**2.3 Necrosis assays**

Necrosis assays are generally used to check for membrane integrity of the cells after treatment. As shown in **Figure 3**, NPs can adhere to membranes causing changes in their structure and function, hence it's important to carry out these assays. Neutral Red (2-amino-3 methyl-7-dimethylaminophenazoniumchloride) uptake assay is a widely used assay used to measure cytotoxicity. The Neutral Red dye is a weak, cationic, cell permeable dye taken up by viable cells and localized in the lysosome. It permeates into the cell by nonionic passive dilution and binds to the phosphate groups found on the lysosome matrix. The dye is extracted from the viable cells and spectrophotometrically measured [74, 75]. It is a sensitive dye that measures the integrity

#### **Figure 3.**

*The toxicity mechanisms induced by nanoparticles [73].*

#### In Vitro*,* In Vivo *and* Ex Vivo *Models for Toxicity Evaluation of Nanoparticles: Advantages… DOI: http://dx.doi.org/10.5772/intechopen.111806*

of the cell membrane and measures the viability of the cells [76]. The Neutral Red uptake assay is very sensitive and requires less equipment. It does not face much interference and does not have unstable reagents like other assays like MTT, XTS, etc. [77]. It is simple and can be used to detect only viable cells. It can also be done together with estimation of total protein content [78]. However, the use of Neutral Red, a fluorescent-based assay, is prone to interference from the NPs, which could lead to quenching of the dye. In a study to check the toxicity effect of silver NPs, Neutral Red, amongst other dyes, were observed to face interference from the particles [79]. Also, when applied on certain compounds that are volatile and insoluble in water, it faces problems in analysis as it mainly works when soluble in water [76]. Some chemicals could also cause the transformation of the dye into insoluble crystals, which could increase the toxicity estimation of the assay, giving a false negative result. When total protein estimation is done after the neutral red uptake assay, it could lead to reduction of the amount of protein estimated [78].

Trypan Blue Exclusion Assay is used to measure cytotoxicity of nanoparticle treatment on cells. The Trypan Blue is a dye that is absorbed by dead cells, leaving out viable cells and giving the actual number of viable cells after treatment.

Trypan Blue Exclusion Assay is very simple and quick to do, and it does not require any technical know-how. However, Trypan Blue assay is less sensitive and less reliable compared to other assays. It can be tedious and time-consuming when done on large number of samples [80]. Because of the use of hemocytometer, the possibility of making counting errors is present. Also, if the cells are not properly diluted or poorly dispersed in the counting chamber, it can contribute to the error [81]. The assay is insensitive as it cannot distinguish between live cells and cells that are gradually losing cell function. Another disadvantage of this assay is it can cause toxicity to mammalian cells [82].

#### **2.4 Oxidative stress assays**

Due to the high surface area to volume ratio of NPs, they elicit the production of reactive oxidative species (ROS) from the cells as shown in **Figure 4**, which could lead to further cellular damage. The ROS produced is measured using some of the oxidative stress assays.

2′,7′-dichlorofluorescein diacetate (DCFH-DA) is a nonionic, nonpolar fluorophore that penetrates the membrane and is sensitive to ROS. When DCFH-DA is internalized by the cell, it is enzymatically hydrolyzed by cytosolic esterases into dichlorofluorescein (DCFH), which remains in the cytosol. The nonfluorescent DCFH is then oxidized by hydroxyl radicals and cellular ROS into highly fluorescent dichlorofluorescein (DCF), which is then analyzed by flow cytometry or fluorescence microscopy [84].

Tritiated borohydride and 2,4-dinitrophenylhydrazine (DNPH) are used to measure protein carbonyl levels, which are indicative of oxidative damage. Protein carbonyls are reduced to alcohols by borohydride. This reduction is measured spectrophotometrically at wavelength absorbance of 340 nm. Also, when protein carbonyls react with DNPH a stable 2,4-dinitrophenyl (DNP) hydrazone product is generated and its absorbance is read between 360 and 390 nm [85]. DNPH is highly sensitive and specific and result from the assay can further be improved with the use of highperformance liquid chromatography (HPLC) or Western blotting [86].

