**3.1 Factors affecting runoff and soil loss**

Table 2 shows the value of soil loss for the three soils. Soil loss decreased with increasing peat contents for all combinations of soil type, rainfall duration and compaction effort. Soil loss also decreased with increasing compaction effort and increased with increasing rainfall duration. Soil loss was consistently highest in the sandy soil, intermediate in the clay soil and lowest in the clay loam soil. Table 3 shows that cumulative runoff increased with increasing rainfall duration and compaction levels but decreased with increasing peat content. Runoff was highest in the clay, followed by clay loam and lowest in the sandy loam soil.

aValues are means of two replicates.

Table 2. Soil lossa (kg) for three soils with and without peat compacted and exposed to four rainfall durations

Table 4 summarizes the mean values of cumulative runoff and soil loss for the different experimental factors. Mean runoff increased with increasing clay content, compaction effort and rainfall duration but decreased with increasing peat content. For soil loss, mean values of soil loss varied from 1.36 kg in the sandy soil to 0.95 kg in the clay loam soil. The analysis of variance (Table 5) showed that the main effects of soil type, peat content, compaction effort and rainfall duration were all significant (P = 0.001) for the two parameters as depicted by the 'F' values. Rainfall duration was the most important factor that affected the two parameters. In addition, the most significant interaction that affected the two parameters was that between soil type and rainfall duration which was significant at 0.1% level. This was followed by the interactions between compaction effort and rainfall duration and between peat content and rainfall duration in that order for the two parameters. These interactions and the main effects will be described below.

Soil Loss-Rainfall Duration Relations as Affected

matter originating from grass.

**3.1.2 Soil type**

runoff rates.

by Peat Content, Soil Type and Compaction Effort 185

1987). Although soil strength is known to increase the resistance of soils to erosion (Rachman et al., 2003; Wuddivira, 2008), the present results further confirm that peat reduces soil loss by increasing infiltration and decreasing runoff during rainfall rather than by strengthening the soil as was obtained for other organic materials like farmyard manure by Wuddivira et al. (2009) and Ekwue et al. (2009). Runoff and soil erosion are important not only for soil and water conservation, but also to reduce nutrient discharge with runoff (Bjorneberg et al., 2000). This means that the reduction of surface runoff and soil erosion by peat will not only aid soil conservation, but also reduce loss of plant nutrients in the soil. The interaction between peat content and rainfall duration (Fig. 3) shows that the effect of peat on soil loss increases with rainfall duration. A similar interaction was reported for soil detachment by Ekwue (1991) in connection to organic

The main effect of soil type was the second most important of all the experimental factors on soil loss and runoff (Table 5). This was almost like the previous paper by Ekwue and Harrilal (2010) where it was the most important factor. This may be as a result of the same process involved in the raindrop erosion measured in the two studies. In a previous study by Ekwue et al. (2009), soil type was the least important factor and this may be because this study measured wash erosion by surface runoff, while the present and the Ekwue and Harrilal (2009) examined the total erosion process of transport of soil particles detached by raindrop which is commonly referred as interrill erosion (Levy et al., 2001). Piarco sandy loam had the largest quantity of mean soil loss and this has been consistent in the with these two recent studies. Although this soil had the least runoff as the rainfall duration increased (Tables 3 and 4), its low percentage clay content (18.1%, Table 1) decreased the soil strength (Table 6), thus decreasing the soil's ability to increase the cohesiveness of the particles. The larger size of the sandy loam soil led to greater presence of large pores which enhanced infiltration. Results show that this led to lower surface runoff. However, decreased soil cohesiveness, the presence of more loose detached sand particles ensured that the soil had greater soil loss than the other soils despite its maintenance of high infiltration and low

With the 46.3% clay content of the Talparo clay soil (Table 1), the soil cohesiveness and soil strength (Table 6) was the greatest as was measured by the soil penetration test. However, due to the low infiltration and high runoff rates recorded for this soil, the Talparo soil still had more soil loss than the Maracas clay loam soil. Although there was little raindrop detachment, due to the quantity of clay in the soils composition, the Talparo clay experienced the lowest infiltration and greatest amount of surface runoff of the three soils (Tables 3 and 4). This quantity of surface runoff was able to produce soil erosion, and as the rainfall duration increased, so too did the runoff and also the quantity of erosion. However, its high clay composition and high soil strength ensured that there was less erosion than the Piarco sandy loam. The Maracas clay loam had the least soil loss. This was mainly its evenly balanced composition of sand, silt and clay (Table 1). The Maracas clay loam had 30.6% clay content which was enough to produce good cohesive nature and soil strength for the particles so as to minimize splash erosion and easy detachment. The sand and silt composition allowed the soil to have steady infiltration throughout the testing period and leading to runoff which was closer to that recorded for the sandy loam soil (Table 4). These


**Author details**

*Sustainability Concept in Developing Countries*

countries.

**Acknowledgements**

improved the manuscript.

The impact of climate change on access to freshwater will be highly visible under the SSP2 scenario, where the population growth is higher than the other two scenarios and no dramatic improvements in infrastructure are taken. However, the SSP1 scenario seems to handle in the best possible way the climate change uncertainty by having low population growth and increased access to water due to investments, while the SSP1 climate change impacts would be closer to the SSP2, as although population increase with lower to SSP2 rates, the investments in fossil fuels will increase CO2 emissions, making the climate change impacts more extreme. In addition to the above, improving standards of living would increase further the water requirements of the citizens widening the gap between demand and supply in all three scenarios. Water scarce will increase competition over water supply driving so the political and societal instability within and between the

Energy and food sectors will also be affected by the climate change as they are both based on water access. Hence, under SSP2 and SSP5, energy and food sectors will be significantly disturbed enhancing potential conflicts and wars between the countries as a result of social pressure. SSP1 seems to be the only case, where the adverse effects of climate change are mitigated as a result of the increased investments in water access and lower use of carbon-intensive technologies, as the oil

This study has potential limitations. The projections taken place in the model are based on international organisations and studies. Moreover, the lack of data constituted the necessary use of proxies of other similar cases, simplifying so, the individuality of each country. Last but not least, the long-term time horizon is accompanied with great uncertainty. The projections are therefore subject to biases and confounding that may have influenced the results of this section. The breakdown in shorter terms of the indicators analysed in this study could give a more

This work is supported by the Decision Analytic Framework to explore the water-energy-food nexus in complex transboundary water resource systems of fast developing countries (DAFNE) project, which has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 690268. Comments from two anonymous reviewers greatly

price is high, and renewable energy gets competitive against fossil fuels.

precise understanding of the future status quo in the two CS.

**50**

Phoebe Koundouri1,2,3,4\* and Lydia Papadaki1,2,5

1 Athens University of Economics and Business, Greece

2 EIT Climate-KIC Hub, Greece

3 United Nations Sustainable Development Network, Greece

4 European Association of Environmental and Natural Resource Economists, Italy

5 UN SDSN, Greece

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 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.


**Introduction**

nerve root avulsions, particularly when all or almost all the brachial plexus nerve roots are avulsed. Some attempts to reimplant avulsed roots have been tried, but clinical results in

This book offers a new look at the field of brachial plexus injuries, taking advantage of the vast experience and knowledge of great figures who treated these dreadful conditions over many years. We hope the reader enjoys the book as much as the authors did writing it.

> **Vicente Vanaclocha** Assistant Professor

**Nieves Sáiz-Sapena** University of Valencia Valencia, Spain

Valencia, Spain

Department of Neurosurgery Hospital General Universitario

humans have been dismal.

