [10]. Silver is effective at low concentrations and promotes wound healing without appreciable toxic risk. However, there is a small risk of developing allergies to silver [22, 23]. In fact, silver and copper ions can disrupt or kill the microbes via different mechanism path. First, the ions can diffuse trough the cell membrane and bond to the enzyme of the cell. The enzymatic activity of the cell is decreased, which inhibit the growth of the cell until the death of the cell. Second, Silver ion can kill microbes by binding to intracellular proteins and inactivating them, can inhibit the synthesis of ATP (Adenosine triphosphate) and lead to DNA (Deoxyribonucleic acid) denaturation [24]. To observe the killing mechanism of silver ion more directly, TEM (Transmission electron microscopy) and X-ray techniques were used to facilitate the investigation. When
Others metals oxides of interest are titanium dioxide (TiO2) and zinc oxide (ZnO). The mechanism of those compounds is believed to be mostly from the generation of reactive oxygen species (ROS). Those compounds prevent the antioxidant defense system and damage the cell membrane of the microbe. This mechanism is catalyzed by ultraviolet light. It is of particular interest as an adjuvant to the UV disinfection, which is of growing usage for disinfection against COVID-19. However, this also means the efficiency of those metal oxides is largely influenced by the environment in which they are used. The efficiency of the metal oxide could be reduced in the presence of antioxidant or pigment, often used in synthetic textile. The morphology of the particle will have a great impact on the stability of the product as well as the antibacterial activity. In general, the greater surface area will provide a greater activity, but decrease the durability for the leaching type.
Currently, silver is used in a large number of antimicrobial commercial textile products at a relatively low cost. The silver is in the form of ultra-fine metallic particles and is mainly applied to polyesters, in the finishing stage. Ruco-BAC®, SilverClear®, UltraFresh®, Silpure®, AlphaSan®, Microfresh®, Solefresh®, GuardYarn®, and SmartSilver® are some of the commercially available antibacterial agents applied on textiles [3, 27, 28]. In the case of synthetic fibers, metal and metal salts particles can be incorporated into the polymer prior to extrusion (or before electrospinning for nanofibers). For example, silver can diffuse into the fiber surface and in the presence of moisture it can form silver ions. Gradual release can lead to an extended period of antibacterial activity. In addition, silver nanoparticles can be padded onto cellulosic and synthetic fabrics, resulting in a durable antibacterial finish [29]. While metals and metal salts has excellent antimicrobial activity, leaching from treated textiles into laundering effluent is problematic. Ionic silver is highly toxic to aquatic organisms, with the EPA setting water quality criteria at 1.9 ppb in salt water and 3.4 ppb in fresh [30]. Effluent from both home laundering and industrial application can transfer silver into sewage treatment facilities, depleting necessary bacterial communities. Research conducted by Geranio et al. found that fabric treated with AgCl released only 2.7 ppb (2.4 ppb for AgCl plus a binder) of total silver per gram of textile after the first wash cycle [31]. As the effectiveness of silver depends on the release of silver ions, too few ions result in a lack of antimicrobial action, and too many yield an excess leading to pollution and waste. Success depends on finding the balance between minimum antimicrobial concentration and effectiveness.
2,4,4′-trichloro-2′-hydroxydiphenyl ether, commonly known as Triclosan is a broad-spectrum antibacterial agent, having a Minimum Inhibitory Concentration (MIC) of less than 10 ppm against most kinds of bacterial species. Triclosan has been used since 1960 in a wide variety of consumer products including toothpastes, hand soaps, deodorants, mouthwashes, shower gels, etc. Its mode of action is inhibiting bacterial growth by blocking biosynthesis of lipids. As a relatively small molecule, triclosan can be used by exhaustion, combined with dyes, or applied after dyeing. Through melt-mixing or suspension polymerization, triclosan can be incorporated directly into synthetic polymers (Figure 1) [5, 32].
Molecular structure of Triclosan.
Triclosan inhibits the growth of microbes by using an electrochemical mode of action to penetrate and disrupt the cell wall of microbes. When incorporated within a polymer, it migrates to the surface and protects the material [3, 33]. When embedded in β-cyclodextrin triclosan forms a complex and can exhibit antimicrobial action with minimum quantities [34]. Some researchers claim that triclosan inhibits a specific function i.e., lipids synthesis in a bacteria [35]. Others claim that lower levels of triclosan resistance by strains of bacteria shows that triclosan inhibits bacterial cell function in multiple ways. A decrease in the antimicrobial efficiency of triclosan treated material when the material is subjected to repeated home wash cycles has been reported by [36]. One of the greatest concerns regarding triclosan is that when exposed to sunlight, it breaks down into 2, 8-dichlorodibenzo-p-dioxin, a chemical related to other harmful polychlorinated dioxins. Therefore, it has raised a lot of concern in European governments, and its application in consumer products is banned in some countries [37, 38].
