**6. Validation of numerical results**

The validation of present results is essential to check the credibility of the numerical solution methodology. The presently computed results are compared with the exact solutions obtained for some limiting cases of the problem. Figures 2 and 3 compare these results, and an excellent correspondence is seen to exist between the two sets of data. In addition, table 1 shows the comparison values of the local skin friction coefficient with those reported by of Khan and Shahzad [22]. It is seen that the comparison is in very good agreement, and thus gives us confidence to the accuracy of the numerical results.


**Acknowledgements**

This work was funded by research grant from the University of Alicante (VIGROB-309). The authors would like to thank Francisco Grimalt Salvá and José Antonio Abellán for their helpful discussions about the color of the feathers in genera of the *Fringillidae* family and flamingos.


**2.2 Digital instrumentation and control**

Although the first closed-loop industrial computer control system was installed by Texaco Company at its Port Arthur refinery in 1959 [1], I&C in nuclear reactors

**Figure 2.** *Signals for a notional RPS.*

### *Cyber-Informed Engineering for Nuclear Reactor Digital Instrumentation and Control DOI: http://dx.doi.org/10.5772/intechopen.101807*

largely remained analog until about 30 years ago when digital transmitters, indicators, controllers and data recorders began replacing analog sensors, indicators, actuators, and pen-based chart recorders. And, while non-safety digital control systems (e.g., feedwater control systems, turbine control systems and reactor control systems) are now commonly installed in nuclear reactors, safety-related digital control systems (e.g., RPS, ESFAS) are much less common, especially in the United States. The United States has been slow to adopt digital technology because of previously unanalyzed risks associated with new and unknown attributes, including common cause failures and cyber risks. International adoption of digital technology in nuclear reactors, including safety-related control systems, has been more aggressive than in the United States. Of course, new advanced reactors are being primarily designed with DI&C.

As described by the Nuclear Energy Institute (NEI), an I&C device in the U.S. power reactor industry is typically considered 'digital' if it contains any combination of hardware, firmware, and/or software that can execute internally stored programs and algorithms without operator action [2]. Hardware includes microelectronics, such as digital or mixed signal integrated circuits, as well as larger assemblies, such as microprocessors, memory chips, and logic chips. Hardware may also include other peripherals, such as expansion drives or communication controllers. Software includes operating systems, platforms, and applications used for process control, human machine interfaces, and other specific programs used for device or system operation. Firmware is software stored in non-volatile memory devices that provides low-level control specific to the hardware. Firmware executes higher-level operations and controls basic functionality of the device, including communication, program execution, and device initialization.

Field sensors and controllers may be standalone, small local systems, or larger distributed control systems. Devices may be connected by physical cables or wireless technology (e.g., WiFi, cellular, satellite, Bluetooth, radio frequency identification). There is also a range of communication protocols used in DI&C depending on the design and manufacturer.

The systems, structure, and components (SSCs) used in U.S. NPP safetyrelated protection systems are categorized as Institute of Electrical and Electronics Engineers (IEEE) class 1E technologies as defined by IEEE 308-1971 (and later) [3]. They must be designed to conform with General Design Criteria (GDC) in 10 CFR 50 Appendix A [4], IEEE 279-1971 [5], IEEE 308-1971 [3], and IEEE Std 603-1991 [6], as applicable based on construction permit dates. Guidance in Regulatory Guide 1.152 [7] and IEEE 7-4.3.2-2003 [8] may also be used to comply with Nuclear Regulatory Commission (NRC) regulations. Internationally, applications or components that perform IEC category A safety-related functions may fall under IEC 61513 [9], International Atomic Energy Agency (IAEA) SSR-2/1 [10], and IAEA SSG-39 [11] requirements.

These general design criteria include conformance requirements for independence and single-failure criterion such as defense-in-depth, diversity (i.e., different technology), redundancy (i.e., secondary equipment that duplicates the essential function), physical separation, and electrical isolation. The purpose of single-failure criterion is to ensure no single failure of a component interferes with the safety function and proper operation of the safety system [6]. Generally, it is impossible to prove that digital systems are error free. And, while common-cause failures can occur with analog equipment, it is more likely that software errors will result in common-cause failures, such as identical software-based logic errors that could cause simultaneous functional failure of all four RPS divisions. Thus, since unanticipated common-cause failures are more likely in digital systems than analog systems, there is increased burden to prove to the regulator that the design adequately meets the general design criteria outlined in the applicable requirements.


