**Chapter 3**

*Current Issues in Knowledge Management*

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**32**


**Preface to "Multibody Systems with Flexible Elements"**

Multibody systems with flexible elements are a field of great contemporary interest in multiple fields, with various industrial applications. Their study and the development of appropriate models represent a desire of current research in the field of applied mechanics. The high interest in the field is also indicated by the publications dealing with multibody systems and the large amount of scientific literature and numerous published monographs. This book aims to contribute to this large flow of scientific information in a representative field of current research. Mechanical systems with elastic elements are considered where the displacements of each or some of the elements of the system are generally large and cannot be neglected in mechanical modeling. The study of these multibody systems covers many industrial fields, but also has applications in medicine, sports, and art. The systematic treatment of the dynamic behavior of interconnected bodies has led to an important number of formalisms for multibody systems within mechanics. This formalism is currently used in many engineering fields, especially robotics and vehicle dynamics. The formalism of multibody systems offers a means of algorithmic analysis, assisted by computers, and a means of simulating and optimizing an arbitrary movement of a possibly high number of elastic bodies in the connection. The domain where researchers apply these methods are robotics, simulations of the dynamics of vehicles, biomechanics, aerospace engineering (helicopters and the behavior of cars in a gravitational field), internal combustion engines, gearboxes, transmissions, mechanisms, the cellulose industry, simulations of particle behavior (granulated particles and molecules), dynamic simulations, military applications, computer games, medicine, and rehabilitation.

We hope that the present volume will present a small but useful contribution in the field of multibody systems.

> **Marin Marin, Dumitru Baleanu, and Sorin Vlase** *Editors*


**5. Discussion**

The alteration of the microbiota appears to be a common ground for both OP and FA. In particular, an altered intestinal barrier can be considered common damage to both diseases. The intestinal barrier is defined as a functional unit that constantly balances the antigenic charge of the intestinal lumen with a complex immunological and non-immunological organization of the intestinal mucosa. It performs two fundamental functions for the survival of the individual: allow the absorption of nutrients and defend the body from the penetration of harmful macromolecules mediated by the tight junctions of the apical epithelial cells. It has recently been observed that tight junctions are regulated in their

functioning by cytokines produced in the intestine and can be altered by various factors, including alcohol consumption, dietary imbalances, and the action of bacterial toxins [91]. Alteration of the intestinal barrier and the gut microbiota cause the development of an important inflammatory substrate in the intestine, which leads to FA and the loss of estrogen typical of primary OP. A study by Li et al. found that depletion of hormones increases inflammation in the intestine through a greater antigenic load that crosses the intestinal barrier [22]. Estrogens seem to have an ambivalent role in promoting the development of allergic diseases and the degranulation of mast cells in association with exposure to allergens. Andrè et al. also found involvement of the use of oral contraceptives in the etiology of urticaria and chronic angioneurotic edema [91].

The intestinal microbiota is also able to influence the estrogens circulating level through the secretion of β-glucuronidase, an enzyme that activates them. The integrity of the intestinal barrier, normally preserved by the presence of four phyla, Bacteriodetes, Firmicutes, Actinobacteria, and Proteobacteria, is altered by dysbiosis, in which the reduction in cell-cell junctions increases intestinal permeability, resulting in bacterial translocation inducing a systemic inflammatory state at the basis of various pathological processes. Furthermore, it is important to underline that dysbiosis leads to a reduced deconjugation of estrogens with a reduction in their circulation, leading to the activation of CD4 + T cells. CD4 + T cells produce RANKL, OPG, and TNFα, promoting osteoclast activation and bone absorption through the OPG- RANK-RANKL signal transduction pathway.

There is evidence that the inflammatory process is at the basis of both OP and FA. Several proinflammatory and anti-inflammatory mediators are involved in the immunopathogenesis of FA, in which allergens can stimulate Th1, Th2, and Th17 cytokines in a heterogeneous way. Kara et al. [92] have hypothesized the monitoring of cytokines such as TNFα and IL-6 in the follow-up of patients with FA, while Nadelkopoulou et al. [93] have investigated the role of IL-10 in the treatment of FA. TNF-a and IL-1 are also the main cytokines involved in bone metabolism and bone loss related to estrogen deficiency. The role of IL-33 is being debated [94], its activity contributing to the development of various allergological conditions through its action on mast cells, eosinophils, Th2 cells, Tregs, natural killers, basophils, dendritic cells, and activated macrophages, but at the same time appears to have a protective role on bone by inhibiting RANKL-dependent osteoclastogenesis [91–99].

Bone health is highly dependent on diet and nutritional style that determines the type of microbiota in host organisms. The intestinal microbiome, in fact, contributes to the production of proteins and enzymes related to digestion and energy metabolism since it ferments undigested nutrients transforming them into SCFA, leading to a decrease in intestinal permeability and greater absorption of minerals such as calcium. There are several factors linked to the microbiota that unite OP and FA; in particular, a key role is played by SCAF with immunomodulatory and anti-inflammatory effects, exercised through the promotion of immune tolerance, the production of IgA and IgM, the reduction in the production of proinflammatory cytokines including IL-1β, IL-6, and IL-17 [41], and the production of anti-inflammatory IFNγ and IL-10 with consequent expansion of Treg cells and suppression of proinflammatory Th17 and Th2 cells.

The strong influence of the diet on the microbiota and consequently on the OP and allergic manifestations has led to evaluate the fundamental role of nutrition. In fact, a diet rich in fat can reduce the absorption of essential elements for bone health, including vitamin D, K, C, and folate. Moreover, the high-fat diet of Western countries is one of the factors that can contribute to the increased prevalence of allergies in Western countries [100].

Diet-induced obesity has been demonstrated to be a factor of increased susceptibility for FA, and in particular, the microbiota associated with the high-fat diet was found to be able to increase the propensity for FA as evidence of the connection between diet-microbiota and FA [101]. It has been reported that heat-killed lactic acid bacteria (LAB) increased the percentage of peripheral CD4 + CD25 + Foxp3 + Treg cells and relieve symptoms in the pollen season when administered to patients with mild Japanese cedar pollinosis. Although not through the microbiota, LAB is thought to act directly on the immune system. In particular, increased Treg, along with SCFA, is considered a promising target for improving both allergies and bone metabolic balance [102]. Roduit et al. analyzed the levels of SCFA in fecal samples of 301 children at the age of 1, reporting that children with the highest levels of butyrate and propionate were less likely to suffer from asthma by the age of 3 and 6 years and showed significantly lower allergic sensitization with a decrease in food allergy risks and allergic rhinitis diagnosis. More recently, Cait et al. examined the role of bacterial butyrate production in the gut during early childhood in the development of atopic disease and concluded that the lack of genes encoding key enzymes for both the breakdown of carbohydrates and the production of butyrate was the basis of allergic sensitization [1].