The antimicrobial agents that belong to this category are chemically bound to the textile substrate. Hence, the antimicrobial can act only on the microbe that are in contact with the treated textile’s surface. By virtue of its binding nature, these antimicrobials are not depleted and therefore potentially may have higher durability than [39]. However, compounds on a treated fabric might get abraded or deactivated with long-term usage and lose their durability [40]. The antimicrobial agents listed under this category are Quaternary Ammonium Compounds (QACs), Polyhexamethylene Biguanide (PHMB), chitosan and N-halamines.
Surface-active agents (surfactants) contain two distinct regions in their molecules: a long chain hydrophobic hydrocarbon tail and a hydrophilic head. Based on the charge of the hydrophilic group, they are classified into cationic, anionic, nonionic, and amphoteric compounds. Among the wide range of these surfactants, the cationic agents (Quaternary Ammonium Compounds or QACs) are known to be the most effective (Figure 2). QACs have significant antimicrobial properties and are excellent for deodorization and hard surface cleaning. They are used as biocides in a variety of consumer products, including toothpaste, mouthwash, shampoo, soap, deodorant, etc. The application of QACs as disinfectants goes back to 1936, where Dunn investigated the antibacterial properties of alkyldimethyl-benzylammonium chloride and found the phenol-coefficients against
General molecular structure of Quaterny ammonium (QAC).
Molecular structures and description of the different generations of quaternary ammonium (QAC).
The attachment of QACs to a textile material is known to be predominantly by ionic interactions between the anionic fiber and the cationic QAC. Therefore, in the case of fabrics that contain sulfonate or carboxylic groups, QACs can be attached to fibers by using an exhaustion dyeing process [45, 46, 47]. In the case of synthetic fibers, which contain fewer reactive sites and are quite resistant to antibacterial finishing modifications (such as Nylon 66); dye molecules can act as bridges to bind the functional molecules to fibers [48]. For example, acid dyes can be used to dye the fabric and then QACs can be applied under alkaline conditions. This ionic bonding between the QAC and the dye is relatively strong and provides a semi-durable antibacterial finishing [47, 48]. Hence a dyed fabric can achieve higher add on levels of QACs and antimicrobial efficacy as compared with undyed fabrics [48]. One commercial QAC- based antibacterial textile is Bioguard®. The active antimicrobial agent is 3-trimethoxysilylpropyldimethyloctadecyl ammonium chloride, also known as AEM 5700 or Dow Corning 5700 Antimicrobial agent, which has an MIC = 10–100 mg/L against Gram-negative and Gram-positive bacteria. This compound is made into aqueous solution and applied by spraying, padding, and foam finishing. During drying, silane forms covalent bonds with the textile, resulting in excellent durability. This compound has been commercially used on nylon, cotton, and polyester. Recently, novel quaternary ammonium functional dyes have been applied on textiles in order to combine antimicrobial finishing and dyeing of textiles in a single step [49, 50, 51].
QACs are active against a broad range of microorganisms such as fungi, Gram-positive and Gram-negative bacteria, and some viruses. QACs have a positive charge on the N atom and inflict a variety of effects on microorganisms, including the disruption of cell membrane, denaturation of proteins, and damage to cell structures. It has been proposed that during the inactivation of bacteria, the quaternary ammonium group remains intact and can retain its antibacterial ability as long as the QAC is attached to the fibers [48]. When a microbe approaches a QAC treated fabric, the free end of the agent’s molecule reacts with the cell wall and causes a leakage of the negatively charged species in the microbe cell. It eventually causes the cell’s death [39, 52]. The cationic ammonium group and the negatively charged bacteria membrane are attracted to each other. Consequently, the interactions result in the formation of a surfactant-microbe complex that interrupts all the normal functions of the membrane [53]. QACs affect bacterial DNA, causing a loss of multiplication ability [5]. If the long hydrocarbon chain is bonded to the cationic ammonium in the structure of the QAC, two types of interactions between the agent and the microorganism can occur: a polar interaction with the cationic nitrogen of the ammonium group and a non-polar interaction with the hydrophobic chain. Penetration of the hydrophobic group into the microorganism consequently occurs, enabling the alkylammonium group to physically interrupt all key cell functions [5]. The efficiency of the quaternary ammonium depends on the generation and chain substitution. It is known that the germicidal power increases with an increase in carbon chain length, while the surface activity also increases in the same way [44]. The QACs with 12–18 carbons have been used extensively as disinfectants. The typical dosage is under 1%, and even under 0,1% for some application.