**5.2. Effect of unloading on the synthesis of collagen**

Several quantitative and qualitative changes in the intramuscular connective tissue contribute to the deteriorated function and biomechanical properties of the immobilized skeletal muscle [6,11,51]. Muscle and tendon collagen and the connective tissue network are known to respond to altered levels of physical activity [51,55]. In contrast to physical loading, immobilization of rat limb leads to a decrease in the activities of collagen synthesizing enzymes both in skeletal muscle and tendon [20,56].

Along with the increased amount of intramuscular connective tissue, the number of capillaries decreases dramatically [57,58]. Each capillary is surrounded by a dense layer of the connective tissue fibres, isolating the capillary from the adjacent muscle fibre, which disturbs the blood supply of the muscle fibres and further increases the muscle fibre atrophy [57,58].

During immobilization, the normal three-dimensional orientation of the collagen fibres is disrupted. The normal orientation constitutes of the fibres running parallel to the muscle fibres on their surface, preventing muscle cells from over-elongation and –contraction. In addition, thin perpendicular fibres connect adjacent muscle fibres to each other [7,10,11]. As a result of decreased loading, the number of longitudinal fibres increases, the crimp angel of the collagen decreases and this diminishes the ability of the muscle to elongate [2,11] and because of that the skeletal muscle shows significantly decreased tensile strength [1,3]. In addition to changes in collagen abundance, alterations in the degree of collagen cross-linking would have a profound effect on the mechanical properties of skeletal muscle, causing a decrease in muscle stiffness [10,17].

In contrast to physical loading, immobilization leads to the decrease in the enzyme activities of collagen biosynthesis, which suggests that the biosynthesis of the collagen network decreases as a result of reduced muscular and tendinous activity [8,56]. The rate of the total collagen synthesis depends mostly on the overall protein balance of the tissue, but it seems to be positively affected by stretch in both muscle and tendon [55].

Collagen expression during immobilization has been shown to be at least partially downregulated at the pretranslational level [55]. Although the relative amount of the connective tissue increases during immobilization, the gene expression of type I and III collagens decreases during the first three days of immobilization [20]. The content of type IV collagen was also reduced as a result of immobilization [23]. The activities of prolyl 4-hydroxylase (P 4-H) and galactosylhydroxylysyl glucosyltransferase (GGT) decrease from the first three days of immobilization up to at least three weeks, suggesting decreased collagen biosynthesis during that time [20,59]. The degradation of collagens has been found to be enhanced during immobilization, as the expression of both MMP-2 and MMP-9 increased after 30 days of immobilization [30]. The quantity of TIMP-1 was also increased after 30 days of immobilization [30].

The collagen concentration increases when expressed both as a function of muscle dry weight or muscle cross-sectional area, but this increase in muscle collagen is primarily due to the muscle atrophy induced by immobilization [13].

#### **5.3. Effect of unloading on the contractile apparatus of skeletal muscle**

As the skeletal muscle is a highly plastic tissue, the conditions associated with the disuse are accompanied by adaptation. A period of time without weight bearing cause modifications of structure and the function of skeletal muscles, of which atrophy and a slow-to-fast transition are the most prominent [60,61]. Many animal models such as the kind limb suspension, immobilization in shortened and lengthened position, spaceflight and denervation show that the removal of a mechanical load produces atrophy and contractile alterations, more evident in the slow muscle soleus than in fast muscles as extensor digitorum longus [60,62,63]. Besides space flight and bed rest experiments, long periods of muscle disuse in relation to a disease or traumatic injuries of the joints or of the bones are relatively common experience for human beings [62,63]. The inactivity causes only small increases in contractile speed and myofibrillar adenosine triphosphatase (ATPase) activity and slight elevations in the percentage of the fast type myosin heavy chain (MyHC) isoforms in fast-twitch muscles, as compared to slow-twitch muscle [64,65].