Although OP and allergies are two conditions with a high prevalence in the general population and the relationship between fracture risk and allergic diseases such as asthma, atopic dermatitis, urticaria, FA is now well established, to date, we do not yet have adequate epidemiological studies on the prevalence of allergies in patients with OP [103–109]. This probably partially depends on the recent recognition of their interrelationship other than the difficulty in including all together so many "allergic" pathologies with such different peculiarities. The currently available data in the literature refer to single pathologies and limited series. Furthermore, the available studies evaluate the presence of OP in allergic diseases and not the prevalence of allergies in patients with OP. Even in recent studies conducted to evaluate the prevalence of comorbidities in subjects with OP, no data relating to allergies emerge [110], probably because there is still not enough awareness of the influence of allergic disease on bone health. Furthermore, the retrospective analysis conducted in Italy on 64.852 subjects at high risk for fracture collected between 2016 and 2020, through the DeFRAcalc79, does not take into account allergic diseases among the variables for calculating the risk of OP [111].

Vitamin D plays an important role in the pathogenesis of OP and also in the regulation of intestinal tight junctions, leading to the hypothesis that its deficiency may compromise the integrity of the barrier or induce alterations in the composition of the intestinal microbiota, increasing the risk of FA and of OP. Vitamin D, in fact, is important in the maintenance of bone health through the regulation of serum calcium homeostasis. The lack of vitamin D increases bone resorption in order to maintain the right serum calcium levels, making up for the lack of calcium reabsorbed by the gut induced by vitamin D deficiency [112].

Sardecka-Milewska et al. found that children with cow's milk allergy have lower serum concentrations of vitamin D than healthy children [113]. The role of vitamin D in the development of FA is further confirmed by Koplin et al., who documented an attenuated association between low serum levels of vitamin D and food allergy only in subjects with polymorphisms associated with lower levels of vitamin D-binding protein. This involvement of vitamin D in FA could be linked to the ability of vitamin D to induce the expression of IL-10 by Treg cells, leading to oral tolerance and its maintenance [114,115].

Finally, it is also essential to remember the relationships between microbiota and microRNA (miRNA). The latter are small non-coding RNAs capable of regulating gene expression. The importance of the role of miRNAs in many pathological conditions [116–118], including allergies [119] and OP [120,121], is emerging. Being able to fully understand the relationship between miRNA and microbiota could allow to have new disease markers and pave the way for new targeted and personalized therapeutic strategies [122].

The importance of the unaltered microbiota is underlined by the fact that the growing tendency to use antibiotics leads to impaired intestinal absorption with a deficiency of minerals important for bone health, thus contributing to the development of OP, on the other hand, the use of antibiotics compromises the development of oral tolerance mechanisms leading to increased development of FA.

On this basis is founded the use of prebiotics and probiotics for beneficial modulation of the intestinal microbiota both in OP and in allergological pathologies. For example, it has been shown that LGG is able to reduce the expression of TNF α, IL-17, and RANKL in cells isolated from the small intestine and bone marrow of mice, decreasing bone resorption, and also have desensitizing effects in cow's milk and peanut allergy [45,88]. The data in this review based on the current literature highlight how the microbiota and some bacterial species can influence the propensity to develop diseases, including allergies and OP. In particular, the supplementation of beneficial bacteria and diet corrections seems to improve the outcome and prevent the onset of these diseases.


*2.2. Cell Culture*

Human fetal NSCs were purchased from Clontech (human neural cortex; catalog number Y40050). Maintenance of hNSCs was performed in Neurobasal medium without phenol red (Thermo Fisher, Waltham, MA, USA) and in the presence of B-27 supplement (1:100; catalog number 12587010; Thermo Fisher), N-2 supplement (1:200; catalog number 17502-048; Invitrogen, San Diego, CA, USA), 20 ng/mL fibroblast growth factor (catalog number 4114-TC-01M; R&D Systems, Minneapolis, MN, USA, 20 ng/mL epidermal growth factor (catalog number 236-EG-01M; R&D Systems), GlutaMAX

(catalog number 35050061; Thermo Fisher), and sodium pyruvate. Cells were prepared in 6-well plates precoated with laminin (10 mg/mL; catalog number L2020; Sigma-Aldrich, St. Louis, MO, USA) and were grown to near confluence (80 to 90%) prior to passage. For passaging, cells were rinsed gently with phosphate-buffered saline (PBS) (without calcium and magnesium) and then treated with TryPLE for 5 min at 37 ◦C for cell detachment (catalog number 12563029; Thermo Fisher). The GSC 387 cell line was prepared and maintained similarly to what has been previously described using the media and detachment conditions used for hNSCs described above [29]. Vero cells (ATCC CCL-81) were purchased from ATCC and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Waltham, MA, USA) with high glucose (4.5 g/L), 10% fetal calf serum (FCS) (Sigma), and 1% Penicillin-Streptomycin (PS) (Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦C and 5% CO2.


*4.2. Urine Sample Preparation for LC-MS/MS*

First morning urine samples were collected from all examined patients. The middle portion of freshly passed morning urine was collected in 10 mL test tubes, centrifuged at 3000 rpm for 15 min, the supernatant was frozen in 1 mL aliquots and stored at −20 ◦C.

Urine aliquots with a volume of 0.1 mL were quickly thawed and 0.5 mL of cold acetone was added to precipitate proteins overnight at −20 ◦C. Then the samples were centrifuged at 20,000× *g* for 10 min, the supernatant was removed, the precipitate was dissolved in 50 μL of 8 M urea/200 mM Tris-HCL, pH 8.5. The proteins were restored with 5 mM dithiotreitol for 30 min at 37 ◦C, alkylated with 20 mM iodoacetamide in the dark for 30 min. Before hydrolysis, 200 μL of deionized water was diluted, trypsin (Trypsin Gold, Promega, Madison, WI, USA) was added in an enzyme-protein ratio of 1:25, incubated overnight at 37 ◦C. The reaction was stopped by adding formic acid to the final concentration of 1%. Peptides were centrifuged at 18,000× *g*, the supernatant was left for desalting. Desalting was carried out by solid-phase extraction using plates (Oasis HLB 96-well Microelution Plate, Waters, Beverley, MA, USA). The eluate was lyophilized and dissolved in 0.1% formic acid to a concentration of 0.5mg/mL for further LC-MS/MS analysis.