To resume, the quaternary ammonium compounds are membrane active agents, their target site is at the inner (cytoplasmic) membrane in the bacteria (or plasma membrane in yeasts) [8, 44]. One of the mechanisms proposed for the antimicrobial action of QACs is in this sequence:
Adsorption and penetration of QAC in the microorganism’s cell wall
Reaction with the lipid or protein cytoplasmic membrane, which will disorganize the membrane
The leakage of low molecular weight intracellular material
Degradation of nucleic acids and proteins
Wall lysis which is caused by the autolytic enzymes.
Without detailing the studies carried out on the toxicity of quaternary ammoniums, different experiences were carried out on their ocular toxicity [54, 55], contact dermatitis [56], their skin sensitizer (human contact allergen) and asthma [57, 58]. Quaternary ammonium compounds are known to cause occupational asthma. It was found that nurses exposed to a class of QAC and all exhibited early or delayed asthma symptoms when handling disinfectant solutions containing QAC. The same study was done with products lacking in QAC and the results were negative [59]. These results have been confirmed by a multitude of studies [57, 58, 59]. In parallel, it has been reported that repeated occupational exposure after handling QACs as powders or solutions could cause sensitization [60]. In conclusion, the studies above all confirm the link between prolonged exposure to quaternary ammonium compounds and asthma. However, regarding ocular and dermal irritation, it seems that the quaternary ammonium compounds allergenicity is likely to be related to the compound’s solubility. Apparently, no quaternary ammonium compounds can be regarded as allergens. In most of the studies that classify these compounds as irritants/allergens, the lipid or water-soluble compounds have been studied, while the non-soluble QACs certainly do not have the same properties.
PHMB is a hetero disperse mixture of polyhexamethylene biguanide (Figure 3). Polyhexamethylene biguanide (PHMB, commercially known as Vantocil) is a broad-spectrum antibacterial agent with low toxicity, having an MIC = 0.5–10 ppm. It has been previously used as a disinfectant in pool sanitizers, mouthwashes, wound dressings, and in the food industry. PHMB can disrupt the integrity of cell membranes [61].
Molecular structure of Polyhexamethylene biguanide (PHMB).
The halide form of PHMB i.e., polyhexamethylene biguanide hydrochloride is applied on cellulosic materials [62]. PHMB is found to form hydrogen bonds with cellulosic fibers. With the increase in the concentration of PHMB there is a dominant increase in hydrogen bond formation between PHMB and fibers [63]. When the fabric treated with PHMB is exposed to a bacterium, the biocide interacts with the surface of the bacteria and is transferred to the cytoplasm and cytoplasmic phospholipids in the bacterial membrane. This biocide is positively charged, and therefore it mainly reacts with negatively charged species and includes aggregation, leading to increased fluidity and permeability. This results in the leakage of inner material from the outer membrane and eventually causes death of an organism [52].
N-halamines are heterocyclic compounds containing one or two covalent bonds formed between nitrogen and halogen [64]. N-halamines contain one or more nitrogen-halogen covalent bonds formed by the chlorination or bromation of imide, amide or amine groups. The halogen, which is usually chloride, is replaced with hydrogen in presence of water or chloroform and acts as biocide (Figure 4) [65]. By using chlorine-containing N-halamine compounds, durable antibacterial finishing can be achieved on textiles. N-halamines are broad-spectrum disinfectants, which have been used previously for water treatment. Their antibacterial activity is known to be due to the oxidative properties of halamine bond (N-Cl). In order to kill the bacteria, N-Cl will be transformed to N-H, which can be recharged with chlorine (during laundering, by using bleach). The product of the reaction is reversible, meaning the N-halamide can be regenerated with the presence of chlorine compound. This function is found in hypochlorite, commonly found in bleach solution. The regeneration with bleach can be done during the washing process. This novel regenerable method was first proposed by Sun and Xu for the treatment of cotton fabric [66]. Since then, many different heterocyclic N-halamines have been applied on polyester, nylon, keratinous fibers, and cotton through covalent bonding. In all these studies, it was demonstrated that regenerable and durable antibacterial activity can be achieved by recharging the fabric in aqueous chlorine solutions.