#### **5.4. Slow-to-fast transition in skeletal muscle during unloading**

tissue fibres, isolating the capillary from the adjacent muscle fibre, which disturbs the blood

During immobilization, the normal three-dimensional orientation of the collagen fibres is disrupted. The normal orientation constitutes of the fibres running parallel to the muscle fibres on their surface, preventing muscle cells from over-elongation and –contraction. In addition, thin perpendicular fibres connect adjacent muscle fibres to each other [7,10,11]. As a result of decreased loading, the number of longitudinal fibres increases, the crimp angel of the collagen decreases and this diminishes the ability of the muscle to elongate [2,11] and because of that the skeletal muscle shows significantly decreased tensile strength [1,3]. In addition to changes in collagen abundance, alterations in the degree of collagen cross-linking would have a profound effect on the mechanical properties of skeletal muscle, causing a decrease in muscle

In contrast to physical loading, immobilization leads to the decrease in the enzyme activities of collagen biosynthesis, which suggests that the biosynthesis of the collagen network decreases as a result of reduced muscular and tendinous activity [8,56]. The rate of the total collagen synthesis depends mostly on the overall protein balance of the tissue, but it seems to

Collagen expression during immobilization has been shown to be at least partially downregulated at the pretranslational level [55]. Although the relative amount of the connective tissue increases during immobilization, the gene expression of type I and III collagens decreases during the first three days of immobilization [20]. The content of type IV collagen was also reduced as a result of immobilization [23]. The activities of prolyl 4-hydroxylase (P 4-H) and galactosylhydroxylysyl glucosyltransferase (GGT) decrease from the first three days of immobilization up to at least three weeks, suggesting decreased collagen biosynthesis during that time [20,59]. The degradation of collagens has been found to be enhanced during immobilization, as the expression of both MMP-2 and MMP-9 increased after 30 days of immobilization [30]. The quantity of TIMP-1 was also increased after 30 days of immobilization

The collagen concentration increases when expressed both as a function of muscle dry weight or muscle cross-sectional area, but this increase in muscle collagen is primarily due to the

As the skeletal muscle is a highly plastic tissue, the conditions associated with the disuse are accompanied by adaptation. A period of time without weight bearing cause modifications of structure and the function of skeletal muscles, of which atrophy and a slow-to-fast transition are the most prominent [60,61]. Many animal models such as the kind limb suspension, immobilization in shortened and lengthened position, spaceflight and denervation show that the removal of a mechanical load produces atrophy and contractile alterations, more evident in the slow muscle soleus than in fast muscles as extensor digitorum longus [60,62,63]. Besides space flight and bed rest experiments, long periods of muscle disuse in relation to a disease or

**5.3. Effect of unloading on the contractile apparatus of skeletal muscle**

be positively affected by stretch in both muscle and tendon [55].

muscle atrophy induced by immobilization [13].

supply of the muscle fibres and further increases the muscle fibre atrophy [57,58].

56 Composition and Function of the Extracellular Matrix in the Human Body

stiffness [10,17].

[30].

The different response can be explained, considering the fact that skeletal muscles in different parts of the body are subjected to different patterns of recruitment and activity [66,67]. The anti-gravitational Sol muscle is recruited for prolonged periods at a moderate level of intensity, whereas the extensor digitorum longus muscle is less frequently recruited, performing short, high-force contractions [68,69]. It is commonly known that inactivity affects the functional and biochemical properties of antigravity muscles, causing a significant decrease in both contrac‐ tion and relaxation times [66,70] and a significant increase in the maximal shortening velocity and myofibrillar ATPase activity [71]. The above-mentioned changes are considered to be a result of the gene expression, especially the genes involved in the fibre type transformation [71–73]. The co-ordinated changes in the gene expression are particularly apparent for myosin and consequently the disuse induces a slow-to-fast transition, as reflected by an increase in fast MyHC isoforms at the expense of slow MyHC in the Sol and a fast-to-faster MyHC shift in the GM muscle [74,75]. An increase in fast MyLC isoforms, an increased proportion of fast troponin subunits and hybrid fibres co-expressing fast and slow MyHC and MyLC appears during slow-to-fast transition in Sol muscle [64,71,76]. The fibre type transition results in a change in muscle metabolism, fuel use, and more fatigable muscle [77]. Several histochemical analyses have also suggested that the functional changes in immobilized muscles are due to an increase in fast-twitch IIa fibres [64]. In addition to above-mentioned facts, the increased sarcoplasmic reticulum calcium-ATPase activity and the preferential loss of thin filaments all contribute to faster contractile properties of the Sol muscle [78]. The increased shortening velocity may be an attempt to compensate for the loss of power generating capacity during unloading caused by weakening [79].