Another means of assessing the production of ROS is through analysis of lipid peroxidation products like malondialdehyde (MDA) and 4-hydroxyl-2-nonenal

**Figure 4.** *Induction of oxidative stress by nanomaterials [83].*

(4-HNE). MDA is formed when large polyunsaturated fatty acids (PUFAs) are broken down and during the metabolism of arachidonic acid (AA) [87]. The byproduct reacts with thiobarbituric acid (TBA) at pH 3.5 to produce MDA-TBA which is measured by a spectrophotometer at wavelengths of 532 nm and fluorescently at 553 nm [88]. 4-HNE is a by-product of AA and peroxidation of PUFAs. It reacts with primary amines to form Schiff 's bases and thiol. It also reacts with amino compounds to form Michael's adduct which is detected by HNE-protein adduct ELISA assays [89, 90].

DCFH-DA, However, is prone to inaccurate interpretation due to nonspecific enzymatic oxidation and photooxidation [84, 91]. With the use of DNPH, larger samples are required for the analysis [92]. The lipid peroxidation is nonspecific since TBA reacts with other molecules other than MDA, which could lead to false positive results.


*3.3. Adaptive-ECMS*

The traditional ECMS method requires tuning the equivalence factor offline for every driving profile to ensure that the battery state of charge remains within the desired/operational limit. However, in real-world applications the future velocity profile is not known. Therefore, it is necessary to tune the ECMS factor in real time to ensure acceptable operation of the energy management system, especially when starting from an unfavorable initial condition. A-ECMS adjusts the equivalence factor based on drive cycle prediction, driving pattern recognition, or feedback from the battery state of charge [19]. In this work a modification of the approach that uses the SoC feedback [20] is adopted. The equivalence factor is adjusted as

$$a\_{\rm eq}(k) = \begin{cases} 0.25 & \text{if } \text{ SoC} < 40\% \\ 0.10 & \text{if } \text{ SoC} > 60\% \\ \max\left(\min\left(a\_{\rm eq}(k-1) + q\left(\text{SoC}(k) - \text{SoC}(k-1)\right), 0.25\right), 0.10\right) & \text{otherwise} \end{cases} \tag{9}$$

where *q* is a constant coefficient. The suitable sampling time for Equation (9) depends on the relative size of the energy storage and energy consumption. Simulations showed that a sampling time of less than 1 min can keep the SoC in 40–60% range for the tested drive cycle. However, a sampling time of 15 s was selected for the experiments for a tighter control over SoC, without adding a significant computational effort. Further investigations into sampling time dependence on drive cycle and vehicle parameters are left for future development. Figure 8 shows the A-ECMS implementation for hardware-in-the-loop experiments presented in the next section. *Pb* represents the battery power.

**Figure 8.** A-ECMS implementation for hardware-in-the-loop experiments.

#### **4. Experimental Setup**

## *4.1. Testbed*

Figure 9 shows a picture of the engine-dynamometer experimental testbed, which includes an AVL AC transient dynamometer, an instrumented 1.6 L Ford EcoBoost engine, and the power split supercharger by EATON. An AVL SESAM-FTIR emission measurement bench is used to measure exhaust gas species and calculate fuel flow rate along with a hot wire air flow rate meter. An AVL battery simulator is used to power the PSS and measure the motor current and voltage. A rapid prototyping electronic control system (RPECSTM) from Southwest Research Institute (SwRI) was used to integrate all engine controllers, the online energy management system, and implement real-time vehicle and driver models.