VIII Preface

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: Brachial Plexus Injuries - Past,**

Although they have a low incidence [1–3], brachial plexus injuries continue to be a cause of serious disability [4]. Their victims are mostly young people in the middle of their lives or newborns, always with worrisome long-term consequences [2–4]. Unfortunately brachial plexus lesions can also be the result of iatrogenic injuries [5]. The quality of life of those

Nowadays, road accidents in young people continue to be the most common cause, particularly when the victim is thrown in the air and lands on the shoulder [2, 5, 10]. This is particularly common in developing Third World countries, where people have to recourse to the motorcycle for their daily transportation [1, 11–14], as a car is an option outside their reach. Other causes are wars [15–17] and assaults [18, 19]. The incidence is higher in males than in females [1, 2, 13, 20], probably related to the highest aggressiveness and violent behavior in the former [21]. In newborns brachial plexus injuries are usually due to problems during vaginal delivery [3, 22], particularly in case of a macrosomic fetus [23, 24], common in diabetic mothers [25]. In the developed countries, the fear of unpleasant medicolegal consequences in case of an obstetric brachial plexus injury has induced a significant increase in the proportion of cesarean section deliveries [26, 27]. The incidence of iatrogenic brachial plexus lesions unfortunately continues to be stable overtime with no signs of reduction [5, 28–30]. These iatrogenic lesions are induced while performing lymph node biopsy [5, 31, 32], vessel catheterization [33, 34], on applying radiotherapy in the treatment of cancer [35, 36] repairing upper limb bone fractures [37, 38], in programmed orthopedic procedures [39, 40], due to inadequate patient positioning [41, 42] or when restraining aggressive patients [43]. Preventing these iatrogenic injuries is particularly important, not only because they might lead to ugly medicolegal consequences [28] but because of our motto "primum non nocere"

affected is devastated, with high personal [4, 6], familial [7], and societal costs [8, 9].

**Introductory Chapter: Brachial Plexus Injuries - Past,**

© 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.

DOI: 10.5772/intechopen.81675

**Present, and Future**

**Present, and Future**

Vicente Vanaclocha and Nieves Saiz-Sapena

Additional information is available at the end of the chapter

Vicente Vanaclocha and Nieves Saiz-Sapena

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81675

**1. Introduction**

### **Introductory Chapter: Brachial Plexus Injuries - Past, Present, and Future Introductory Chapter: Brachial Plexus Injuries - Past, Present, and Future**

DOI: 10.5772/intechopen.81675

Vicente Vanaclocha and Nieves Saiz-Sapena Vicente Vanaclocha and Nieves Saiz-Sapena Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81675


**8. Complications**

Full arch rehabilitation treatments with dental implants can have complications and failures. In general, complications may be related to the patient's systemic compromise, increased functional demand, surgical technique, post-operative care, design and type of prosthesis, etc. The overall success rate for dental implants is between 90–100% according to the study [48].

The most frequent prosthetic complications after the placement of an implantsupported prosthesis are: mucositis, loosening or fracture of the abutment screw or prosthetic components, and fracture of the acrylic or porcelain structure. Although most complications resolve favorably in follow-up appointments, it is essential to establish an adequate surgical and prosthetic management protocol to achieve predictable and successful long-term results [49, 50].


*<IP_ADDRESS>. Other new methods*

#### **Bacteriophages**

more than 300 genes and employed 70 high-throughput screening campaigns over a period

It must be admitted that target-based genomic approach has not yielded satisfactory results, nevertheless, retooled target-based strategies can still play an important role in discovery process. Most antibiotic targets are limited to peptidoglycan synthesis, ribosomal protein synthesis, folate synthesis, and nucleic acid synthesis and topoisomerization. In the future we could continue to discover new antibiotics for these old targets through improvement of the existing scaffolds or even finding new scaffolds. For instance, Lipid II is a membraneanchored cell-wall precursor that is essential for bacterial cell-wall biosynthesis; it is not on‐ ly classical target for several old antibacterial classes, but is also targeted by the new antibiotics, such as lantibiotics, mannopeptimycins and ramoplanin [71]. Grouping targets by a common inhibitor scaffold rather than by function may lead to new targets; and as

mentioned above, insights from outside the antibiotic arena are also important [59].

rum to obviate the essentiality of FAS II enzymes in vivo [72].

Compared with the fruitless target-based genomic approach, traditional whole-cell assays are more effective in antibiotic discovery. Just because it is not necessary to worry about cell permeability of a novel scaffold in the development process if whole-cell assays are used. As most of the existing libraries have already been used to screen for antibacterial drugs, libra‐ ries with new chemical diversity are extremely important in this approach. Sometimes, look for libraries that don't belong to antibacterial development areas may be useful. In fact, most pharmaceutical companies of other therapeutic areas have invested considerable resources in synthesizing small molecule libraries [59]. Candidates with a strong hit in a whole-cell an‐ tibacterial assay should be tested in the right animal model early in development, because In vitro experiment results are not always reliable. For example, Antimicrobial drug target type II fatty acid synthesis (FASII) is reported to be essential for their efficacy against infections caused by multiresistant Gram-positive bacteria. But another study showed that Streptococ‐ cus agalactiae and S. aureus could take up sufficient unsaturated fatty acids from human se‐

Antibacterial spectrum is a major consideration when selecting a target for lead optimiza‐ tion. Permeability and target distribution determine the pectrum [73]. That is to say, the drug candidates should possess two properties at the same time: one is penetrating the cell and evading efflux pump systems, another is retaining potent activity at the molecular tar‐ gets. However, since almost all targets of the antibacterials in clinical use are present in all bacteria, the antibacterial drug spectra are determined largely by the ability of permeability. Therefore, some compounds are just Gram-positive organism-selective and have no effect against Gram-negative pathogens which have a second membrane acting as a permeability barrier [74; 75]. Efflux pump inhibitors (EPIs) have been explored for broadening the anti‐

of 7 years, but unfortunately did not create a clinical used antibacterial [70].

*<IP_ADDRESS>. New targets*

300 Drug Discovery

*<IP_ADDRESS>. Forward is back*

*<IP_ADDRESS>. Focus on spectrum*

Bacteriophages and their fragments could kill the bacteria. They have been developed as an‐ tibacterials in humans, poultry and cattle industries, aquaculture and sewage treatment. This approach has novel mechanism of action that is completely different from current anti‐ microbials, but the problems are that quality control and standardization are difficult. Phage lysins, which are produced late in the viral infection cycle, can bind to cell wall peptidogly‐ can and rapidly induce Gram-positive bacteria lysis [80]. The sequencing of phages genomes may identify more proteins suitable for novel antibacterials [81; 82].

Other methods to find new drugs could be modulating immunity, developing monoclonal antibody for specific bacteria, designing antibacterial peptides (including antimicrobial pep‐ tides and compounds from animals and plants, the natural lipopeptides of bacteria and Fun‐ gi [83; 84]), and so on.


**GAME THEORY - APPLICATIONS IN LOGISTICS AND ECONOMY**

#### **Game Theory - Applications in Logistics and Economy**

http://dx.doi.org/10.5772/intechopen.72482 Edited by Danijela Tuljak-Suban

#### **Contributors**

Torkel Bjørnskau, Baseem Khan, Jingtao Shi, Jianbo Zhang, Alain Chateauneuf, Vassili Vergopoulos, Gershon Wolansky, Penelope Hernandez, Adriana Alventosa, Deyu Lin, Quan Wang, Pengfei Yang

#### **© The Editor(s) and the Author(s) 2018**

The rights of the editor(s) and the author(s) have been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights to the book as a whole are reserved by INTECHOPEN LIMITED. The book as a whole (compilation) cannot be reproduced, distributed or used for commercial or non-commercial purposes without INTECHOPEN LIMITED's written permission. Enquiries concerning the use of the book should be directed to INTECHOPEN LIMITED rights and permissions department (permissions@intechopen.com). Violations are liable to prosecution under the governing Copyright Law.

Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html.

#### **Notice**

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in London, United Kingdom, 2018 by IntechOpen eBook (PDF) Published by IntechOpen, 2019 IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, The Shard, 25th floor, 32 London Bridge Street London, SE19SG – United Kingdom Printed in Croatia

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library

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Game Theory - Applications in Logistics and Economy Edited by Danijela Tuljak-Suban p. cm. Print ISBN 978-1-78923-897-6 Online ISBN 978-1-78923-898-3 eBook (PDF) ISBN 978-1-83881-691-9


**2. Association between Nut Consumption and Cardiometabolic Disorders**

Globally, cardiometabolic diseases including type 2 diabetes mellitus (T2DM), coronary heart disease (CHD), coronary artery disease (CAD), and stroke are leading causes of morbidity and mortality [10]. Most of these diseases could be prevented by changing behavioral risk factors such as suboptimal diet [11].

Tree nuts and peanuts, because of their healthy antioxidant biochemical profile, can improve the lipid profile, increase insulin sensitivity and metabolism, and favorably influence other cardiometabolic risk factors [12].