Muscle disuse is often accompanied by increased fatigability, which is caused by the reduced oxidative capacity of disused muscles [65,80,81]. Capillary loss and reduction in blood flow might contribute to the increased fatigability by an impaired supply of energy substrates and oxygen to the muscle [82].

#### **5.5. Formation of muscle atrophy during unloading**

While immobilization at shortened length induces atrophy, immobilization in lengthened position induces hypertrophy, which is largely attributable to addition of sarcomeres in the longitudinal direction [26,83]. Immobilization in the shortened position, e.g. hindlimb suspension, induces preferential transcription of fast MyHC isoforms, reminiscent of the slowto-fast transition observed in other models of disuse [26,84]. The disuse atrophy is character‐ ized by the loss of muscle mass and decrease of muscle diameter. In the case of muscle atrophy, there some noticeable changes in the muscle cell at the cellular level including sarcomere dissolution and endothelial degradation, a marked reduction in the number of mitochondria, the accumulation of the connective tissue, the elimination of apoptotic myonuclei and a decrease in capillary density [85–87]. Selective susceptibility of fibre types to immobilization seems to exist, while the red muscle fibres show the greatest atrophy. The decreased synthesis of protein and increased protein degradation are characteristic features to muscle atrophy. At least half of the total muscle protein is myofibrillar protein, and this fraction is lost at a faster rate than other muscle proteins during atrophy [84]. Three major proteolytic systems to skeletal mass protein loss are the cytosolic calcium-dependent calpain system, the lysosomal proteases and the ATP-dependent ubiquitin-proteasome system, which work as partners during muscle proteolysis rather than one system being used exclusively during atrophy [88]. Recent advances in cellular biology show the oxidative stress to be an important regulator of pathways leading to muscle atrophy during periods of disuse, increasing the expression of the key components of the proteasome proteolytic system. This proteolytic system is a prominent contributor to protein breakdown in skeletal muscle during periods of inactivity [37,84,86].

### **5.6. Effect of unloading on the skeletal muscle MyHC composition**

Prolonged periods of time spent with a diminished or no-weight bearing have a deleterious effect on skeletal muscle with the decreased protein synthesis, the loss of muscle mass and alterations of biochemical parameters [74,77]. The main findings confirmed that the proportion of slow MyHC isoforms decreased and the proportion of MyHC fast isoforms increased in consequence of altered functional conditions [46,64,89].

Clinical observations show that the atrophy of the skeletal muscle occurs as a result of immobilization and is caused by the changed functional conditions in the muscular system. Skeletal muscle function depends on the intact proprioceptive activity, motor innervation, mechanical load, and joint mobility. If one of these factors is altered, the muscle will undergo adaptation. As an increased muscular activity leads to the enhancement of the structures involved in contraction, inactivity or disuse is followed by the reduction of the muscle mass [8,77].

Alterations of biochemical parameters and changes at the ultrastructural level of the contractile apparatus are considered to be characteristic of atrophied muscles. The effect of disuse on the skeletal muscle depends on the fibre type composition of the muscle. The degenerative changes in disused muscles at the ultrastructural level have been shown to be most severe in slow oxidative muscle fibres. It is suggested that the most vulnerable muscles were antigravity muscles crossing a single joint [90].

The properties of muscle contraction which depend on the MyHC isoform composition decrease in atrophied skeletal muscle [46,89,91]. Contractile activity can induce differential expression of myosin protein isoforms in skeletal muscle. MyHC composition has an important regulatory role in myosin ATPase activity and muscle fibre shortening velocity [92]. A prolonged activity causes alterations in the MyHC composition. A decrease in the mechanical load stimulates the conversion of slow myosin in muscles of mixed fibre type composition, whereas a decrease in the weight-bearing load results in a decrease in slow myosin content [46,92].