**7. References**

180 Pulmonary Embolism

(a) (b)

(c) (d)

defect score was 52.5.

**6. Conclusion**

Fig. 7. Pulmonary embolism with right ventricular dysfunction. (a) Dual-energy CT angiography (DECTA) shows filling defect in right main pulmonary artery. (b) DECTA shows enlarged right ventricle (RV/LV ratio > 1). (c & d) Lung iodine images show

perfusion defect in right lung except superior segment and posterior basal segment of right lower lobe. Multifocal wedge shaped perfusion defect in also noted in subpleural portion of left lung. The relative volume of pulmonary perfusion defect was 47.8% and perfusion

This chapter focused on the prognostic value of RV dysfunction as measured by chest CT in patients with hemodynamically stable PE. Although echocardiographic assessment of RV dysfunction is a generally accepted imaging modality, the lack of a clear echocardiographic definition of RV dysfunction, and the poor RV images, reduces its diagnostic capabilities. RV dysfunction assessed by CT, including RV enlargement, RV/LV ratio, interventricular septal bowing, pulmonary obstruction score, and pulmonary perfusion defect have been associated with poor patient outcomes. Risk stratification of patients with stable PE based on chest CT findings may comparable in results to echocardiography and may be useful.

Risk Stratification of Submassive Pulmonary Embolism:

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**9**

*Sweden*

**Quantitative Ventilation/Perfusion**

**for Pulmonary Embolism Diagnosis**

*Lund University and Skåne University Hospital, Lund*

Marika Bajc and Jonas Jögi *Department of Clinical Physiology*

**Tomography: The Foremost Technique**

The value of perfusion scintigraphy in the detection of pulmonary embolism (PE) was demonstrated as early as 1964 by Wagner et al. PE causes perfusion defects that conform to the anatomical distribution of the pulmonary vascular bed. Perfusion defects in acute PE are therefore of sub-segmental, segmental or lobar character. Ventilation is normally preserved in these areas and the observed wedge shaped mismatch between ventilation and perfusion is typical for PE. Planar ventilation/perfusion scintigraphy (V/P scan) was until the 1990s the method of choice for studying patients with suspected PE. However, the large Prospective Investigation of Pulmonary Embolism Diagnosis (PIOPED I), showed a high number of non-diagnostic examinations (65%) with V/P scan and the probabilistic interpretation criteria were confusing to the clinicians (Gray et al., 1993; The PIOPED Investigators, 1990). Planar imaging has become obsolete, particularly when the issue is

identification and quantification of focal or regional aberration of organ function.

defects down to the sub-segmental level (Bajc et al., 2002b).

cardiopulmonary diseases.

The advantage of three dimensional tomography over planar imaging for PE detection had already been shown in 1983 in a study on dogs (Osborne et al., 1983). Furthermore, Magnussen et al. (1999) used a computerized model of PE to highlight the advantage of SPECT over planar imaging in the assessment of the size and location of perfusion defects. Using a dual head camera, Palmer et al. (2001), developed a fast and efficient method for ventilation/perfusion tomography (V/P SPECT ) for clinical practice with total acquisition time of only 20 minutes. Moreover, they developed an algorithm to calculate the quotient between ventilation and perfusion and to present it as V/Pquotient images. This facilitated PE diagnosis and the quantification of PE extension, which led to the use of the term quantitative V/P SPECT. Using a porcine model, Bajc et al. validated V/P SPECT for diagnosis of PE and confirmed the superior value of tomography over planar imaging with excellent interobserver agreement of

The objective of this chapter is to acquaint readers with the latest methodological approach of V/P SPECT in the diagnosis of PE, in accordance with the new guidelines of the European Association of Nuclear Medicine (Bajc et al., 2009a, b). In this chapter we also discuss the value of V/P SPECT in the follow up after acute PE and in the diagnosis of other

**1. Introduction**

Zhang, L. J., Yang, G. F., Zhao, Y. E., Zhou, C. S., & Lu, G M. (2009). Detection of pulmonary embolism using dual-energy computed tomography and correlation with cardiovascular measurements: a preliminary study. *Acta Radiol* Vol. 50 (8): 892-901.


**3.4 Mechanisms to negotiate uneven terrain**

The system should allow for significantly more maneuverability on uneven (offcamber) terrain than the standard wheelchair. The suspension would manage the off-camber surfaces through autonomous adjustments (**Figure 3**), when the user

**161**

**Figure 4.**

*Independent pass over obstacle [18].*

*Development of a Rough Terrain Wheelchair Design DOI: http://dx.doi.org/10.5772/intechopen.91267*

safely done in a conventional wheelchair [18].

the system work even in a stationary situation as well [17].

is on an incline of around 10 degrees. As the right shock compresses and the left relaxes, consequently enabling the seat to level itself, offering greater stability to the wheelchair structure seat. The determination of shocks ought to take into consideration autonomous control of spring rate and damping ratio, consequently enabling

Different obstacles may require the suspension to work autonomously, for instance, a block of wood or a crack in the walkway can be effectively explored with the same suspension framework. As given in **Figure 4**, the right side of the wheelchair must articulate upwards so as to keep up all the four wheels in contact with the ground surface, as the wheelchair moves over the obstructions. The right rear shock compresses, while the others adjust to equilibrium, allowing for all four wheels to

The operators explore obstructions in their everyday life. A number of these snags require autonomous wheel articulation, while others may require the shocks to work in unison.  Assuming the user is moving on a path perpendicular to the curb, the wheels will act in unison as the back end of the chair rolls off of the curb, with the suspension absorbing the force. This is not a navigation that could be