Molecular structure of N-halamines.
N-halamine compounds, of which N-chloramine is one form, can provide instant and complete kill of a broad-spectrum of microorganisms. The antibacterial property is based on active chlorine, Cl+. Two mechanisms can be used to explain the antibacterial activity of N-chloramine. One mechanism is that free chlorine is released into water and then forms HClO or ClO−. The other is that chlorine binds directly to acceptor regions in bacteria and greatly influences their enzymatic and metabolic processes [64]. It was found that the antibacterial activity mainly attributed to the second mechanism because the dissociated chlorine is limited [67]. N-halamines possess stability that is suitable for long-term use, storage and regeneration. N-chloramine can be achieved by the reaction between sodium hypochlorite solution and imide, amide or amine groups. N-halamines have been used in water treatment and incorporated into cellulose-containing fabrics, polyester fibers and polyamide [68, 69, 70]. Although no research has directly addressed N-halamine in wound dressing, it has been grafted onto fibers or fabrics so it may be used in wound dressing [69, 70].
Halamine can be applied on different textile including cellulose, polyamide and polyester fibers [71, 72, 73, 74]. It has also been found to have extraordinarily durable biocidal functions in a series of laundering tests [75]. However, N-halamine materials are found to be decomposed upon exposure to ultraviolet irradiation as in direct sunlight [76]. The main problem with N-halamines was that they result in a significant amount of absorbed chlorine (or maybe other halogens), which can remain on the fabric surface, resulting in unpleasant odor and fabric discoloration. The use of bleach and the presence of strong oxidizing degrade the dye on the textile, which leads to discoloration of the textile. This antimicrobial technology is best used on bleach resistant textile. One method known to resolve this problem is using a reduction step to remove the residual unbounded halogen from the surface of fabric [75, 76, 77, 78, 79]. An alternative antibacterial finishing agent is known to be peroxyacids (such as peroxy acetic acid, which is extensively used in hospitals.) Peroxyacids should convert to carboxylic acid in order to deactivate bacteria, but can be regenerated by reacting with an oxidant (such as hydrogen peroxide). Despite the stability of the peroxyacids on the fabric during prolonged periods, the antibacterial activity reduces largely after a number of washing and recharging cycles [73, 74].
Chitosan is derivatized by the deacetylation of Chitin, the main component of shrimp, crab, and lobster shells. Chitin, a poly (β-(1–4)-N-acetyl-D-glucosamine) is a natural polysaccharide. Chitin is synthesized by many living organisms. Chitin is the second most abundant polysaccharide in nature after cellulose [80]. When chitin is acetylated to at least about 50%, then it is called chitosan [28]. Chitosan (Figure 5) contains three reactive sites including a primary amine and two primary or secondary hydroxyl groups per glucosamine unit. As a result, it is readily subject to chemical modification. The structural characteristics of chitosan mimic glycosaminoglycan components of the extracellular matrix, so the biocompatibility, biodegradability, antibacterial, hemostatic and antioxidant activities and muco-adhesive properties impart versatility [81]. Chitosan’s good antibacterial activity along with its biodegradability, biocompatibility, and most importantly nontoxicity makes it an ideal biocidal agent in food science, pharmaceuticals, medicine, and textile applications. Despite all these advantages, chitosan lacks good solubility above pH 6.5. Its applications in a commercial context are not as wide as might be expected [82]. One of the potential problems with an effective chitosan based antimicrobial agent is that chitosan is insoluble in water and possesses high molecular weight. The high molecular weight increases the viscosity of the medium and causes detrimental effect on the hand and feel of the fabric [83]. Chitosan can be used to spin antimicrobial fibers or as a finishing agent for surface modification. Therefore, researchers are exploring chitosan derivatives that are soluble in water over a wide pH range for expanding the chitosan applications.
Molecular structure of chitosan.
Given that chitosan does not dissolve in aqueous media at neutral and alkaline pH’s and its antimicrobial activity likewise is not particularly good in neutral or alkaline solutions, so there is many causes to chemically modify chitosan. These modifications were made with the aim of proposing more soluble chitosan derivatives better suited for textiles. Recent researchers reported that chitosan derivatives have better water solubility, antibacterial and antioxidant properties [84]. Chitosan can be modified to include quaternary ammonium groups, alkyl and aromatic groups, substituents having free amino or hydroxyl groups, carboxyalkyl groups and amino acids and peptides [85]. And different applications have been found for these chitosan derivatives. Among the derivatives of chitosan we cite: Carboxymethyl Chitosan, N,N,N,-trimethyl chitosan (TMC) and Chitosan nanoparticles (CSNP).