**3. Biomechanics of heart valve prostheses**

During their motion natural valve leaflets imposed to different mechanical loading and corresponding stress fields. Mechanical bending is developed during opening especially at sites near their attachments to valvular ring, while shear stress is gradually developed at their sides faced blood flow. A near parabolic velocity profile is produced in fully developed central axial blood flow across the valve which fast stabilizes leaflets in a position parallel to its axis. Upon starting of closing phase of the valves a reverse leaflet movement is

Prosthetic Aortic Valves: A Surgical and Bioengineering Approach 69

**ΕΗ (MPa)**

**COLLAGEN MODULUS**

BPN BPG

0,1 0,5 1 5 10 20 **Cyclic Frequency (Hz)**

Fig. 7. (a) Typical stress-strain diagram of a pericardial tissue under uniaxial tensile cyclic loading 0.1 Hz demonstrating non linear mechanical behaviour. EH is the characteristic high

Pathology of natural heart valve calcification causes valve dysfunction (stenosis, regurgitation, tissue rupture). A lot of possible aetiologies and mechanisms seem to be implemented in its initiation and development in human body tissues. Chronic pathologies, infections, metabolism, long term drag therapies and age related tissue degeneration, as well biochemical compounds involved in the structure of implanted biomaterials may contribute in different ways in growth of different types of calcium phosphate crystals from ions of electrolytes diluted in biological fluids, that finally deposited in valve tissue structure (Gross, 2003; Schoen & Levy, 2005). However, despite the complicated aetiologies and mechanisms of tissue calcification, crystal growth is basically a physicochemical process of calcium and phosphate ion crystallization under certain physicochemical conditions that

Calcium phosphate deposition on implants may result from the presence of high phosphorus levels in the biological fluids in contact with the implanted surface. Due to their very low solubility products, a number of phosphate scale minerals may form in aqueous supersaturated solutions. In the order of decreasing solubility, they are listed in the following table 2. At high solution supersaturations it is possible that a number of precursor phases may be formed, depending on the solution pH, which finally transform into the thermodynamically more stable HAP, in accordance to Ostwald's rule of stages which predicts that the least stable phase having the highest solubility is formed preferentially during a stepwise precipitation process. It is well established that kinetic factors may be more important in determining the nature and, hence, the characteristics of the solid deposits formed during the precipitation process than the respective equilibrium consideration complications that may arise from the formation of mixed solid phases,

(collagen) modulus, the tangential modulus of the second linear phase. (b) Collagen modulus of fresh natural (BPN) and glutaraldehyde treated (BPG) bovine pericardial tissue

(a) (b)

**EH**

(mean ± SE) at different cyclic loading rates

0 0,05 0,1 0,15 0,2 0,25 **Strain ΔL/L0**

may be satisfied during the function of a living organism.

**4.1 Biomineralization: Physicochemical background**

caused of the overgrowth of one crystalline phase over another.

**4. Heart valve calcification**

**BPG 0,1 Hz #DHML5B**

0 0,2 0,4 0,6 0,8 1 1,2

**Stress MPa**

performed with changing stress fields applied to their structure, till the closing position during which these membranous tissues, supported at their sites of attachment on valvular rings and their coaptation areas, are imposed in surface tensile loading, while obstructing blood backflow and big pressure differences between their sides. As a result of all these different mechanical loadings imposed, leaflet tissue is remodelled in a multilaminate, anisotropic (see diagram in figure 6) reinforced composite biomaterial, demonstrating a structure of multilayered 3D collagen and elastin fibrous networks, integrated and moved into an amorphous organic protein matrix, filled with cells and extracellular water, electrolytes and soluble proteins of low molecular weight. This tissue structure, different in individual valve leaflets, is specifically able to function at the specific anatomic position. Been in a different position, like, for example, from pulmonary artery to aorta as in Ross procedure, tissue remodelling started to make the pulmonary valve leaflets able to resist higher mechanical loading in aortic position, compared with that of pulmonary circulation.

Fig. 6. Diagram (from measurements in histological sections) of the laminate and total leaflet thickness changed under different pressure levels applied during their fixation with glutaraldehyde. In dynamic mode, the valve was under normal function during fixation. Anisotropic deformation of the different tissue laminates is demonstrated as pressure increases

Natural heart valve leaflets exhibit non linear viscoelastic mechanical behaviour under mechanical loading (figure 7a). A similar mechanical behaviour is demonstrated by tissue heart valves of all species, as well all membranous soft tissues. Chemical modification of bioprosthetic heart valves, needed for removing antigenic factors and stabilization against enzyme biodegradation, resulted in significant stiffening of leaflet tissue compared with its natural state, as demonstrated by increase of high (collagen) modulus (figure 7b).

Other viscoelastic mechanical parameters of valve leaflet tissue, like low (elastin) modulus (the slope of the first linear part of the loading stress-strain curve (fig. 7a)) and relaxation index may also changed, although hysteresis, another viscoelastic characteristic of membranous soft tissues (defined as the ratio of dissipated to the loading energy in every loading-unloading cycle) demonstrating energy dissipation inside the tissue during cyclic deformation, measured at 20/35% of loading energy depending on cyclic frequency seems to be unchanged after chemical modification.

Fig. 7. (a) Typical stress-strain diagram of a pericardial tissue under uniaxial tensile cyclic loading 0.1 Hz demonstrating non linear mechanical behaviour. EH is the characteristic high (collagen) modulus, the tangential modulus of the second linear phase. (b) Collagen modulus of fresh natural (BPN) and glutaraldehyde treated (BPG) bovine pericardial tissue (mean ± SE) at different cyclic loading rates

## **4. Heart valve calcification**

68 Aortic Valve Surgery

performed with changing stress fields applied to their structure, till the closing position during which these membranous tissues, supported at their sites of attachment on valvular rings and their coaptation areas, are imposed in surface tensile loading, while obstructing blood backflow and big pressure differences between their sides. As a result of all these different mechanical loadings imposed, leaflet tissue is remodelled in a multilaminate, anisotropic (see diagram in figure 6) reinforced composite biomaterial, demonstrating a structure of multilayered 3D collagen and elastin fibrous networks, integrated and moved into an amorphous organic protein matrix, filled with cells and extracellular water, electrolytes and soluble proteins of low molecular weight. This tissue structure, different in individual valve leaflets, is specifically able to function at the specific anatomic position. Been in a different position, like, for example, from pulmonary artery to aorta as in Ross procedure, tissue remodelling started to make the pulmonary valve leaflets able to resist higher mechanical loading in aortic position, compared with that of pulmonary circulation.

2 mm Hg 5 mm Hg 16 mm Hg 45 mm Hg 90 mm Hg dynamic

Fig. 6. Diagram (from measurements in histological sections) of the laminate and total leaflet

Other viscoelastic mechanical parameters of valve leaflet tissue, like low (elastin) modulus (the slope of the first linear part of the loading stress-strain curve (fig. 7a)) and relaxation index may also changed, although hysteresis, another viscoelastic characteristic of membranous soft tissues (defined as the ratio of dissipated to the loading energy in every loading-unloading cycle) demonstrating energy dissipation inside the tissue during cyclic deformation, measured at 20/35% of loading energy depending on cyclic frequency seems

thickness changed under different pressure levels applied during their fixation with glutaraldehyde. In dynamic mode, the valve was under normal function during fixation. Anisotropic deformation of the different tissue laminates is demonstrated as pressure increases Natural heart valve leaflets exhibit non linear viscoelastic mechanical behaviour under mechanical loading (figure 7a). A similar mechanical behaviour is demonstrated by tissue heart valves of all species, as well all membranous soft tissues. Chemical modification of bioprosthetic heart valves, needed for removing antigenic factors and stabilization against enzyme biodegradation, resulted in significant stiffening of leaflet tissue compared with its

natural state, as demonstrated by increase of high (collagen) modulus (figure 7b).

**Laminate and total thickness of aortic valve leaflet**

**(Circumferencial)** Fibrosa

Spongiosa Ventricularis

**Fixation pressure**

to be unchanged after chemical modification.