Comparing the changes of proportion of MyHC isoforms in the contractile apparatus during unloading among human subjects and experimental animals, we can see that they are similar in their direction and amplitude. In conclusion, the adaptation of the mammalian skeletal muscles to the unloading depends on the contractile and metabolic characteristics of skeletal muscle and is not dependent on the species of the mammal. As the certain connections exist between the contractile and metabolic characteristics of skeletal muscle it is understandable why the specific atrophy causes the decrease of the main function of the skeletal muscle.


**6. Final considerations**

It was presented and tested a method based on modelling an simulation by Petri Nets and Throughput Diagram for the calculation of two important indicators in manufacturing management: in process inventory and lead time. With the simulation results (provided by the Petri Net model outputs and the throughput diagram) the manufacturing process can be predicted, as well as some modification can be measured and analyzed to optimize the production. As well as save money on alterations that could produce losses in production processes and often, in the real world, are hard to be perceived.

Measurement of Work-in-Process and Manufacturing Lead Time by Petri Nets Modeling and Throughput Diagram 105

Boucher, T.; Jafari, M.; Meredith, G. Petri net control of an automated manufacturing cell. Computers and Industrial Engineering, Tarry Town, NY, USA, v. 17, n.1, p.459-463,

Dallery, Y.; Gershwin, S. Manufacturing flow line systems: a review of models and

Dicesare, F.; Harhalakis, G.; PROTH, J. M.; Silva, M.; Vernadat, F. B. Practice of Petri nets in

Drath, R.; Schwuchow, S. Modellierung diskretkontinuierlicher Systeme mit Petri-Netzen.

Braunschweig: Technische Universität Braunschweig, Institut für Regelungs-und-

Fung, R.; Jiang, Z.; Zuo, M.; TU, P. Adaptive production scheduling of virtual production systems using object-oriented Petri nets with changeable structure. International

Govil, M.; FU, M. Queuing theory in manufacturing: a survey. Journal of Manufacturing

Jeng, M. A Petri net synthesis theory for modeling flexible manufacturing systems. IEEE Transactions on Systems, Man and Cybernetics Part B, v. 27, n. 2, p.169-183, 1997. Jeng, M.; Xie, X.; Huang, Y. Manufacturing modeling using process nets with resources. In: IEEE International Conference on Robotics and Automation. April. 2000, San Francisco,

Law, A.; Kelton, W. Simulation modeling and analysis. New York: McGraw-Hill, 1991. Lee, D.; Dicesare, F. Scheduling flexible manufacturing systems using Petri nets and heuristic search. IEEE Transactions on Robotics and Automation, New York, NY, v.10,

Maciel, P.; Lins, R.; Cunha, P. Introdução às redes Petri e aplicações. Campinas: Instituto de

Martinez, J.; Muro, P.; Silva, M. Modeling, validation and software implementation of production systems using high level Petri nets. IEEE International Conference on Robotics and Automation. Março – Abril, 1987, Raleigh, North Carolina. Proceedings…

Murata, T. Petri Nets: Properties, Analysis and Applications. Proceedings of the IEEE,

Papadopoulos, H. T.; Heavey, C.; Browne, J. Queuing theory in manufacturing systems

Papadopoulos, H.; Heavey, C. Queuing theory in manufacturing systems analysis and design: a classification of models for production and transfer olines. European Journal

Peterson, J. Petri Net Theory and the modelling of system. Englewood Cliffs, NJ: Prentice-

Proth, J.; Wang, L.; XIE, X. A class of Petri nets for manufacturing system integration. IEEE Transactions on Robotics and Automation, Valbonne, v.13, n.3, p.317-326, 1997.

analytical results. Queuing Systems, v. 12, n.1-2, p. 3-94, 1992.

Journal of Production Research, v. 40, n.8, p.1759-1785, 2002.

USA. Proceedings… San francisco, USA: IEEE, 2000.

Raleigh, North Carolina: I3E Comput. Soc., v.4, p. 1180-1185.

Chicago, IL, USA, v. 77, n.4, p.541-580, 1989.

of Operational Research, v.92, n.1, p.1-27, 1996.

analysis and design. London: Chapman & Hall, 1993.

In: Schneider, E. (Org.). Entwurf komplexer Automatisierungssyteme.

manufacturing. London: Chapman & Hall, 1993.

Automatisierungstechnick, 1997.