The modification of the structure of chitosan by the addition of carboxymethyl in the structure of chitosan allows the manufacture of carboxymethyl chitosan (CMC). Compared to chitosan, CMC is characterized by high solubility at neutral and alkaline pHs. This modification does not affect its characteristic properties [86]. In addition, CMC has superior antimicrobial activity, biocompatibility and safety for humans. Usually, there are O-CMC, N-CMC, N, singlet O and N, N-dicarboxymethyl chitosan that have different chemical structures (Figure 6). For antimicrobial properties, the antimicrobial activity of different types of CMCs against
As example, some carboxymethyl derivatives of chitosan [
The second chitosan derivative fairly presented in the literature is TMC (Figure 7). It is a partially quaternized derivative [89]. It is obtained by nucleophilic substitution of the primary amino group in position C-2 by a quaternary amino group [89]. This modification facilitates the aqueous solubility of TMS at neutral and basic pH due to the presence of a permanent positive charge independent of pH (the quaternary amino amino groups) [90, 91]. This high positive charge density is responsible for the high antibacterial performance reported for TMC compared to Chitosan [92, 93, 94]. It is essential to point out that this modification takes place under alkaline conditions using sodium iodide as catalyst and N-methyl-2-pyrrolidone as solvent [95]. In addition, it can take place by reaction with dimethyl sulfate [96, 97] or dimethylformamide [98].
Molecular structure of N,
Among the derivatives of Chitosan, which have a higher antimicrobial activity than the chitosan solution, there are the chitosan nanoparticles (CSNP). For the moment, there is no clear explanation for this high efficiency, but one of the hypotheses given is based on the high specific surface area of nanoparticles as well as their better affinity for the microbial cell wall [99]. Unlike TMC, CNSPs are prepared with simple methods, without organic solvent or high shear force. Among these methods there are: emulsion crosslinking, coacervation (precipitation), ionic gelation, spray drying, microemulsion, diffusion of emulsifying solvent and polyelectrolyte complex [100]. The degree of deacetylation of chitosan and its molecular weight are factors that affect the formation and size of CNSPs [101].
Chitin is a film-forming polymer with antibacterial and fungi-static property. It triggers the defensive mechanism in host inducing certain enzymes like phytoalexins, chitinases, pectinases, glucanases, and lignin in plants [28]. Chitosan and its derivatives have been studied as antimicrobial agents against bacteria, fungi and viruses, in experiments involving in vivo and in vitro interactions with chitosan in different forms (solutions, nanoparticles, films, fibers and composites). Chitosan can react in two different mechanisms, killing or inhibiting the growth of microorganisms (biocide or Biostatic). However, its action often takes place without distinction between activities [102]. The antimicrobial performance of Chitosan or one of its derivatives depends largely on its molecular structure and its properties such as molecular weight [103], the degree of deacetylation and its water solubility [104, 105, 106, 107, 108]. In addition, its pH and its concentration in solution affect its effectiveness against microorganisms [109]. Chitosan has MIC of 0.05–0.1% (w/v) against most common kinds of bacteria. The mode of its antibacterial action is not yet fully understood [110, 111, 112], but it is possible that the amine groups provide positive charges which can react with the negatively charged surface of microbes; therefore, they can change the cell permeability, which finally leads to intracellular substance leakage [113, 114]. Chitosan acts primarily as a disruptor of the outer membrane and not as a penetrating agent. Using transmission electron microscopy, impaired membrane function was demonstrated by shrinkage, implying that water and ion leakage had occurred. However, other studies have proposed a mechanism by encapsulation where chitosan forms a polymeric substance around the bacterial cell preventing nutrients from entering the cell and its subsequent death [115]. In addition, a third mechanism based on metal chelation, removal of spore elements and binding to nutrients essential for microbial growth is proposed. This mechanism is based on the strong binding capacities known for chitosan to metals. This absorption of cations occurs by metal chelation favored by the amino groups present in the chitosan molecules [115]. The efficiency of this mechanism depends largely on the pH of the medium. A high pH favors this absorption mechanism by the fact that the amino groups of the chitosan will not be protonated under these conditions. This will allow the pair of electrons on the nitrogen atom to be available for donation to metal ions. Some studies have assumed that the metal can behave as an electron acceptor which connects via –NH2 functions to one or more chitosan chains, forming bridges with hydroxyl groups [116].