**Thickness (1/100 mm)**

Pathology of natural heart valve calcification causes valve dysfunction (stenosis, regurgitation, tissue rupture). A lot of possible aetiologies and mechanisms seem to be implemented in its initiation and development in human body tissues. Chronic pathologies, infections, metabolism, long term drag therapies and age related tissue degeneration, as well biochemical compounds involved in the structure of implanted biomaterials may contribute in different ways in growth of different types of calcium phosphate crystals from ions of electrolytes diluted in biological fluids, that finally deposited in valve tissue structure (Gross, 2003; Schoen & Levy, 2005). However, despite the complicated aetiologies and mechanisms of tissue calcification, crystal growth is basically a physicochemical process of calcium and phosphate ion crystallization under certain physicochemical conditions that may be satisfied during the function of a living organism.

#### **4.1 Biomineralization: Physicochemical background**

Calcium phosphate deposition on implants may result from the presence of high phosphorus levels in the biological fluids in contact with the implanted surface. Due to their very low solubility products, a number of phosphate scale minerals may form in aqueous supersaturated solutions. In the order of decreasing solubility, they are listed in the following table 2. At high solution supersaturations it is possible that a number of precursor phases may be formed, depending on the solution pH, which finally transform into the thermodynamically more stable HAP, in accordance to Ostwald's rule of stages which predicts that the least stable phase having the highest solubility is formed preferentially during a stepwise precipitation process. It is well established that kinetic factors may be more important in determining the nature and, hence, the characteristics of the solid deposits formed during the precipitation process than the respective equilibrium consideration complications that may arise from the formation of mixed solid phases, caused of the overgrowth of one crystalline phase over another.

Prosthetic Aortic Valves: A Surgical and Bioengineering Approach 71

DCPD

OCP

HAP

TCP

1/

ν

∞ *s* (2)

(1)

*o*

refer to solution and equilibrium conditions respectively, *α* denote

4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

pH

Fig. 8. Solubility isothems for calcium phosphate phases at 25°C, Ionic strength 0.1M NaCl Spontaneous precipitation investigations have been faced with the problem that at high supersaturations the ACP forming initially results in a rapid decrease of the calcium and phosphate ion activities which fall bellow the values needed for the spontaneous formation of other calcium phosphate phases. A careful analysis of the precipitation of calcium phosphate over the pH range 5.0-8.0 suggested that the limiting ion activity product for ACP was constant as expected for a discrete mineral phase. Moreover it has also been shown that the presence of magnesium in the precipitation medium promoted the formation

The driving force for the formation of a solid phase in a continuous aqueous phase is the solution supersaturation which can be developed in many ways including temperature fluctuation, pH change, mixing of incompatible waters, increasing the concentration by evaporation or solid dissolution etc. Although supersaturation is the driving force for the formation of a salt, the exact values in which precipitation occurs are quite different from salt to salt and as a rule, the degree of supersaturation needed for a sparingly soluble salt is orders of magnitude higher than the corresponding value for a soluble salt. For sparingly

of DCPD at the expense of ACP (Feenstra & de Bruyn, 1979; Posner et al., 1984).

( ) ( )

ν

*a IP S a a K* α

+ − α

*m*

( ) ( )

Δμ = − μ μ

ν

*m a M s s*

+ −

*s M A*

the activities of the respective ions and *ν++ν-*= *ν*. *IP* and *o Ks* are the ion products in the

The fundamental driving force for the formation of a salt from a supersaturated solution is the difference in chemical potential of the solute in the supersaturated solution from the

+ − Α + − ∞ ∞ ⎧ ⎫ ⎪ ⎪ ⎛ ⎞ = ⎨ ⎬ = ⎜ ⎟ ⎜ ⎟ ⎪ ⎪ ⎝ ⎠ ⎩ ⎭

ν

ν

soluble salts Mν+Aν- the supersaturation ratio, *S*, is defined as:

∞

supersaturated solution and at equilibrium respectively.

log ( Ca

where subscripts *s* and

respective value at equilibrium:

t / M )

0.0

Table 2. Calcium phosphate crystalline phases, formulae and corresponding thermodynamic solubility products. \*(Hench & Wilson, 1991), \*\*(Eanes et al., 1965), \*\*\*(LeGeros et al., 1975), \*\*\*\*(Betts & Posner, 1974)

The tendency for a particular calcium phosphate phase to form in supersaturated aqueous media may be determined from the solubility phase diagrams such as the diagram shown in figure 8. It has been reported that when calcium phosphate is precipitated from highly supersaturated solutions forms an unstable precursor phase. This phase is characterized by the absence of peaks in the powder x-ray diffraction pattern and is known as the amorphous calcium phosphate (ACP). The composition of ACP appears to depend upon the precipitation conditions and is usually formed in supersaturated solutions at pH >7.0 (Betts & Posner, 1974; Eanes et al., 1965; LeGeros et al., 1975; Newesely, 1966). In slightly acidic calcium phosphate solutions the monoclinic DCPD is formed (Bets & Posner, 1974; Brown & Lehr, 1959). OCP is formed by the hydrolysis of DCPD in solutions of pH 5-6 and may also be precipitated heterogeneously upon TCP (Brown et al., 1957). HAP is the thermodynamically most stable phase and often, when precipitated in the presence of foreign ions, substitution of calcium, phosphate and/or hydroxyls by some of these ions take place. Thus, substitutions of OH- by F- or Cl- ions, of the phosphate by sulfate and carbonate and of the calcium by Sr2+, Mg2+ and Na+ ions have been reported (Heughebaert et al., 1983; Legeros R.Z & Legeros J.P., 1984; Moreno & Varughese, 1981; Nathan, 1984).

A considerable amount of the work done for the identification of calcium phosphate minerals which precipitates spontaneously has been based on the stoichiometric molar ratio of calcium to phosphate calculated from the respective changes in the solutions. This ratio has been found in several cases to be 1.45±0.05 which is considerably lower than the value of 1.67 corresponding to HAP which is generally implied as the precipitating mineral. A number of different precursor phases have been postulated to be formed including TCP (Montel et al., 1981; Narasaraju & Phebe, 1996; Walton et al., 1967), OCP (Eanes & Posner, 1968, Posner, 1969) and DCPD (Furedi-Milhofer et al., 1976). On the basis of the analysis of the induction times preceding the spontaneous precipitation of calcium phosphate, it was concluded that at high solution supersaturations the initially forming ACP was converted into an apatitic mineral through an OCP precursor phase formation (Fransis & Webb, 1971).

**Product**

1.87x10-7(mol L-1)\*

9.2x10-7 (mol L-1)\*

2.8x10-9 (mol L-1)\*\*

2.5x10-99(mol L-1)\*\*\*

5.5x10-118 (mol L-1)\*\*\*\*

**Solid Phase Abbrev. Formula Therm.Solub.**

CaHPO4.2H2O

CaHPO4

Ca3(PO4)2

(o≤x≤2)

Ca8H2(PO4)6.5H2O

Ca10-x(HPO4)x(PO4)6-x(OH)2-x

The tendency for a particular calcium phosphate phase to form in supersaturated aqueous media may be determined from the solubility phase diagrams such as the diagram shown in figure 8. It has been reported that when calcium phosphate is precipitated from highly supersaturated solutions forms an unstable precursor phase. This phase is characterized by the absence of peaks in the powder x-ray diffraction pattern and is known as the amorphous calcium phosphate (ACP). The composition of ACP appears to depend upon the precipitation conditions and is usually formed in supersaturated solutions at pH >7.0 (Betts & Posner, 1974; Eanes et al., 1965; LeGeros et al., 1975; Newesely, 1966). In slightly acidic calcium phosphate solutions the monoclinic DCPD is formed (Bets & Posner, 1974; Brown & Lehr, 1959). OCP is formed by the hydrolysis of DCPD in solutions of pH 5-6 and may also be precipitated heterogeneously upon TCP (Brown et al., 1957). HAP is the thermodynamically most stable phase and often, when precipitated in the presence of foreign ions, substitution of calcium, phosphate and/or hydroxyls by some of these ions take place. Thus, substitutions of OH- by F- or Cl- ions, of the phosphate by sulfate and carbonate and of the calcium by Sr2+, Mg2+ and Na+ ions have been reported (Heughebaert et al., 1983; Legeros R.Z & Legeros J.P., 1984; Moreno & Varughese, 1981; Nathan, 1984). A considerable amount of the work done for the identification of calcium phosphate minerals which precipitates spontaneously has been based on the stoichiometric molar ratio of calcium to phosphate calculated from the respective changes in the solutions. This ratio has been found in several cases to be 1.45±0.05 which is considerably lower than the value of 1.67 corresponding to HAP which is generally implied as the precipitating mineral. A number of different precursor phases have been postulated to be formed including TCP (Montel et al., 1981; Narasaraju & Phebe, 1996; Walton et al., 1967), OCP (Eanes & Posner, 1968, Posner, 1969) and DCPD (Furedi-Milhofer et al., 1976). On the basis of the analysis of the induction times preceding the spontaneous precipitation of calcium phosphate, it was concluded that at high solution supersaturations the initially forming ACP was converted into an apatitic mineral through an OCP precursor phase formation (Fransis & Webb, 1971).