**Figure 9.** Engine-dynamometer experimental testbed.


**1. Introduction**

Mesenchymal stem cells (a.k.a. mesenchymal stromal cells, MSCs) hold enormous promise as a durable, sustainable, and novel cell-based biologic therapeutic for a diverse range of clinical applications aimed at preventing or reversing organ injury and promoting tissue regeneration.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Substantial data have accumulated regarding the safety of administering both autologous and allogeneic MSCs to patients with a broad array of diseases. In addition, it is increasingly clear that MSCs exert anti-fibrotic, pro-angiogenic, regenerative, and immunomodulatory effects, and therefore, offering therapeutic potential in a wide range of presently untreatable conditions. The growing evidence supporting the use of MSCs as therapeutic strategy includes their relative ease of isolation and expansion in culture, multilineage differentiation capacity, immunomodulatory, anti-inflammatory, anti-microbial, and regenerative effects, homing and migratory capacity to injury sites, safety profile in allogeneic transplantation, and few ethical considerations [1, 2]. The use of large animal models in preclinical studies has been instrumental in deciphering the underlying mechanisms of action of MSC therapy [3]. Moreover, substantial human phenotypic data has demonstrated that MSC therapy is safe [4–10] and holds the potential for repair and regeneration of diverse organ systems and amelioration of multiple chronic illnesses for which there is currently no cure [4, 6, 7, 9–24]. There are currently various MSC sources under investigation in preclinical and clinical studies, namely bone marrow, adipose tissue, umbilical cord blood, umbilical cord, and amniotic membranes/placenta (**Figure 1**). Multiple mechanisms of action underlie successful MSC therapy, including MSC engraftment and differentiation, and more importantly, the secretion of bioactive paracrine molecules that inhibit apoptosis, fibrosis, and inflammation and promote neovascularization/neo-angiogenesis and endogenous stem cell recruitment, proliferation, and differentiation [25–27] (**Figure 2**). In particular, cell-cell interactions between MSCs

and endogenous host cells within stem cell niches provide structural support and produce the soluble signals that regulate stem cell function in tissues[1, 28–30] (**Figure 1** inset). An indepth molecular understanding of how MSCs produce the therapeutic benefits demonstrated in numerous clinical trials is critical for the development and design of new clinical trials as well as for the development of newer generations of MSC products that have greater efficacy and sustainability. This chapter will review the current state of knowledge in the use of MSCs

Mesenchymal Stromal Cells as a Therapeutic Intervention

http://dx.doi.org/10.5772/intechopen.78586

145

MSCs are non-hematopoietic stem cells with multilineage potential that originate from the mesodermal germ layer. The pioneering studies conducted by Friedenstein et al. provided the first evidence that these fibroblast-like cells, described as spindle-shaped and clonogenic in culture conditions could be isolated from bone marrow via their inherent adherence to plastic in culture [31, 32]. MSCs are an integral part of the stromal microenvironment and support hematopoietic stem cells and regulate hematopoiesis, although they comprise only ~0.01–0.001% of the total nucleated cells in the bone marrow [33, 34]. Moreover, MSCs have been isolated from virtually every tissue type, including adipose tissue, liver, lung, skeletal and heart muscle, synovial membrane, amniotic fluid, placenta, umbilical cord blood, and

as a therapeutic strategy for organ structural and functional repair.

dental pulp, suggesting that they reside in all organs [35–37].

**2. Biology of mesenchymal stem cells**

**Figure 2.** Mechanism of action of mesenchymal stem cell therapy.

**Figure 1.** Mesenchymal stem cell tissue sources, ex vivo expansion, and role in stem cell niche. Initially identified in bone marrow, MSCs can be isolated from various tissues in the body. To isolate MSCs from a bone marrow biopsy, first the mononuclear cells are isolated from red blood cells by Ficoll density centrifugation, and subsequently, the MSCs are separated from the mononuclear cells by plastic adherence in culture. Inset: the constituents of a stem cell niche are depicted in this schematic. ECM extracellular matrix. *Adapted from Wagers AJ et al., Cell Stem Cell, 2012*.