Systems, v. 18, n.3, p.210-214, 1999.

n.2, p.123-133, 1994.

Hall Editions, 1981.

Computação - UNICAMP, 1996.

1989.


*3.2. TCD of Fresh and Dried Pomegranate Arils*

Storage of pomegranate fruit contributed to the changes in the TCD of fresh arils, and subsequently had a significant effect on the TCD of dried arils. A notable variation was observed in the TCD with increased storage period, with the highest TCD being 11.2 after the 12-week storage period (Table 1). For dried arils processed with hot-air and freeze-dryers, there was a significant (*p* < 0.0001) interaction in TCD (Table 2). Hot-air drying had the least (3.02), while freeze-dried arils had the highest (23.6) TCD after the 12-week storage period (Table 2). A change in TCD is an important attribute of a dried product, expressing the capacity of the human eye to distinguish between various colours attributed to different products [27]. Coklar et al. [39] reported similar findings where hawthorn fruit dried using a freeze dryer had a better colour appearance than fruit dried with oven and microwave dryers. Ali et al. [40] reported that freeze-dried guava fruit preserved its colour the best compared to sunlight and convective oven dryer. The colour change of dried arils could be influenced by the drying method involved and also by the naturally occurring biochemical changes happening during storage of pomegranate fruit.

**Table 2.** Changes in the physicochemical properties of dried pomegranate arils during 12 weeks of cold storage at 7 ± 0.3 ◦C, 92 ± 3% RH (w.b).

TSS, total soluble solids; TA, titratable acidity; TCD, total colour difference. Data presented as means ± SE in each row followed by different letters are significantly different (*p* < 0.05) according to Fisher's LSD.


*3.3.3 Simulation*

*Simulation* (1/28), or real-world simulation, is a group of games, including vehicle driving or particular physical challenges. However, unlike action games, simulation is often more focused on navigation experience and related tasks. In 2019, one VR drone racing game called "Brain eRacing" (VR DR) was reported with 30 healthy participants [56]. However, unlike other games using MI for navigation control, MI is only used to accelerate the drone's speed. The final average game time is 54.88s, while the group with the last average time is 69.6s.


**Author details**

Dane Tadeu Cestarolli and Elidia Maria Guerra\* Department of Chemistry, Biotechnology and Bioprocess Engineering, Federal University of São João Del Rei, Campus Alto Paraopeba, Ouro Branco, MG, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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.


1). The two most informative studies have been published in recent months and have not been included in previous reviews. Each of these recent studies found that yoga improved function more than usual care or self-care. The Sherman et al. study employed a three group design, and thus, provided important "comparative effectiveness" data by comparing yoga to an exercise program led by physical therapists. It is notable that was not statistically superior to this exercise intervention. The only other study that used a comparison group of proven efficacy was Evans et al who also found no significant differences on function/disability. Thus, we conclude that yoga may not be superior to other nonpharmacological interventions with moderate effects sizes. However, attendance and satisfaction rates were higher among the yoga group in the Sherman study, indicating that it may be a more attractive intervention to many individuals.

With the exception of a few smaller studies, yoga interventions have been shown to reduce pain severity or the "bothersomeness" of pain, when compared with usual care, or information alone (See Table 2). In looking at the recent RCTs, the Sherman study found that yoga patients were less bothered by low back pain than self-care patients, while the Tilbrook study found no significant differences in back pain severity. The Tilbrook finding is a bit surprising, even though self-reported pain severity is different than pain bothersomeness. Sherman et al. specifically chose to measure pain bothersomeness because self-reported pain severity may be harder to measure reliably across various groups. It is also possible that the design of the Tilbrook study (13 different private practices across the UK) may have affected the results obtained. This may have resulted in greater heterogeneity among the instructors, intervention, or participants, and it is unclear whether the statistical analysis accounted for clustering with the 13 cohorts.

Our review also suggests that yoga can reduce reliance on pain medication when compared with usual or self-care. However, only a few studies have published results on these outcomes so conclusions remain more tentative (See Table 3). With the exception of the Williams at el study, very little information is provided on how medication was measured. Measuring medication use poses its own challenges, with self-reported interview data often differing from medical record information or pill counts. Future research should consider other methods for measuring medication usage including the use of medical record information when possible.