Ca10(PO4)6(OH)6

Table 2. Calcium phosphate crystalline phases, formulae and corresponding thermodynamic solubility products. \*(Hench & Wilson, 1991), \*\*(Eanes et al., 1965), \*\*\*(LeGeros et

DCPD

DCPA

TCP

OCP

HAP

al., 1975), \*\*\*\*(Betts & Posner, 1974)

Dicalcium phosphate dihydrate Dicalcium phosphate anhydrous β.Tricalcium phosphate Octacalcium phosphate Hydroxyapatite Defect Apatites

Fig. 8. Solubility isothems for calcium phosphate phases at 25°C, Ionic strength 0.1M NaCl

Spontaneous precipitation investigations have been faced with the problem that at high supersaturations the ACP forming initially results in a rapid decrease of the calcium and phosphate ion activities which fall bellow the values needed for the spontaneous formation of other calcium phosphate phases. A careful analysis of the precipitation of calcium phosphate over the pH range 5.0-8.0 suggested that the limiting ion activity product for ACP was constant as expected for a discrete mineral phase. Moreover it has also been shown that the presence of magnesium in the precipitation medium promoted the formation of DCPD at the expense of ACP (Feenstra & de Bruyn, 1979; Posner et al., 1984).

The driving force for the formation of a solid phase in a continuous aqueous phase is the solution supersaturation which can be developed in many ways including temperature fluctuation, pH change, mixing of incompatible waters, increasing the concentration by evaporation or solid dissolution etc. Although supersaturation is the driving force for the formation of a salt, the exact values in which precipitation occurs are quite different from salt to salt and as a rule, the degree of supersaturation needed for a sparingly soluble salt is orders of magnitude higher than the corresponding value for a soluble salt. For sparingly soluble salts Mν+Aν- the supersaturation ratio, *S*, is defined as:

$$S = \left| \frac{\left(a\_{M^{m+}}\right)\_{s}^{\nu+} \left(a\_{A^{a-}}\right)\_{s}^{\nu-}}{\left(a\_{M^{m+}}\right)\_{\infty}^{\nu+} \left(a\_{A^{a-}}\right)\_{\infty}^{\nu-}} \right| = \left(\frac{IP}{K\_{s}^{\circ}}\right)^{1/\nu} \tag{1}$$

where subscripts *s* and ∞ refer to solution and equilibrium conditions respectively, *α* denote the activities of the respective ions and *ν++ν-*= *ν*. *IP* and *o Ks* are the ion products in the supersaturated solution and at equilibrium respectively.

The fundamental driving force for the formation of a salt from a supersaturated solution is the difference in chemical potential of the solute in the supersaturated solution from the respective value at equilibrium:

$$
\Delta \mu = \mu\_{\text{o}} - \mu\_{\text{s}} \tag{2}
$$

Prosthetic Aortic Valves: A Surgical and Bioengineering Approach 73

biological environment but in accelerated process conditions, in order to study calcification mechanisms. In vitro calcification models have also proposed. These models have the advantage of studying the role of isolated or group of parameters that may contribute in calcification. Both models are very useful especially as pre-screening methods for studying the efficacy of various anticalcification treatments proposed (Bailey et al., 2004; Gross, 2003;

**OUT**

**Magnetic Stirrer**

**H H**

**T**

**H H**

**T N**

**G S**

**Electric Burette**

**A**

**B C**

The use of various model experimental procedures has resulted in the suggestion that the calcification is initiated at the matrix vesicles (Anderson, 1983; Wuthier, 1982), by acid phospholipids (Boskey, 1981; Boskey & Posner, 1977) or by heterogeneous nucleation of various calcium phosphate phases in the body fluids considered as aqueous solutions supersaturated with respect to the salts formed (Kim & Trump, 1975; Dallas et al., 1989;, Schoen et al., 1985; Brown et al., 1988; Grott et al., 1992). Despite intensive research, it seems that there is no agreement on the mechanism of formation of calcific deposits. As a result, the evaluations of bioprosthetic materials used for cardiac valve replacements are often met

Fig. 9. Experimental set-up for the quantitative investigation of the calcification of heart valves in vitro. (A) Automatic titrator apparatus (B) Mounting of heart valves (C) Lid of the

**Glass / SCE electrode**

**Reactor**

**Supersaturated solution**

**Thermostated water IN**

Kapolos et al., 1997; Krings et al., 2009; Schoen & Levy, 2005).

**pH meter**

**Impulsomat**

reactor (Kapolos et al., 1997)

Since the chemical potential is expressed in terms of the standard potential and the activity, *α*, of the solute:

$$
\mu = \mu^o + RT\ln a \tag{3}
$$

where *R* and *T* are the gas constant and the absolute temperature respectively. Substitution of eq. (3) to eq.(2) gives for the driving force for solid deposition (Mullin, 1993):

$$\frac{\Delta\mu}{RT} = \ln\left(\frac{a\_s}{a\_\infty}\right) = \ln S \tag{4}$$

For electrolyte solutions the mean ionic activity is taken:

$$a = a\_{\pm}^{\ \nu} \left( \nu = \nu\_{+} + \nu\_{-} \right) \tag{5}$$

and

$$\frac{\Delta\mu}{RT} = \ln\left(\frac{\alpha\_{\pm,s}}{\alpha\_{\pm,o}}\right)^{\frac{1}{\nu}} = \ln S \tag{6}$$

#### **4.2 Crystal growth in heart valves**

Degeneration of the leaflet tissue, together with calcification constitutes the principal reasons for bioprosthetic valve failure (Schoen et al., 1992). Calcification consists as already mentioned in the formation of sparingly soluble salts of calcium phosphate due to the presence of high levels of calcium and phosphate in blood serum (Schneck, 1995). Although the calcific deposits consist of apatitic calcium phosphate (HAP containing mainly carbonate, fluoride, magnesium and sodium) the formation of transient precursor phases such as DCPD and OCP is possible, as in vitro studies have shown (Brown et al., 1957; Heughebaert et al., 1983; Moreno & Varughese, 1981). The formation of calcium phosphates on porcine heart valves to a percentage of 30-50% is responsible for their dysfunction after 12-15 years due to stenosis or insufficiency (Narasaraju & Rao, 1979).

Despite the fact that the thermodynamic driving force in blood serum for nucleation and growth is sufficiently high for the homogeneous formation of calcium phosphates, the process is believed to be heterogeneous as dead cell remnants, lipids or degenerative collagen fragments may provide active sites for heterogeneous nucleation. Chemical treatment of porcine and pericardial bioprosthetic valves with glutaraldehyde is considered as one of the main causes of valve calcification (Hammersmeister et al., 1993; Schoen & Levy, 1992). Despite intensive research of the past few decades, the mechanism of initiation and development of calcific deposits on tissues in contact with blood is still poorly understood. As a result the development and production of biological valves resistant to calcification is still a major challenge (Grabenwoger et al., 1996; Schoen et al, 1987). Biological, chemical and mechanical factors seem to play a significant role in the kinetics of the process of calcification (Zipkin, 1970).