**Figure 2.** Mechanism of action of mesenchymal stem cell therapy.

Substantial data have accumulated regarding the safety of administering both autologous and allogeneic MSCs to patients with a broad array of diseases. In addition, it is increasingly clear that MSCs exert anti-fibrotic, pro-angiogenic, regenerative, and immunomodulatory effects, and therefore, offering therapeutic potential in a wide range of presently untreatable conditions. The growing evidence supporting the use of MSCs as therapeutic strategy includes their relative ease of isolation and expansion in culture, multilineage differentiation capacity, immunomodulatory, anti-inflammatory, anti-microbial, and regenerative effects, homing and migratory capacity to injury sites, safety profile in allogeneic transplantation, and few ethical considerations [1, 2]. The use of large animal models in preclinical studies has been instrumental in deciphering the underlying mechanisms of action of MSC therapy [3]. Moreover, substantial human phenotypic data has demonstrated that MSC therapy is safe [4–10] and holds the potential for repair and regeneration of diverse organ systems and amelioration of multiple chronic illnesses for which there is currently no cure [4, 6, 7, 9–24]. There are currently various MSC sources under investigation in preclinical and clinical studies, namely bone marrow, adipose tissue, umbilical cord blood, umbilical cord, and amniotic membranes/placenta (**Figure 1**). Multiple mechanisms of action underlie successful MSC therapy, including MSC engraftment and differentiation, and more importantly, the secretion of bioactive paracrine molecules that inhibit apoptosis, fibrosis, and inflammation and promote neovascularization/neo-angiogenesis and endogenous stem cell recruitment, proliferation, and differentiation [25–27] (**Figure 2**). In particular, cell-cell interactions between MSCs

144 Stromal Cells - Structure, Function, and Therapeutic Implications

**Figure 1.** Mesenchymal stem cell tissue sources, ex vivo expansion, and role in stem cell niche. Initially identified in bone marrow, MSCs can be isolated from various tissues in the body. To isolate MSCs from a bone marrow biopsy, first the mononuclear cells are isolated from red blood cells by Ficoll density centrifugation, and subsequently, the MSCs are separated from the mononuclear cells by plastic adherence in culture. Inset: the constituents of a stem cell niche are

depicted in this schematic. ECM extracellular matrix. *Adapted from Wagers AJ et al., Cell Stem Cell, 2012*.

and endogenous host cells within stem cell niches provide structural support and produce the soluble signals that regulate stem cell function in tissues[1, 28–30] (**Figure 1** inset). An indepth molecular understanding of how MSCs produce the therapeutic benefits demonstrated in numerous clinical trials is critical for the development and design of new clinical trials as well as for the development of newer generations of MSC products that have greater efficacy and sustainability. This chapter will review the current state of knowledge in the use of MSCs as a therapeutic strategy for organ structural and functional repair.


Author details

Kai On Chu and Calvin C.P. Pang\*

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong


**Abstract**

The right to education is enshrined in international law and its fulfillment appears every day as a citizen demand. In general, the States recognize the right to education when they signed the Covenant on Economic, Social and Cultural Rights. Social and Cultural Rights in which it is materialized in a concrete reality that must be expressed in education itself. It is necessary to understand that education is the result of a nondemocratic model imposed by a dictatorship, the purpose of which was to protect capital, with which everything remained a prisoner of a neoliberal conception. Of course, education is a necessity both individually and collectively, so that efforts for quality higher education are necessarily related to the democratization of societies. Finding such an answer is everyone's task and this forces the analysis of laws and behaviors.

**Keywords:** right to education, pre-eminence, inequality, economic, social and cultural rights, teaching, research and extension


**Author details**

Hnin Wuit Yee Kyaw1,2\* and Phone Pyai Tun2

1 Konstanz University of Applied Sciences, Konstanz, Germany

2 University of Forestry and Environmental Sciences, Nay Pyi Taw, Myanmar

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.