Even though, chitosan has already been utilized for the treatment of fibrous materials, a comprehensive research on their use for antimicrobial functionalization of viscose fibers for development of modern medical textiles for applications in medical devices is still missing. There is a lack of information regarding the behavior of different chitosans in contact with the cellulose materials. In addition, in-depth knowledge of their physical, chemical, and biological properties is missing [117, 118].
For textiles, chitosan and its derivatives could constitute one of the products that can be used for the finishing processes [116]. In addition, they could be used for the production of raw materials such as chitin and chitosan fibers [116]. The latter are widely used, alone or in admixture with other products such as viscose, in the medical field for the manufacture of non-woven, which can be used for dressings, or as a carrier for medicaments in the form of hollow fibers [119, 120]. Microbiological tests showed antimicrobial activity, no cytotoxicity was detected for a chitosan-polypropylene nonwoven [119]. Viscose tampons treated with chitosan were utilized for maintaining the physiological pH of vagina and acting as moisturizing agent, while simultaneously providing antimycotic and antibacterial activity [121]. Textiles treated in such a way were effective against gram-negative and gram-positive bacteria [122]. Some studies have shown that chitosan-based products provide rapid healing and less dense skin lesions compared to standard products [122, 123, 124]. By way of example, it has been found that the treatment of cotton with a carboxymethyl chitosan derivative at a concentration of 0.1% provides antibacterial protection against
In addition to chitin and chitosan, antibacterial performance has been identified for other naturally occurring products such as honey. The latter and because of its antibacterial activity allows the treatment of certain burns [127, 128, 129]. This antibacterial performance, against certain large bacteria-infecting wounds such as
At the same time, we must not forget the products of plant origin (
The antimicrobial efficacy of textiles could be characterized by different methods of analysis. These methods are standardized and divided into two categories: 1) qualitative, such as AATCC TM147, AATCC TM30 (American Association of Textile Chemists and Colorists Test Method), ISO 20645, ISO 11721 (International Organization for Standardization) and SN 195920, SN 195921 (Swiss standard) and 2) quantitative, such as AATCC TM100, ISO 20743, SN 195924, JIS L 1902 (Japanese industry standards) and ASTM E 2149 (or its modification).
Qualitative methods are characterized by their speed and simplicity. They are mainly based on the agar diffusion test. As diffusion through agar occurs at different rates depending on the textiles and the nature of the antimicrobial agents used, this category of methods is not suitable for all types of textiles. Some differences could be identified between the different qualitative methods. As an indication, we can mention that the textile is laid on an inoculated agar plate for AATCC TM147. While it is place between two agars plates, with one side inoculated for ISO 20645. Usually the qualitative method has an incubation period. After this period (24–48 h) depending on the type of microorganisms tested, the plates are examined for bacterial growth directly underneath the fabric and around its edges (zone of inhibition). The appearance of the zone of inhibition depends on the ability of the antimicrobial agent to diffuse into the agar and it is binding to the textiles. The appearance of a zone of inhibition and its size are indicative of the rate of release of the active agent and its antimicrobial efficacy. It is important to specify that zone of inhibition does not necessarily imply that microorganisms have been killed; they might have only been prevented from growing. By qualitative methods, the efficacy of different agents cannot be compared [137].