#### **4.3 Models of heart valve calcification**

Heart valve calcification is a slow process, difficult to be studied in vivo in humans. Various animal models, like the rat subcutaneous implantation, have been used for simulation of

Since the chemical potential is expressed in terms of the standard potential and the activity,

where *R* and *T* are the gas constant and the absolute temperature respectively. Substitution

ln ln *s a S*

1 , , ln ln *s S*

ν

∞ Δ ⎛ ⎞ = = ⎜ ⎟ ⎝ ⎠

*a a* ( ) ν = = + ± ν ν ν

> α

± ± ∞ Δ ⎛ ⎞ = ⎜ ⎟ = ⎜ ⎟ ⎝ ⎠

Degeneration of the leaflet tissue, together with calcification constitutes the principal reasons for bioprosthetic valve failure (Schoen et al., 1992). Calcification consists as already mentioned in the formation of sparingly soluble salts of calcium phosphate due to the presence of high levels of calcium and phosphate in blood serum (Schneck, 1995). Although the calcific deposits consist of apatitic calcium phosphate (HAP containing mainly carbonate, fluoride, magnesium and sodium) the formation of transient precursor phases such as DCPD and OCP is possible, as in vitro studies have shown (Brown et al., 1957; Heughebaert et al., 1983; Moreno & Varughese, 1981). The formation of calcium phosphates on porcine heart valves to a percentage of 30-50% is responsible for their dysfunction after

Despite the fact that the thermodynamic driving force in blood serum for nucleation and growth is sufficiently high for the homogeneous formation of calcium phosphates, the process is believed to be heterogeneous as dead cell remnants, lipids or degenerative collagen fragments may provide active sites for heterogeneous nucleation. Chemical treatment of porcine and pericardial bioprosthetic valves with glutaraldehyde is considered as one of the main causes of valve calcification (Hammersmeister et al., 1993; Schoen & Levy, 1992). Despite intensive research of the past few decades, the mechanism of initiation and development of calcific deposits on tissues in contact with blood is still poorly understood. As a result the development and production of biological valves resistant to calcification is still a major challenge (Grabenwoger et al., 1996; Schoen et al, 1987). Biological, chemical and mechanical factors seem to play a significant role in the kinetics of

Heart valve calcification is a slow process, difficult to be studied in vivo in humans. Various animal models, like the rat subcutaneous implantation, have been used for simulation of

α

= + (3)

(4)

(6)

+ − (5)

ο μ μ

of eq. (3) to eq.(2) gives for the driving force for solid deposition (Mullin, 1993):

*RT* μ

12-15 years due to stenosis or insufficiency (Narasaraju & Rao, 1979).

*RT a* μ

ln *RT a*

For electrolyte solutions the mean ionic activity is taken:

**4.2 Crystal growth in heart valves**

the process of calcification (Zipkin, 1970).

**4.3 Models of heart valve calcification**

*α*, of the solute:

and

biological environment but in accelerated process conditions, in order to study calcification mechanisms. In vitro calcification models have also proposed. These models have the advantage of studying the role of isolated or group of parameters that may contribute in calcification. Both models are very useful especially as pre-screening methods for studying the efficacy of various anticalcification treatments proposed (Bailey et al., 2004; Gross, 2003; Kapolos et al., 1997; Krings et al., 2009; Schoen & Levy, 2005).

Fig. 9. Experimental set-up for the quantitative investigation of the calcification of heart valves in vitro. (A) Automatic titrator apparatus (B) Mounting of heart valves (C) Lid of the reactor (Kapolos et al., 1997)

The use of various model experimental procedures has resulted in the suggestion that the calcification is initiated at the matrix vesicles (Anderson, 1983; Wuthier, 1982), by acid phospholipids (Boskey, 1981; Boskey & Posner, 1977) or by heterogeneous nucleation of various calcium phosphate phases in the body fluids considered as aqueous solutions supersaturated with respect to the salts formed (Kim & Trump, 1975; Dallas et al., 1989;, Schoen et al., 1985; Brown et al., 1988; Grott et al., 1992). Despite intensive research, it seems that there is no agreement on the mechanism of formation of calcific deposits. As a result, the evaluations of bioprosthetic materials used for cardiac valve replacements are often met

Prosthetic Aortic Valves: A Surgical and Bioengineering Approach 75

where *kg* is the rate constant for crystal growth, *f(S)* a function of the total number of the growth sites available and *n* the apparent order of the crystal growth process. Logarithmic plots according to eq.7 yielded the kinetics shown in figure 12. The practical implications of this finding is that the deposition of the mineral phase on the membrane matrix is controlled

Fig. 11. Scanning electron micrographs of : (a) surface of the valves (b) OCP deposited on the

Possible strategies aiming at the retardation of the calcification process should therefore rely on the alteration of the surfaces so as to make surface integration more difficult. An additional feature revealed by the kinetics plots at constant supersaturation (figure 12) is that the glutaraldehyde treated porcine valves are substrates favoring the mineral

Fig. 12. Logarithm of the rate of OCP formation on glutaraldeyde treated porcine valves (black circles) as a function of the relative solution supersaturation; pH 7.4, 37° C, 0.15M NaCl. Open squares and triangles refer to literature results. Experiments performed in our laboratory with the same methodology on fresh, untreated porcine valves have shown that these tissues failed to induce any formation of calcium phosphate deposits although they

were kept in the mineralizing solution for as long as four days

valves at higher relative supersaturation and (c) OCP+HAP on valves at low relative

by the diffusion of the growth units on the OCP nuclei been formed.

**a b c**

supersaturation

nucleation and growth.

with not unfounded criticism. One of the most successful experimental models presented, which is appropriate for the in vitro investigation of mineralization processes is the constant supersaturation model introduced and further developed by Nancollas and co-workers (Thomson & Nancollas, 1978; Amzad et al., 1978). This methodology allows for the solution supersaturation to be kept constant by the addition of titrant solutions. The concentrations of the reagents in these solutions are calculated so that full replacement of the precipitated mass is ensured. In the case of tests performed on the mineralization of heart valves the rates obtained from this system were proportional to the solution's supersaturation.

An in vitro model, based in the constant supersaturation model was introduced in 1997 in our laboratories for the investigation of heart valve calcification in vitro. The experimental set-up is shown in figure 9. Porcine aortic heart valves, treated with glutaraldehyde were sutured on Plexiglas® frames to be kept in flat position during stirring. The frames were immersed in supersaturated solution with respect to calcium and phosphate ions in near physiological concentrations, at 37°C. Solution's pH drop, associated with initiation of crystal growth deposited out of solution, triggered titrant addition with movement of computer controlled syringe pumps.

Fig. 10. Titrant addition for maintaining constant supersaturation during the course of mineralization of artificial heart valves. The rates were increased with increasing solution supersaturation (σ=0.72 , 1.09, 1.25 from the lower to the upper curve respectively)

Figure 10 presents experimental recordings of titrant volume added with time, in order to maintain solution's supersaturation constant. The signal from a pH drop sensitive device resulted from the growth of calcium phosphate crystals precipitated out of the solution, triggered titrant addition in small time steps. The diagrams correspond to different solution supersaturations. The mineral phase deposited on the glutaraldehyde treated porcine valves was identified as OCP, hydrolysed partially to HAP. The extent of hydrolysis was larger at the lower supersaturations, while at higher supersaturations with respect to OCP, this phase was stabilized, as may be seen in the scanning electron micrographs shown in figure11a-c. The characteristic plate like crystals are clearly seen in figure 11b.The rates, calculated from the titrants addition rates and normalized per unit geometric surface area of the exposed valves, were fitted to the semi empirical equation:

$$R\_{\chi} = k\_{\chi} f(S) \sigma^n \tag{7}$$

with not unfounded criticism. One of the most successful experimental models presented, which is appropriate for the in vitro investigation of mineralization processes is the constant supersaturation model introduced and further developed by Nancollas and co-workers (Thomson & Nancollas, 1978; Amzad et al., 1978). This methodology allows for the solution supersaturation to be kept constant by the addition of titrant solutions. The concentrations of the reagents in these solutions are calculated so that full replacement of the precipitated mass is ensured. In the case of tests performed on the mineralization of heart valves the

An in vitro model, based in the constant supersaturation model was introduced in 1997 in our laboratories for the investigation of heart valve calcification in vitro. The experimental set-up is shown in figure 9. Porcine aortic heart valves, treated with glutaraldehyde were sutured on Plexiglas® frames to be kept in flat position during stirring. The frames were immersed in supersaturated solution with respect to calcium and phosphate ions in near physiological concentrations, at 37°C. Solution's pH drop, associated with initiation of crystal growth deposited out of solution, triggered titrant addition with movement of

rates obtained from this system were proportional to the solution's supersaturation.