In some cases, there can be need to attach a set of wide auxiliary wheels or wheel extensions to the standard wheelchair for it to be stable when used off road on sandy, loose or unstable terrain. Of late, a couple of expansive tire-like plastic or elastomeric treads are connected and fitted onto the back wheels of the wheelchair to give a substitute handhold grip to the user in a way similar to mounted standard wheels arrangement above [19]. The treads are additionally marginally recessed inward from the original tire, being of a smaller diameter than the back wheel, to allow the wheelchair to ride exclusively on the tire on hard surfaces and make drive on such surfaces equivalent to one would typically anticipate. The treads act together with a second pair of lightweight plastic wheels which are adjusted to fit on either side of every one of the two stanchion mounted front wheels, the combination acting to give an broad bearing surface to better steadiness, load bearing capacity and simpler controlling of said vehicle when utilized on said sandy, loose or shaky landscapes. The wheelchair may likewise be fitted with an anti-tip over mechanical assembly to prevent flipping in reverse when navigating difficult landscape and to allow the wheelchair to be moved in an upward angled position which puts the most substantial portion of the

have some type of contact, and a resulting good stability is achieved [18].

weight on the back wheel, increasing grip on the shaky terrain [19].

**Figure 3.** *Independent suspension travel [18].*

#### *Development of a Rough Terrain Wheelchair Design DOI: http://dx.doi.org/10.5772/intechopen.91267*

*Public Health in Developing Countries - Challenges and Opportunities*

**3.4 Mechanisms to negotiate uneven terrain**

The system should allow for significantly more maneuverability on uneven (offcamber) terrain than the standard wheelchair. The suspension would manage the off-camber surfaces through autonomous adjustments (**Figure 3**), when the user

**160**

**Figure 3.**

**Figure 2.**

*Rock obstructions [7].*

*Independent suspension travel [18].*

is on an incline of around 10 degrees. As the right shock compresses and the left relaxes, consequently enabling the seat to level itself, offering greater stability to the wheelchair structure seat. The determination of shocks ought to take into consideration autonomous control of spring rate and damping ratio, consequently enabling the system work even in a stationary situation as well [17].

Different obstacles may require the suspension to work autonomously, for instance, a block of wood or a crack in the walkway can be effectively explored with the same suspension framework. As given in **Figure 4**, the right side of the wheelchair must articulate upwards so as to keep up all the four wheels in contact with the ground surface, as the wheelchair moves over the obstructions. The right rear shock compresses, while the others adjust to equilibrium, allowing for all four wheels to have some type of contact, and a resulting good stability is achieved [18].

The operators explore obstructions in their everyday life. A number of these snags require autonomous wheel articulation, while others may require the shocks to work in unison.  Assuming the user is moving on a path perpendicular to the curb, the wheels will act in unison as the back end of the chair rolls off of the curb, with the suspension absorbing the force. This is not a navigation that could be safely done in a conventional wheelchair [18].

In some cases, there can be need to attach a set of wide auxiliary wheels or wheel extensions to the standard wheelchair for it to be stable when used off road on sandy, loose or unstable terrain. Of late, a couple of expansive tire-like plastic or elastomeric treads are connected and fitted onto the back wheels of the wheelchair to give a substitute handhold grip to the user in a way similar to mounted standard wheels arrangement above [19]. The treads are additionally marginally recessed inward from the original tire, being of a smaller diameter than the back wheel, to allow the wheelchair to ride exclusively on the tire on hard surfaces and make drive on such surfaces equivalent to one would typically anticipate. The treads act together with a second pair of lightweight plastic wheels which are adjusted to fit on either side of every one of the two stanchion mounted front wheels, the combination acting to give an broad bearing surface to better steadiness, load bearing capacity and simpler controlling of said vehicle when utilized on said sandy, loose or shaky landscapes. The wheelchair may likewise be fitted with an anti-tip over mechanical assembly to prevent flipping in reverse when navigating difficult landscape and to allow the wheelchair to be moved in an upward angled position which puts the most substantial portion of the weight on the back wheel, increasing grip on the shaky terrain [19].

**Figure 4.** *Independent pass over obstacle [18].*

Push-bar outfitted gadget permits structures that allow people of varying physical strength to move the wheelchair along a wide scope of landscapes, including delicately tricky terrain. The push-bar enable the users to initiate mobility without hand to wheel system. The unique design drives the chair forward with a forward movement of the drive bars while pulling back on the push-bars effects the braking mechanism. Turning is accomplished with a forward movement on one push-bar and a backward movement on the other push-bar. The off-road seat of this design offers the operator a multi-speed equipped favorable advantage over traditional wheelchairs. Changing into lower gears for very adverse terrain such as sandy environment, gives the operators the opportunity to go where they have not had the option to go with conventional wheelchairs. Just changing into higher gears considers allows for movement on pavement and other hard surfaces. The push-bars are removable and may consequently be put away with so that the wheelchair might be moved utilizing traditional hand-to-wheel pushing [20]. This component enables the user to deal with a wide range of landscapes and conditions that are normally restrictive to operators of ordinary wheelchairs and power wheelchairs to be used particularly in open air situations and elements for example forest floors, uneven and harsh terrain. Further, it very well may be utilized to conquer numerous every day snags, for example, curbs and high thresholds [21]. It is advantageous for people with less upper body and arm strength. Accordingly, it accommodates more independent participation in open air exercises, such as hunting, chasing, and so forth.

The review above points toward one thing that current rough terrain wheelchairs do not provide total stability ("not ideal"). Thus, there is still a need to come up with a wheelchair that will prevent the user either toppling backward, forward or sideways.

### **4. Materials and methods**

The design development process entailed coming up with a design that improves the wheelchair stability mechanism, safety precautions as well as reduce powering requirements. Some investigations were done on existing all-terrain stability mechanisms, including the possibility of redesigning the wheels and adjusting the center of gravity for improved stability. The safety of the operator, the machine and influence to the nearby environment were considered among other key parameters. Some effort was made to ensure that the final framework of the wheelchair would address the issues of user safety.

The researchers carried out a study on current wheelchair powering methods as well as noting their efficiencies. Power transmission mechanism was fundamental in achieving speed and power. Some engagement with different disability organizations was done to acquire first hand expectations of users.

The generated design concepts were evaluated and analyzed for suitability for adoption. The chosen concept was further developed to come up with dimensions of the wheelchair, sizing of the wheelchair parts, material selection and detailed drawing of the final design. The detailed drawings were drawn using AutoCAD (2007) and Autodesk Inventor (2017) was used to simulate the strength of the wheelchair frame. The bill of materials was drawn up and the cost estimate and the feasibility of fabricating the prototype unit in the local workshop were done.