Depression and other indicators of the impact of yoga on psychological outcomes were assessed in a small number of studies (See Table 4). Significant effects were found in a few of the smaller studies, but only non-significant trends were found for the SF-12 MCS in the fullscale trial in which they were measured. The Sherman study did not publish data on psychological variables in their initial manuscript but these results may be forthcoming. Given the cognitive and relaxation components of yoga, along with the higher rates of depression among individuals with chronic low back pain, further research in this area is very important.

We also reviewed the safety of yoga for some of the larger randomized controlled trials. Data from the largest and most recent trials suggests that two serious adverse events occurred among a combined total of 243 participants. These events were related to increased back pain, one being a herniated disc. Another 10% of these yoga participants experienced non-serious adverse events that were almost exclusively increases in back pain. Thus, participating in yoga interventions by persons with chronic low back pain is not without risk, but the vast majority of participants had no problems and experienced considerable benefit for a chronic debilitating condition.

Table 2. Studies that examined the effect of yoga on pain.

Yoga as a Treatment for Low Back Pain: A Review of the Literature 349

Table 3. Studies that examined the effect of yoga on medication use.

Table 3. Studies that examined the effect of yoga on medication use.

Table 2. Studies that examined the effect of yoga on pain.

Table 4. Studies that examined the effect of yoga on psychological health.

Yoga as a Treatment for Low Back Pain: A Review of the Literature 351

Overall, our results are similar to and confirmatory of those published in a review article in 2011. This prior review was useful, but was released before the results of the two largest randomized controlled trials that have been published to date. Thus, we believed it was important to add these studies to the body of existing literature. We conclude that yoga interventions impact multiple outcomes that are important to the health and well-being of people afflicted with chronic low back pain. Recent, high quality evidence suggests that yoga provides comparable effects to, and is more appealing than, formal stretching and strengthening programs led by physical therapists. The recent data suggest that it would behoove health care organizations and the members they serve, to have yoga as an available

Anderson, G. B. J. "The Epidemiology of Spinal Disorders." In *The Adult Spine: Principles and Practice.*, edited by JW Frymoyer, 93-141. New York: Raven Press, 1997. Barnes, P. M., E. Powell-Griner, K. McFann, and R. L. Nahin. "Complementary and Alternative Medicine Use among Adults: United States, 2002." *Adv Data*, no. 343 (2004): 1-19. Barnes, P. M., B. Bloom, and R. L. Nahin. "Complementary and Alternative Medicine Use

Broad, W. J. "How Yoga Can Wreck Your Body." *New York Times*, January 5, 2012 2012. Choo, P. W., C. S. Rand, T. S. Inui, M. L. Lee, E. Cain, M. Cordeiro-Breault, C. Canning, and

Chou, R., A. Qaseem, V. Snow, D. Casey, J. T. Cross, Jr., P. Shekelle, and D. K. Owens.

Chou, R., and L. H. Huffman. "Medications for Acute and Chronic Low Back Pain: A Review

Clinical Practice Guideline." *Ann Intern Med* 147, no. 7 (2007): 505-14. Chou, R., and L. H. Huffman . "Nonpharmacologic Therapies for Acute and Chronic Low

Clinical Practice Guideline." *Ann Intern Med* 147, no. 7 (2007): 492-504. Cox, H., H. Tilbrook, J. Aplin, A. Semlyen, D. Torgerson, A. Trewhela, and I. Watt. "A

Canadian Population." *Pain* 107, no. 1-2 (2004): 54-60.

Results of a Pilot Study." *Complement Ther Clin Pract* 16, no. 4 (2010): 187-93. Currie, S. R., and J. Wang. "Chronic Back Pain and Major Depression in the General

Deyo, R. A., S. K. Mirza, and B. I. Martin. "Back Pain Prevalence and Visit Rates: Estimates

Evans, D. D., M. Carter, R. Panico, L. Kimble, J. T. Morlock, and M. J. Spears. "Characteristics

from U.S. National Surveys, 2002." *Spine* 31, no. 23 (2006): 2724-7.

Pilot Study." Altern Ther Health Med 10, no. 2 (2004): 56-9.

among Adults and Children: United States, 2007." *Natl Health Stat Report* 10, no. 12

R. Platt. "Validation of Patient Reports, Automated Pharmacy Records, and Pill Counts with Electronic Monitoring of Adherence to Antihypertensive Therapy."