Fig. 10. Titrant addition for maintaining constant supersaturation during the course of mineralization of artificial heart valves. The rates were increased with increasing solution supersaturation (σ=0.72 , 1.09, 1.25 from the lower to the upper curve respectively)

Figure 10 presents experimental recordings of titrant volume added with time, in order to maintain solution's supersaturation constant. The signal from a pH drop sensitive device resulted from the growth of calcium phosphate crystals precipitated out of the solution, triggered titrant addition in small time steps. The diagrams correspond to different solution supersaturations. The mineral phase deposited on the glutaraldehyde treated porcine valves was identified as OCP, hydrolysed partially to HAP. The extent of hydrolysis was larger at the lower supersaturations, while at higher supersaturations with respect to OCP, this phase was stabilized, as may be seen in the scanning electron micrographs shown in figure11a-c. The characteristic plate like crystals are clearly seen in figure 11b.The rates, calculated from the titrants addition rates and normalized per unit geometric surface area of the exposed

σ

(7)

computer controlled syringe pumps.

valves, were fitted to the semi empirical equation:

() *n R kfS g g* =

where *kg* is the rate constant for crystal growth, *f(S)* a function of the total number of the growth sites available and *n* the apparent order of the crystal growth process. Logarithmic plots according to eq.7 yielded the kinetics shown in figure 12. The practical implications of this finding is that the deposition of the mineral phase on the membrane matrix is controlled by the diffusion of the growth units on the OCP nuclei been formed.

Fig. 11. Scanning electron micrographs of : (a) surface of the valves (b) OCP deposited on the valves at higher relative supersaturation and (c) OCP+HAP on valves at low relative supersaturation

Possible strategies aiming at the retardation of the calcification process should therefore rely on the alteration of the surfaces so as to make surface integration more difficult. An additional feature revealed by the kinetics plots at constant supersaturation (figure 12) is that the glutaraldehyde treated porcine valves are substrates favoring the mineral nucleation and growth.

Fig. 12. Logarithm of the rate of OCP formation on glutaraldeyde treated porcine valves (black circles) as a function of the relative solution supersaturation; pH 7.4, 37° C, 0.15M NaCl. Open squares and triangles refer to literature results. Experiments performed in our laboratory with the same methodology on fresh, untreated porcine valves have shown that these tissues failed to induce any formation of calcium phosphate deposits although they were kept in the mineralizing solution for as long as four days

Prosthetic Aortic Valves: A Surgical and Bioengineering Approach 77

generation biologic valve for patients at about 60 years of age derive improved life expectancy and event-free expectancy regardless of the need for concomitant coronary artery surgery (Birkmeyer et al., 2000). Of course, for special patient groups the indications should be changed. Patients who do need a long-term anticoagulation such as those with chronic atrial fibrillation, intracardiac thrombus, history of thromboembolic events, hypercoagulable state or low ejection fraction, should receive a mechanical valve regardless of age. In the contrary, patients with contraindication to anticoagulants, with bleeding disorders, women of child-bearing age, should receive a biologic valve (Bonow et al., 2006). Patients with chronic renal failure have a higher risk of earlier bioprosthetic valve degeneration, and also an increased incidence of anticoagulation-related complications. For that reason, the current ACC/AHA guidelines (2006) do not recommend the routine use of a

Artificial devices designed and manufactured for mid and long term implantation in patients has to satisfy quality criteria of biocompatibility and function during all implantation time expected. This time period is varied, from a few months, for temporary used prostheses, like some orthopaedic fixation plaques and screws, to long life function, as in the case of prosthetic heart valves. Despite of their evolution and future trends, even if medical technology could make implants satisfying that criteria, prosthetic devices made of, in its best, biologically inert biomaterials cannot meet a serious clinical problem: they cannot follow changes in patient's body from the time of implantation to end of their expected life. In other words, they cannot grow up and remodelled with patient. Tissue engineering is a recent technological approach in the construction of artificial implants that can be gradually remodelled into the patient in real living tissue and organs, following regeneration and auto repair capabilities similar to that of the other natural patient body components (Kretlow & Mikos, 2008; Zilla et al., 2008). Attempts for the construction of TE implants are spread to different tissues and organs, like dermal parches, cartilage, bone and cardiovascular implants and TE or hybrid organs like pancreas or liver. Design and construction of cardiovascular TE implants, like heart valves and blood vessels, is still a challenge because

As a general rule TE valves composed of two groups of biomaterials. One group is composing the scaffold, a structure having the morphology of natural heart valves, usually a biodegradable flexible composite synthetic membrane of a polymeric fibber network embedded in amorphous organic matrix. Different structure of valve parts like valve ring, wall stent and leaflets give to the synthetic valve mechanical strength and flexibility identical to function like natural heart valves. However, scaffolds have a temporary role as, in addition and in parallel with their normal physiological function as heart valves, they may have the ability to support cell adhesion in their structure. Different cells, like fibroblasts, smooth muscle cells and endothelia may adhere, proliferate, stimulated and function into the scaffold valve structure to produce different tissue components that will synergy to compose suitable valvular tissue. Some of these valve cells are transported into scaffold material prior implantation, followed by in vitro cell culture and parallel mechanical valve function in special designed devices, bioreactors. A hybrid structure of synthetic and living biomaterials is made in vitro, which by implantation in the living organism is expected to continue remodelling into real living tissue and organ. As the final

mechanical prosthesis (Bonow et al., 2006).

**6. The future: Tissue engineering (TE)**

of numerous worldwide needs and the severity of possible failure.

Comparison with data obtained for OCP synthetic crystals (Tomazic et al., 1989) and collagen of various types (Heughebaert & Nancollas, 1984; Combes, 1996) showed that glutaraldehyde treated porcine valves accelerated the formation of OCP. The significantly larger value of the rate constant suggested that in this case the number of active sites is not possibly a function of the available surface area. Structural factors on a molecular level should also be considered. Calcium phosphate crystal growth and crystal phases transformations obtained from the results of our in vitro model have been well correlated with similar results obtained from microscopic examinations, chemical analysis and spectrophotometric characterization of crystal phases on samples from calcified natural and bioprosthetic heart valves (Mikroulis et al., 2002). From the composition, the morphology and the size of the developed crystals their nature was determined by comparison with reference synthetic calcium phosphate phases. With this technique it was possible to determine the morphology of CDs developed at the internal sites of the tissue at high magnifications. It seems that in cases of natural heart valves the CDs are mixture of HAP (Ca:PO4=1.67) and OCP (Ca:PO4=1.33), while in bioprosthetic CDs the percentage of OCP is higher than of native in which the ratio Ca:PO4 (1,82) is close to the Ca:PO4 composition of mature physiological biomineral in bone (1,75).

As an overall conclusion from combined studies examined CDs from calcified valve leaflets in vitro, in animal models and in vivo, a model of development of calcification by crystal growth through the formation of precursor phases, which are gradually hydrolyzed in smaller in size, thermodynamically more stable crystal formations may be introduced. According to this, initiation of calcification may be supposed to take place in sites of heterogenous nucleation, formed in different tissue deficiencies, together with local changes in already highly supersaturated body fluids. This model can be very useful in the introduction of anticalcification therapies or techniques for better biomaterials.