#### **5. Design concept generation**

The concepts were formulated based on the ability of the design to meet the objective criteria which were: reliability, maintainability, efficiency, robustness,

**163**

**Figure 5.**

*Cambered rear wheels.*

*Development of a Rough Terrain Wheelchair Design DOI: http://dx.doi.org/10.5772/intechopen.91267*

**5.1 Cambered rear wheels concept**

**5.2 Extended front caster wheels concept**

following sections.

machinability, stability, safety, cost, ease of operation, and weight. Three possible design solutions were generated for the off road wheelchair as explained in the

The wheelchair is operated like a normal conventional wheelchair, by pushing the wheels forward. Sideways stability is affected by wheelchair width (**Figure 5**). The rear wheels are tilted inwards to increase the base width thus stabilizing the chair. A back-caster wheel is fitted to prevent the wheelchair from falling backwards.

The further out to the side of the wheelchair base, the more the chair l resists tipping over sideways. The camber pushes the wheels nearer to the wheelchair user and more in accordance with the operator's forward push stroke, therefore making it simpler to push. This can be particularly useful for ladies, who normally have smaller shoulders yet more extensive hips than men. Additionally, with cambering, traction is improved when navigating slopes. The wheelchair provides more stability, comfortable seating and it is better for overweight people. On the other hand, a wide wheelchair is more difficult to get through narrow pathways as camber increases the width of the wheelchair. Most anti-tip designs restrict the wheelchair's ability to travel over uneven surfaces (such as kerbs or dips). It is also hard to push.

The user propels the wheelchair forward by pushing the rear wheels to the front. A back-caster wheel is fitted to prevent the wheelchair from tipping backwards.

machinability, stability, safety, cost, ease of operation, and weight. Three possible design solutions were generated for the off road wheelchair as explained in the following sections.


**4. Manipulation of adipose tissue and ADSCs**

#### **4.1. Introduction**

adiponectin, angiotensin, cathepsin D, penetraxin, pregnancy zone protein and retinol binding protein, as well as stromal cell-derived growth factor (CXCL12) have been found in the conditioned media of ADSCs differentiating towards the adipocyte lineage. ADSCs secrete also oher different well characterized cytokines (GM-CSF, TGF-β, PGE2, IGF-1) and their release can be modulated by exposure to different agents, such as b-FGF and EGF or inflam‐ matory stimula, like lipopolysaccharide (LPS). The role of these and other factors has been investigated by multiple studies regarding one or more possible applications of ADSCs in the field of regenerative medicine. Brain Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Glial Derived Neurotrophic Factor (GDNF) are thought to be important molecules secreted by ADSCs mediating neurotrophic effects and modulating in animal models of Parkinson Disease the recovery after hypoxic-ischemic injuries. Hepatocyte Growth Factor (HGF) and Vascular Endothelial Growth factor (VEGF) are the most important factors capable of inducing angiogenesis in areas that have undergone ischemic episodes and their importance is particularly relevant in wound healing. In cardiac regeneration, IGF-1 and VEGF mediate respectively an anti-apoptotic and angiogenic action, to which is attributed the capacity of ADSCs to have beneficial effects when transplanted/injected in different animal models of myocardial infarction/failure. In conclusion, most of ADSCs secreted factors act through mechanisms that mediate protection against cell death or, alternatively, induce cell migration and proliferation. Alternatively, they can indirectly act on the targeted cell popula‐ tions: by promoting vascularization they can be indirectly linked to an increase of oxygen and nutrients in the affected areas, which may in turn promote local regenerative processes. Indeed, up to now most reports have focused on a limited set of known factors but it is expected that

other molecules are responsible for the regenerative effects of ADSCs.

The regenerative potential of ADSCs has been related also to their immune-modulatory abilities. ADSCs have been shown to be an immune-privileged site, preventing severe graftversus-host response after transplantation procedures in vitro and in vivo. A concern of fundamental importance is the interplay between ADSCs and the host tissue, with particular focus on the immune system. Several studies have shown that ADSCs can be used either for autologous or allogenic cell transplants: this feature would be a major advantage for the employment of adipose tissue as a source for cell-based therapies. Furthermore ADSCs seem to act also as modulators of the immune system. The allogenic potential of these cells could be explained by the property of ADSCs to decrease the expression of hematopoietic markers and HLA-DR after subsequent passages. In addition, it has been observed that ADSCs only express HLA class I, but not HLA class II molecules: the latter can only be induced in ADSCs after incubation with IFN-γ. Furthermore, several experiments have proved that ADSCs do not stimulate lymphocyte proliferation and they do not elicit a response by Mixed Lymphocyte Reaction (MLR): in addition, they can also inhibit phyohemagglutinin (PHA)-stimulated lymphocyte proliferation. These immune-suppressive effects are likely mediated by soluble factors, among which PGE-2 seems to be the most important. Notably, the secretion of cytokines by ADSCs can be modulated not only by the inflammatory stimulus but also by the

*3.2.3. Immunomodulatory properties of ADSCs*

186 Regenerative Medicine and Tissue Engineering

Human subcutaneous adipose tissue provides an ideal alternative source of autologous pluripotent stem cells showing several advantages compared with other sources. As a matter of fact it is ubiquitous and commonly easily obtainable in large quantity with minimal invasive harvesting procedures or methods (either liposuction aspirates or subcutaneous adipose tissue fragments), limited patient discomfort and minimal ethical considerations: it may be trans‐ planted safely and efficaciously. The abundance of stem cells available enables the direct therapeutic adoption of primary cells without any need for culture expansion. Moreover, adipose tissue is also uniquely expandable: currently available procedures for cell isolation yield a high amount of stem cells with remarkable properties of stable proliferation and potential differentiation in vitro, being attractive candidates for clinical applications offering protocols that may provide alternative therapeutic solutions in cell-based therapies and tissue engineering to repair or regenerate damaged tissues and organs. The technologies for adipose tissue harvesting, processing, and transplantation have substantially evolved in recent years together with appropriate commercial development and with updated refinements and information regarding extraction, isolation, storage, options for cultures, growth and differ‐ entiation, cryopreservation and its effect on survival and proliferation of isolated ADSCs, also related to their adoption in tissue-engineered constructs involving biomaterials and scaffolds. Inconsistencies in literature regarding the handling of ADSCs require more extensive inves‐ tigations and controls, in particular in the in vitro processing and differences between the regenerative properties of freshly-processed heterogeneous stromal vascular fraction cells and of culture-expanded relatively homogeneous ADSCs, or the related risk of complications and possible adverse events. There is a need for stronger evidence of the safety, reproducibility and quality of the ADSCs prior to a more extensive use in clinical applications. As a matter of fact, despite the clinical use of adipose tissue grafts and ADSCs worldwide has dramatically increased, questions concerning the safety and efficacy of these treatments are still opened and currently the use of isolated ADSCs for medical indications in a clinical setting has been approved only in selected cases and few countries.