On the other hand, quantitative methods can be used for the majority of antimicrobial agents and textile supports. However, they require a longer time compared to qualitative methods. In addition, they are more expensive because they involve a real count of the microbes to measure the antimicrobial effectiveness. This method of measurement makes it easier to compare the effectiveness of different antimicrobial agents on the same textile support, for example [138, 139, 140, 141]. Quantitative methods are much more specific depending on the mechanism of action of the antibacterial agent. For example, ISO 20645 can be tested with only leaching types because the configuration does not allow observation under the textile [141]. AATCC TM100 and JIS L 1902 can be tested with leaching and non-leaching type’s antimicrobial. These methods (AATCC TM100, ISO20743 and JIS L 1902) are based on similar principle: specified amount (weight, size, and surface area) of sample swatches or substrate are inoculated with a specified number of microorganisms [142]. The inoculum is put in contact with the treated surface via three different methods: absorption, transfer and printing (ISO 20743). The absorption method uses an inoculated broth with a standardized species and concentration. The broth is absorbed by the textile sample. The sample is incubated in different condition depending on the method, to promote the bacterial growth. This method allows testing a leaching, non-leaching or a combination of antibacterial textile as well as bacteriostatic and bactericidal. However, this method is not recommended for textile with hydrophobic treatment or low absorption capacities [143]. While, the transfer method uses an agar plate who is inoculated with the tested bacteria. The contaminated plate is put in contact with the textile for 60 seconds, and after the sample is incubated. This method is used to replicate the contact of the antimicrobial textile with a contaminated surface. Whereas, the printing method apply the bacteria via a printer. This method allows faster incubation time (1 to 4 h, ISO 20743) and faster sample preparation with the automated printer (ISO20743). Finally, the dynamic shake flask method (ASTM E 2149 (or its modification) is particularly appropriate for non-leaching antimicrobials whilst the dynamic contact conditions are applied to the samples [140]. It can be used to assess the activity of the antimicrobial textile as a qualitative test. This method has been used for testing antimicrobial activity of cotton fabrics (or cellulose fibers) treated with the nanoparticles [144, 145], as well as functionalized wool [146], cotton and viscose fibers coated with chitosan [147, 148], and some other fabrics.
Once the microorganism and incubation application protocol are applied according to the desired method, the microorganism count will take place via two different techniques: the plate count method and the luminescence method. The count plate method consists of recovering the microorganism from the broth by re-plating and the number of surviving organisms. The number of colonies forming unit (CFU) is counted and the bacteria concentration is obtained by multiplying the dilution rate. The ATP concentration is quantified via a spectrophotometer according to the luminescence method. This measurement will be compared with a calibration curve prepared according to the ATP standard. The quantification of the ATP of the inoculum is carried out before and after exposure to the antimicrobial treatment. The number of surviving organisms is counted as CFU and results are usually presented as percentages or log10 reduction in contamination relative to the initial inoculum of microorganisms or the untreated control.
It should be noted that antimicrobial analysis methods are quite sensitive to contaminate. For this reason, tests are usually done under tightly controlled conditions to ensure reproducibility of results. However, carrying out tests in such a standardized environment does not reflect the reality of using textiles treated with antimicrobial agents [137]. Another factor that affects these tests is the efficiency of microbial extractions from the sample tissues. In addition, the absence of an absolute standard of effectiveness facilitates changes in the protocols applied creating inconsistencies between laboratories at national and international levels. Taking into account all the factors affecting the effectiveness of antimicrobial tests, certain additional methods are applied in complementarity. These methods include colorimetric analyzes [149], viability test [150], viability staining and microscopy [151], and fluorescent staining coupled with flow cytometry [152]. Despite the advancements made to date, the poor reproducibility of test results is the Achilles point of these tests. Over time, some attempts to establish a correlation between the different analytical techniques have taken place. For example, AATCC TM147 and JIS L 1902 were found to give the same result for a textile sample with a non-leaching antimicrobial [137]. Nevertheless, the strong differences are always an obstacle.
In conclusion, the microorganism presence on textile can be eliminated or the growth can be slowed by treating with a variety of antimicrobial agent. Multiple antimicrobial families were presented in this chapter, including synthetic and natural chemicals (Table 2). Textiles are susceptible to microorganism growth because of the structure and the ability to retain moisture of the textile. Therefore, the microorganism growth can generate multiple undesired consequence, such as hosting and transmitting harmful microbe, creating odor, mold, degradation, discoloration and biofouling for example. Textile can be treated with antimicrobial agent to reduce, slow or eliminate the microbial growth and spread. The antimicrobial was categorized in two types in this chapter, leaching and non-leaching. The non-leaching types are bound to the textile and react with the microbes upon direct contact. On the other side, the leaching types release antimicrobial in the environment at a controlled rate to disrupt the microorganism at proximity of the textile. A summary of the common reagents discussed in this chapter is gathered in the Table 3.
Mechanism | Reagent | Fiber | Remarks |
---|---|---|---|
Nylon, Wool, Polyester and Regenerated- cellulose | Slow release, durable, depletion of Ag might occur | ||
Polyester, Nylon, Acrylic, Polypropylene and Cellulose- acetate | Breaks down into toxic dioxine, large amount needed, bacterial resistance, banned in some European countries | ||
Cotton, wool, Nylon, Polyester and Acrylic | Very durable, covalent bonding, possible bacterial resistance | ||
Nylon, Polyester and Cotton | Large amount needed, bacterial resistance | ||
Wool, Nylon, Cotton and Polyester | Requires regeneration, unpleasant odor from residual Cl | ||
Cotton and Polyester | Poor durability, requires regeneration | ||
Wool, Polyester and Cotton | Low durability, adverse effect on fabric handle |
Conventional reagents used in the antimicrobial finishing of textiles.