#### **5. Conclusion**

Heart valve calcification is still a serious complication for a great number of patients, especially in economical active ages and the elderly. Although anticalcification therapies and procedures have been introduced for valve repair, valve replacement, especially that of aortic and mitral valves is the last choice. Unfortunately, till today there is no "ideal" aortic valve prosthesis. The latter would be easy to be implanted, possess long-term durability, would have no thrombogenicity, maximum effective orifice area, without haemolysis, "resistant" to infective endocarditis, and produce minima noise (Birkmeyer et al., 2000). Currently, available options for the patient include mechanical valve, stented or stentless biologic heterograft valves, allograft valves and pulmonary autograft valves. For the selection between mechanical and biologic valve the surgeon should balance the risks and benefits of each model. The mechanical valve has a long-term durability (till 35-40 years), but on the other hand its thrombogenicity is high, 2-4% per year. In addition, the administered anticoagulation has a significantly increased risk of bleeding. The biologic valve has an increased risk of degeneration, as its durability lasts not more than 10-12 years after implantation (Siddiqui et al., 2009). After this time, a significant regurgitation demands its replacement with an increased operative mortality in comparison to the initial implantation (about two-fold higher). On the other hand, the thrombogenicity of the biologic valve is lower than of mechanical ones, about 1% per year (Puvimanasinghe et al., 2003). Analyses based on mathematical models of data suggest that the selection of 3rd-

Comparison with data obtained for OCP synthetic crystals (Tomazic et al., 1989) and collagen of various types (Heughebaert & Nancollas, 1984; Combes, 1996) showed that glutaraldehyde treated porcine valves accelerated the formation of OCP. The significantly larger value of the rate constant suggested that in this case the number of active sites is not possibly a function of the available surface area. Structural factors on a molecular level should also be considered. Calcium phosphate crystal growth and crystal phases transformations obtained from the results of our in vitro model have been well correlated with similar results obtained from microscopic examinations, chemical analysis and spectrophotometric characterization of crystal phases on samples from calcified natural and bioprosthetic heart valves (Mikroulis et al., 2002). From the composition, the morphology and the size of the developed crystals their nature was determined by comparison with reference synthetic calcium phosphate phases. With this technique it was possible to determine the morphology of CDs developed at the internal sites of the tissue at high magnifications. It seems that in cases of natural heart valves the CDs are mixture of HAP (Ca:PO4=1.67) and OCP (Ca:PO4=1.33), while in bioprosthetic CDs the percentage of OCP is higher than of native in which the ratio Ca:PO4 (1,82) is close to the Ca:PO4 composition of

As an overall conclusion from combined studies examined CDs from calcified valve leaflets in vitro, in animal models and in vivo, a model of development of calcification by crystal growth through the formation of precursor phases, which are gradually hydrolyzed in smaller in size, thermodynamically more stable crystal formations may be introduced. According to this, initiation of calcification may be supposed to take place in sites of heterogenous nucleation, formed in different tissue deficiencies, together with local changes in already highly supersaturated body fluids. This model can be very useful in the

Heart valve calcification is still a serious complication for a great number of patients, especially in economical active ages and the elderly. Although anticalcification therapies and procedures have been introduced for valve repair, valve replacement, especially that of aortic and mitral valves is the last choice. Unfortunately, till today there is no "ideal" aortic valve prosthesis. The latter would be easy to be implanted, possess long-term durability, would have no thrombogenicity, maximum effective orifice area, without haemolysis, "resistant" to infective endocarditis, and produce minima noise (Birkmeyer et al., 2000). Currently, available options for the patient include mechanical valve, stented or stentless biologic heterograft valves, allograft valves and pulmonary autograft valves. For the selection between mechanical and biologic valve the surgeon should balance the risks and benefits of each model. The mechanical valve has a long-term durability (till 35-40 years), but on the other hand its thrombogenicity is high, 2-4% per year. In addition, the administered anticoagulation has a significantly increased risk of bleeding. The biologic valve has an increased risk of degeneration, as its durability lasts not more than 10-12 years after implantation (Siddiqui et al., 2009). After this time, a significant regurgitation demands its replacement with an increased operative mortality in comparison to the initial implantation (about two-fold higher). On the other hand, the thrombogenicity of the biologic valve is lower than of mechanical ones, about 1% per year (Puvimanasinghe et al., 2003). Analyses based on mathematical models of data suggest that the selection of 3rd-

introduction of anticalcification therapies or techniques for better biomaterials.

mature physiological biomineral in bone (1,75).

**5. Conclusion**

generation biologic valve for patients at about 60 years of age derive improved life expectancy and event-free expectancy regardless of the need for concomitant coronary artery surgery (Birkmeyer et al., 2000). Of course, for special patient groups the indications should be changed. Patients who do need a long-term anticoagulation such as those with chronic atrial fibrillation, intracardiac thrombus, history of thromboembolic events, hypercoagulable state or low ejection fraction, should receive a mechanical valve regardless of age. In the contrary, patients with contraindication to anticoagulants, with bleeding disorders, women of child-bearing age, should receive a biologic valve (Bonow et al., 2006). Patients with chronic renal failure have a higher risk of earlier bioprosthetic valve degeneration, and also an increased incidence of anticoagulation-related complications. For that reason, the current ACC/AHA guidelines (2006) do not recommend the routine use of a mechanical prosthesis (Bonow et al., 2006).

## **6. The future: Tissue engineering (TE)**

Artificial devices designed and manufactured for mid and long term implantation in patients has to satisfy quality criteria of biocompatibility and function during all implantation time expected. This time period is varied, from a few months, for temporary used prostheses, like some orthopaedic fixation plaques and screws, to long life function, as in the case of prosthetic heart valves. Despite of their evolution and future trends, even if medical technology could make implants satisfying that criteria, prosthetic devices made of, in its best, biologically inert biomaterials cannot meet a serious clinical problem: they cannot follow changes in patient's body from the time of implantation to end of their expected life. In other words, they cannot grow up and remodelled with patient. Tissue engineering is a recent technological approach in the construction of artificial implants that can be gradually remodelled into the patient in real living tissue and organs, following regeneration and auto repair capabilities similar to that of the other natural patient body components (Kretlow & Mikos, 2008; Zilla et al., 2008). Attempts for the construction of TE implants are spread to different tissues and organs, like dermal parches, cartilage, bone and cardiovascular implants and TE or hybrid organs like pancreas or liver. Design and construction of cardiovascular TE implants, like heart valves and blood vessels, is still a challenge because of numerous worldwide needs and the severity of possible failure.

As a general rule TE valves composed of two groups of biomaterials. One group is composing the scaffold, a structure having the morphology of natural heart valves, usually a biodegradable flexible composite synthetic membrane of a polymeric fibber network embedded in amorphous organic matrix. Different structure of valve parts like valve ring, wall stent and leaflets give to the synthetic valve mechanical strength and flexibility identical to function like natural heart valves. However, scaffolds have a temporary role as, in addition and in parallel with their normal physiological function as heart valves, they may have the ability to support cell adhesion in their structure. Different cells, like fibroblasts, smooth muscle cells and endothelia may adhere, proliferate, stimulated and function into the scaffold valve structure to produce different tissue components that will synergy to compose suitable valvular tissue. Some of these valve cells are transported into scaffold material prior implantation, followed by in vitro cell culture and parallel mechanical valve function in special designed devices, bioreactors. A hybrid structure of synthetic and living biomaterials is made in vitro, which by implantation in the living organism is expected to continue remodelling into real living tissue and organ. As the final

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**Part 3**

**Aortic Root Replacement**


**About the Editors**

#### **Luigi Gurreri**

Luigi Gurreri is a research fellow in chemical engineering at the University of Palermo. His research activities concern modelling and experimental characterization of membrane processes for various applications, including desalination and salinity gradient power, with focuses on fluid dynamics, transport phenomena, and process simulation. He has participated in many EU-funded projects in this area.

#### **Alessandro Tamburini**

Alessandro Tamburini is an assistant professor of chemical process control at the University of Palermo. He has carried out research activities focused on the experimental and numerical analysis of complex systems including multiphase stirred tanks and membrane-based units. He has participated in many EU-funded and national projects on water desalination and renewable energy technologies as well as salinity gradient power processes.

#### **Giorgio Micale**

Giorgio Micale is a full professor of chemical process and plant design at the University of Palermo. His core research topics are the study of conventional and renewable energy desalination processes, salinity gradient power processes, computational fluid dynamics, mixing and multiphase flows, and computer-aided process engineering. He leads his research group and is responsible for several EU projects.


*Edited by Vlassios Hrissanthou and Konstantinos Kaffas*

In the first section of this book on soil erosion, an introduction to the soil erosion problem is presented. In the first part of the second section, rainfall erosivity is estimated on the basis of pluviograph records and cumulative rainfall depths by means of empirical equations and machine learning methods. In the second part of the second section, a physically-based, hydrodynamic, finite element model is described for the computation of surface runoff and channel flows. In the first part of the third section, the soil erosion risk is assessed in two different basins. In the second part of the third section, the soil erosion risk management in a basin is evaluated, and the delimitation of the areas requiring priority planning is achieved.

ISBN 978-1-78985-195-3

Soil Erosion - Rainfall Erosivity and Risk Assessment

Published in London, UK © 2019 IntechOpen © rasikabendre / iStock

## Soil Erosion Rainfall Erosivity and Risk Assessment

*Edited by Vlassios Hrissanthou and Konstantinos Kaffas*