#### **4.2. Origins and delivery of adipose tissue grafts**

Adipose tissue have been used for long time for reconstructive purposes through fat grafting or autologous fat transfer, a method according to which fat from the patient is removed from one area of the body ad reinserted into the desired recipient location. [9] Fat grafting has shown to be beneficial as a reconstructive and cosmetic procedure for patients with volume losses to soft tissues due to disease, trauma, congenital defects or aging. Even so, outcomes of these techniques are often unpredictable and rates of graft reabsorption may be disappointing. As a matter of fact, fat tissue is re-vascularized at the transplantation site within 48 hours from the surgical procedure, in the meantime being fed by diffused materials from surrounding free plasma. The survival rates of the graft are dependent on size of transplanted fat particles and on surface area from which these cells could re-establish their blood supply. In order to minimize reabsorption, studies have demonstrated the efficacy of less traumatic methods of harvesting, processing and injecting. Microinjection of fat by means of the "lipostructure technique" known also as Coleman's technique has been adopted by many plastic surgeons. [10] This technique distributes fat grafts in small aliquots by meticulous injection through multiple access sites, from which the graft fans out into various subcutaneous layers. The abundance of stem cells obtaina‐ ble in many common procedures, such as liposuction and liposculture, enables their direct therapeutic adoption without any need for culture expansion. [11] Even so, precursor cells can be purified by a variety of processes and enzymatic techniques may be adopted to obtain an ADSC-rich stromal vascular fraction (SVF). This issues are currently investigat‐ ed as adjuvants to free fat transfer in order to increase yield of graft retention (cellassisted lipotransfer). The ADSCs contained in the stromal vascular fraction have been applied clinically as early as 2004 for the treatment of perianal fistulas in Crohn's dis‐ ease. [12] However, it is worth pointing out that, even though harvesting and first processing steps overlap, fat grafts SVF cells and ADSCs represent three different therapeu‐ tic options. Fat grafts are obtained directly after centrifugation of lipoaspirates. They contain predominantly mature adipocytes and are poor in ADSCs. The stromal vascular fraction, as mentioned above, is obtained by digestion with collagenase of the lipoaspi‐ rate sample and a subsequent centrifugation step: its cellular composition is heterogene‐ ous, being rich in ADSCs but containing also circulating blood cells, fibroblasts, pericytes and endothelial cells. The adoption of a pure ADSC population requires plating of the SVF and expansion of the stem cell population and thus, differently from the previous two options, ADSCs cannot be harvested and implanted in a one step-procedure. Even if all these approaches exploit to some extent the regenerative potential of adipose tissue they

are quite different procedures having also different therapeutic indications. Thus, atten‐ tion has to be paid in order to avoid confusion. As for harvesting of ADSCs, several factors related to the patient, such as Body Mass Index (BMI) and age, have been analyzed for their impact on cell viability and number. Results are controversial, there is no evidence of a strong correlation of BMI with stem cells viability, number or size. Instead, there seems to be a negative correlation between age and rates of pre-adipocytes proliferation or differentiation, with higher lipolitic activity in the younger population and lower levels of apoptosis. [13] The body region of the donor site is another important variable patientdependent. The abdomen, according to some studies, seems to be the best harvest site, while medial thigh and knee seem to have the lowest levels of viability of ASDCs. [14] These differences have not been proved in other studies. Effects of infiltration of local anesthetics during harvesting have also been investigated: lidocaine and adrenaline seem to have no effects on adipocyte viability. The method of harvest can affect not only viability of ADSCs but also their level of adhesiveness to extracellular matrix proteins. Standard liposuction allows the harvest of larger volumes of adipose tissue but it might result in up to 90% rate of adipocyte rupture. For this reason this technique is not ideal for fat grafting, while it could be more appropriate for ADSCs harvesting. An equivalent damage to preadipocytes has been measured comparing syringe aspiration with fat surgical excision. It is accepted that a larger cannula diameter at harvest correlates with improved cell viability. Partial purification of lipoaspirate can be carried out in the operatory room. The first step is centrifugation, which separates harvested fat into three layers: infra-natant (lowest layer composed of blood, tissue fluid and local anesthetics), middle portion (mostly composed by fatty tissue) and supra-natant (least dense upper layer including lipids). Infra-natant components can be ejected from the base of the syringe, while supra-natant can be poured off and soaked up using absorbent materials. While this technique is the most practical and today commonly used for fat grafting, it may not produce the best fraction of ADSCs possible. Several studies have been conducted on this issue, revealing that gentle centrifu‐ gation produces the highest cell viability, while long periods of centrifugation lead to isolation of the most proliferative cell type. When comparing decantation, washing and centrifugation, stem cells concentration results greater in washed lipoaspirates and pellets contained at the bottom of the centrifuged samples contain the highest concentration of stem cells.