Test | Method | Title | Principle | Antimicrobial type | Uses |
---|---|---|---|---|---|
ASTM E2149 | 20 | Standard Test Method for Determining the Antimicrobial Activity of Antimicrobial Agents Under Dynamic Contact Conditions | Dynamic shake flask test | Non-leaching type | Qualitative: Screening, routine quality-control |
AATCC 147 | Parallel Streak | Antibacterial Activity of Textile Materials: Parallel Streak | Zone diffusion assay: Agar | Leaching-types | Qualitative: Screening, routine quality-control |
AATCC 100 | — | Test Method for Antibacterial Finishes on Textile Materials: Assessment of | Cell suspension intimate contact test | Leaching and non-leaching | Quantitative |
XP G 39–010 | — | Propriétés des étoffes - Étoffes et surfaces polymériques à propriétés antibactériennes - Caractérisation et mesure de l’activité antibactérienne | Cell suspension intimate contact test | Leaching and non-leaching | Quantitative |
JIS L 1902 | Absorption method | Testing Method for Antibacterial Activity of Textiles Quantitative Test | Cell suspension intimate contact test | Leaching and non-leaching | Quantitative |
JIS L 1902 | Transfer method | Testing Method for Antibacterial Activity of Textiles Quantitative Test | Transferred Agar plate contact test | Leaching-types | Quantitative |
JIS L 1902 | Printing method | Testing Method for Antibacterial Activity of Textiles Qualitative Test | ‘Dry’ inoculum intimate contact test | Non-leaching | Quantitative |
JIS L 1902 | Halo Method | Testing Method for Antibacterial Activity of Textiles Qualitative Test | Zone diffusion assay: Agar | Leaching-types | Qualitative: Screening, routine quality-control |
ISO 20645 | Agar diffusion plate test | Textile fabrics — Determination of antibacterial activity — Agar diffusion plate test | Zone diffusion assay: Agar | Leaching-types only | Qualitative: Screening, routine quality-control |
ISO 20743 | Absorption Method | Textiles - Determination of antibacterial activity of antibacterial finished products: Absorption Method | Cell suspension intimate contact test | Leaching and non-leaching | Quantitative |
ISO 20743 | Transfer Method | Textiles - Determination of antibacterial activity of antibacterial finished products: Transfer Method | Cell suspension intimate contact test | Leaching-types | Quantitative |
ISO 20743 | Printing Method | Textiles - Determination of antibacterial activity of antibacterial finished products: Printing method | ‘Dry’ inoculum intimate contact test | Non-leaching | Quantitative |
Comparison of antimicrobial test method for textile.
The antimicrobial efficiency, the durability and skin compatibility of the treated textile must be assessing during the development of an antimicrobial treatment to minimize the risk. The antimicrobial activity testing can be categorized by quantitative and qualitative method. The qualitative method is useful for routine quality control and for the screening of multiples iterations during the development of a product, such as determining the wash durability. However, this method can lead to subjective determination. Instead, the quantitative method eliminates the possible subjectivity with plate count and luminescence technique. In addition, the skin should not be harmed be the treated textile. The safety for the skin can be evaluated with cytotoxicity test to human cell and irritation test in-vitro and in-vivo. The properties of the antimicrobial should be assessed before commercialization of an antimicrobial treated product.
Antimicrobial resistance should also be a concern when developing an antimicrobial treatment for a textile because of the large quantity of antimicrobial agent required achieving the antimicrobial activity and durability. The risk/reward should always be considered before applying antimicrobial product to a textile. The risk of antimicrobial resistance can be minimized. First off, the antimicrobial should not come close to the minimal inhibitory concentration (MIC) of the treatment during the useful life of the product to guarantee an effective product. The MIC of the antimicrobial product can be reached because of poor wash durability of the antimicrobial product. Second of, the synergy, mechanism of different antimicrobial product can be combined to reduce the resistance of a gene to a pathway. A complex antimicrobial mechanism is believed to be more efficient and more complex for the microbe to develop a set of successful mutated gene against the antimicrobials [153].
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) (COVPJ 553781 - 20), and the MEI-Québec (Ministère de l\'Économie et de l\'Innovation).