#### **4.3. Origins and delivery of ADSCs**

of culture-expanded relatively homogeneous ADSCs, or the related risk of complications and possible adverse events. There is a need for stronger evidence of the safety, reproducibility and quality of the ADSCs prior to a more extensive use in clinical applications. As a matter of fact, despite the clinical use of adipose tissue grafts and ADSCs worldwide has dramatically increased, questions concerning the safety and efficacy of these treatments are still opened and currently the use of isolated ADSCs for medical indications in a clinical setting has been

Adipose tissue have been used for long time for reconstructive purposes through fat grafting or autologous fat transfer, a method according to which fat from the patient is removed from one area of the body ad reinserted into the desired recipient location. [9] Fat grafting has shown to be beneficial as a reconstructive and cosmetic procedure for patients with volume losses to soft tissues due to disease, trauma, congenital defects or aging. Even so, outcomes of these techniques are often unpredictable and rates of graft reabsorption may be disappointing. As a matter of fact, fat tissue is re-vascularized at the transplantation site within 48 hours from the surgical procedure, in the meantime being fed by diffused materials from surrounding free plasma. The survival rates of the graft are dependent on size of transplanted fat particles and on surface area from which these cells could re-establish their blood supply. In order to minimize reabsorption, studies have demonstrated the efficacy of less traumatic methods of harvesting, processing and injecting. Microinjection of fat by means of the "lipostructure technique" known also as Coleman's technique has been adopted by many plastic surgeons. [10] This technique distributes fat grafts in small aliquots by meticulous injection through multiple access sites, from which the graft fans out into various subcutaneous layers. The abundance of stem cells obtaina‐ ble in many common procedures, such as liposuction and liposculture, enables their direct therapeutic adoption without any need for culture expansion. [11] Even so, precursor cells can be purified by a variety of processes and enzymatic techniques may be adopted to obtain an ADSC-rich stromal vascular fraction (SVF). This issues are currently investigat‐ ed as adjuvants to free fat transfer in order to increase yield of graft retention (cellassisted lipotransfer). The ADSCs contained in the stromal vascular fraction have been applied clinically as early as 2004 for the treatment of perianal fistulas in Crohn's dis‐ ease. [12] However, it is worth pointing out that, even though harvesting and first processing steps overlap, fat grafts SVF cells and ADSCs represent three different therapeu‐ tic options. Fat grafts are obtained directly after centrifugation of lipoaspirates. They contain predominantly mature adipocytes and are poor in ADSCs. The stromal vascular fraction, as mentioned above, is obtained by digestion with collagenase of the lipoaspi‐ rate sample and a subsequent centrifugation step: its cellular composition is heterogene‐ ous, being rich in ADSCs but containing also circulating blood cells, fibroblasts, pericytes and endothelial cells. The adoption of a pure ADSC population requires plating of the SVF and expansion of the stem cell population and thus, differently from the previous two options, ADSCs cannot be harvested and implanted in a one step-procedure. Even if all these approaches exploit to some extent the regenerative potential of adipose tissue they

approved only in selected cases and few countries.

188 Regenerative Medicine and Tissue Engineering

**4.2. Origins and delivery of adipose tissue grafts**

Embryonic stem cells have an enormous multilineage potential but many ethical and political issues accompany their use. Therefore researchers have directed their attention on pluripotent adult stem cells. Adult stem cells were initially thought to have the differentiation capacity limited to their tissue of origin, however, as already mentioned above, many studies have now demonstrated that stem cells have the capacity to differentiate into cells of mesodermal, endodermal and ectodermal origin. MSCs from the bone marrow show extensive proliferative capacity and a multilineage differentiation potential into several lineages, including osteo‐ blasts, chondrocytes, adipocytes and myoblasts. However, pain, morbidity and low cell numbers upon harvest represent an obstacle to their extensive clinical application. The harvesting of adipose tissue, in comparison, is much less expensive than bone marrow. ADSCs can be isolated both from tissue samples and from lipoaspirate with less invasive procedures and are available in greater quantities (5 x 105 stem cells from 400 to 600 mg tissue). [15] ADSCs can be easily cultured and expanded, retaining their stem cell phenotypes and mesenchymal pluripotency still after several passages, features that make them an ideal source of stem cells for clinical applications.

**•** Isolation and culture of ADSCs

Since Rodbell's description of isolated pre-adipocytes from adipose tissue, a variety of methods have been developed. [16] Today, most laboratories use several common steps to process cells from adipose tissue. These methods include: washing, enzymatic digestion/mechanical disruption, centrifugal separation for isolation of cells which can be used directly, after cryopreservation, or after culture expansion for the generation of ADSCs. Still, despite the extensive use of ADSCs for research purposes, there is no any widely-accepted unique standard protocol for isolating and culturing these cells. For enzymatic digestion most laboratories use collagenases of different subtypes, trypsin, or a mixture of both, at various concentrations with an average incubation time of one hour, at 37°C, in constant shaking. The optimal centrifugation speed is considered to be around 1200g for 5 to 10 minutes. Some additional purification procedures can include filtration through nylon meshes and incubation with an erytrocyte-lysing buffer, usually Krebs Ringer Buffer (KRB) or NH4Cl. This procedure, however, seems to have a negative influence on the growth of ADSCs. Some investigators, after the identification of ADSCs surface immunophenotype, use immune-magnetic beads or flow cytometry to purify the stem cell population directly from the heterogeneous sample, using the CD34+ antigen. The most used culture medium are α-Modified Eagle's Medium (α-MEM), or Dulbecco's Modified Eagle's Medium (DMEM), after addition of fetal bovine/calf serum, (FBS/FCS), L-glutamine, penicillin and streptomycin.

**•** Cryopreservation of ADSCs

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. [17] Cryopreservation is regarded as a promising technique and many studies have focused on this procedure. Other protocols investigated drying (anhydrobiosis) and freeze drying (lyophilization). The majority of in vitro studies agree that cryopreservation of adipocytes in liquid nitrogen, preferably using a set cooling and re-warming protocol, provides the lowest damage to cell viability. These results have been replicated in vivo (murine models) showing that grafts frozen in liquid nitrogen and stored at -35°C had a similar viability and histology compared to fresh tissue: in addition, this method obtained better results than freeze drying and immersion in glycerol. Recently, in order to increase the yield of adipose-derived stem cells post-thawing, the use of cryoprotective agents, such as dimethyl sulphoxide (DMSO) has been examined: samples frozen with DMSO achieved better outcomes than unprotected ones. Thus, cryoprotective agents are now considered as an essential part of any cryopreservation protocol aiming to provide appropriate conditions for the survival of ADSCs and adipocytes.

#### **4.4. Safety concerns**

harvesting of adipose tissue, in comparison, is much less expensive than bone marrow. ADSCs can be isolated both from tissue samples and from lipoaspirate with less invasive procedures and are available in greater quantities (5 x 105 stem cells from 400 to 600 mg tissue). [15] ADSCs can be easily cultured and expanded, retaining their stem cell phenotypes and mesenchymal pluripotency still after several passages, features that make them an ideal source of stem cells

Since Rodbell's description of isolated pre-adipocytes from adipose tissue, a variety of methods have been developed. [16] Today, most laboratories use several common steps to process cells from adipose tissue. These methods include: washing, enzymatic digestion/mechanical disruption, centrifugal separation for isolation of cells which can be used directly, after cryopreservation, or after culture expansion for the generation of ADSCs. Still, despite the extensive use of ADSCs for research purposes, there is no any widely-accepted unique standard protocol for isolating and culturing these cells. For enzymatic digestion most laboratories use collagenases of different subtypes, trypsin, or a mixture of both, at various concentrations with an average incubation time of one hour, at 37°C, in constant shaking. The optimal centrifugation speed is considered to be around 1200g for 5 to 10 minutes. Some additional purification procedures can include filtration through nylon meshes and incubation with an erytrocyte-lysing buffer, usually Krebs Ringer Buffer (KRB) or NH4Cl. This procedure, however, seems to have a negative influence on the growth of ADSCs. Some investigators, after the identification of ADSCs surface immunophenotype, use immune-magnetic beads or flow cytometry to purify the stem cell population directly from the heterogeneous sample, using the CD34+ antigen. The most used culture medium are α-Modified Eagle's Medium (α-MEM), or Dulbecco's Modified Eagle's Medium (DMEM), after addition of fetal bovine/calf

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. [17] Cryopreservation is regarded as a promising technique and many studies have focused on this procedure. Other protocols investigated drying (anhydrobiosis) and freeze drying (lyophilization). The majority of in vitro studies agree that cryopreservation of adipocytes in liquid nitrogen, preferably using a set cooling and re-warming protocol, provides the lowest damage to cell viability. These results have been replicated in vivo (murine models) showing that grafts frozen in liquid nitrogen and stored at -35°C had a similar viability and histology compared to fresh tissue: in addition, this method obtained better results than freeze drying and immersion in glycerol. Recently, in order to increase the yield of adipose-derived stem cells post-thawing, the use of cryoprotective agents, such as dimethyl sulphoxide (DMSO) has been examined: samples frozen with DMSO achieved better outcomes than unprotected ones. Thus, cryoprotective agents are now considered as an essential part of any cryopreservation protocol aiming to

for clinical applications.

**•** Isolation and culture of ADSCs

190 Regenerative Medicine and Tissue Engineering

**•** Cryopreservation of ADSCs

serum, (FBS/FCS), L-glutamine, penicillin and streptomycin.

provide appropriate conditions for the survival of ADSCs and adipocytes.

Inconsistencies in literature regarding the handling of ADSCs require more extensive inves‐ tigations and controls. In particular, a focus should be placed on in vitro processing as well as differences between the regenerative properties of freshly-processed heterogeneous adipose cells and those of culture-expanded relatively homogeneous ADSCs. Related risks of compli‐ cations and possible adverse events like fat necrosis, seromas, oncological recurrences, should be accurately considered. In addition, adiponectin is implicated in the pathogenesis of insulinresistant states, such as obesity and diabetes type 2. In particular, several studies reported that differentiated WAT cells and WAT resident progenitors may promote cancer growth and metastasis by means of a variety of different mechanisms (endocrine, paracrine, autocrine interactions). The main cellular component of WAT are adipocytes, the large cells accumulat‐ ing tryglicerides in lipid droplets. In particular, in conditions like obesity, adipocytes in WAT may eventually became under oxygenated, leading to hypoxia, increased oxidative stress, recruitment of inflammatory leukocytes and eventually fibrosis. In recent experimental models, some adipokines showed to be able to promote tumor growth along with fatty acids released by adipocytes. High levels of adiponectin have been associated with the development of endometrial carcinoma and breast cancer. Leptin has been identified in regulation of cell proliferation and neo-vascularization in malignant and normal cells of different origins, including lung, gastric, colonic, kidney, leukemic, hematopoietic and epithelial cells. Notably, these molecules can enhance proliferation and survival of malignant cells and/or of tumor vasculature. So far, studies investigating the role of WAT in cancer have predominantly focused on pro-tumorigenic effect of ADSCs. In fact the increased proliferation and survivor of malignant cells may result from the engagement of perivascular ADSCs into angiogenesis and vascular maturation, resulting in improved tumor blood perfusion. Cytokines such as adiponectin, leptin, interleukin-6, and TNF alfa seem to be responsible for a chronic low-grade inflammation. Furthermore, mesenchymal cells are known to suppress the activation of Tkiller cells: this finding suggests that also ADSCs may help tumors to evade the host immune response. Thus, adipocytes may be able to produce adipokines and several secretions which could potentially induce cancer reappearance by "fueling" dormant breast cancer cells in tumor bed true "tumor-stroma interaction": even so, up to now, especially for grafting of adipose tissue after breast cancer treatment, there is no strong clinical evidence or international agreement on this topic. [18-19] Depending on country, the safety of adipose tissue grafting is still a controversial issue. In 2009, the American Society of Plastic surgeons Fat Graft task Force concluded that no reliable studies could confirm definitely the oncologic safety of lipofilling in breast cancer patients. A more accurate point of view is provided by a large multicentric observational study on adipose tissue grafting in patients previously affected by breast cancer: considered parameters included the complication rate of the technique, the risk of modification of mammography and a rigorous long-term clinical/instrumental follow-up. [20] At the moment no studies on the effects of lipotransfer on human cancer breast cells in vivo are available. We cannot provide the definitive proof of the safety of lipofilling in terms of cancer recurrence or distant metastasis, but until then, should be performed in experienced hands, and a cautious oncologic follow-up protocol is advised.


**2. Conclusions**

The eight papers published in this collection represent a non-exhaustive list of the most recent leading topics in the fields of statistical seismology and pattern recognition applied to the spatiotemporal evolution of seismicity. Given the complexity of the topic, a rigorous methodology to the approach to the study of spatiotemporal properties of seismicity is needed. Descriptions of methods and implementations of modern and advanced methodologies from multidisciplinary approaches are needed for a shared understanding of the topic and as a starting point for new research. Therefore, this collection stands as an important starting point to outline the issues of interest as well as new challenges in this field.

**Conflicts of Interest:** The authors declare no conflict of interest.