"Diagnosis and Treatment of Low Back Pain: A Joint Clinical Practice Guideline from the American College of Physicians and the American Pain Society." Ann

of the Evidence for an American Pain Society/American College of Physicians

Back Pain: A Review of the Evidence for an Aps/American College of Physicians

Randomised Controlled Trial of Yoga for the Treatment of Chronic Low Back Pain:

and Predictors of Short-Term Outcomes in Individuals Self-Selecting Yoga or Physical Therapy for Treatment of Chronic Low Back Pain." *Pm R* 2, no.11 (2010): 1006-15. Galantino, M. L., T. M. Bzdewka, J. L. Eissler-Russo, M. L. Holbrook, E. P. Mogck, P. Geigle,

and J. T. Farrar. "The Impact of Modified Hatha Yoga on Chronic Low Back Pain: A

health service option at little or no cost.

(2009): 1-23.

*Med Care* 37, no. 9 (1999): 846-57.

Intern Med 147, no. 7 (2007): 478-91.

**6. References**

Overall, our results are similar to and confirmatory of those published in a review article in 2011. This prior review was useful, but was released before the results of the two largest randomized controlled trials that have been published to date. Thus, we believed it was important to add these studies to the body of existing literature. We conclude that yoga interventions impact multiple outcomes that are important to the health and well-being of people afflicted with chronic low back pain. Recent, high quality evidence suggests that yoga provides comparable effects to, and is more appealing than, formal stretching and strengthening programs led by physical therapists. The recent data suggest that it would behoove health care organizations and the members they serve, to have yoga as an available health service option at little or no cost.

#### **6. References**

350 Low Back Pain

Table 4. Studies that examined the effect of yoga on psychological health.

Groessl, E. J., T. G. Ganiats, and A. J. Sarkin. "Sociodemographic Differences in Quality of Life in Rheumatoid Arthritis." *Pharmacoeconomics* 24, no. 2 (2006): 109-21. Groessl, E. J., K. R. Weingart, K. Aschbacher, L. Pada, and S. Baxi. "Yoga for Veterans with Chronic Low-Back Pain." *J Altern Complement Med* 14, no. 9 (2008): 1123-29. Groessl, E.J., K. R. Weingart, N. Johnson, and S. Baxi. "The Benefits of Yoga for Women Veterans with Chronic Low Back Pain." *J Altern Complement Med* (in press). Johnson, Andrew, and Richards Marged. "Celebrity Power Yoga: The New Craze from over

Liu, H., C. E. Golin, L. G. Miller, R. D. Hays, C. K. Beck, S. Sanandaji, J. Christian, T.

Luo, X., R. Pietrobon, S. X. Sun, G. G. Liu, and L. Hey. "Estimates and Patterns of Direct

Posadzki, P., and E. Ernst. "Yoga for Low Back Pain: A Systematic Review of Randomized

Saper, R. B., K. J. Sherman, D. Cullum-Dugan, R. B. Davis, R. S. Phillips, and L. Culpepper.

Sherman, K. J., D. C. Cherkin, R. D. Wellman, A. J. Cook, R. J. Hawkes, K. Delaney, and R. A.

Sullivan, M. J., K. Reesor, S. Mikail, and R. Fisher. "The Treatment of Depression in Chronic Low Back Pain: Review and Recommendations." *Pain* 50, no. 1 (1992): 5-13. Tekur, P., S. Chametcha, R. N. Hongasandra, and N. Raghuram. "Effect of Yoga on Quality of Life of Clbp Patients: A Randomized Control Study." Int J Yoga 3, no. 1 (2010): 10-7. Tekur, P., C. Singphow, H. R. Nagendra, and N. Raghuram. "Effect of Short-Term Intensive

Telles, S., M. Dash, and K. V. Naveen. "Effect of Yoga on Musculoskeletal Discomfort and Motor Functions in Professional Computer Users." *Work* 33, no. 3 (2009): 297-306. Tilbrook, H. E., H. Cox, C. E. Hewitt, A. R. Kang'ombe, L. H. Chuang, S. Jayakody, J. D.

Williams, K. A., J. Petronis, D. Smith, D. Goodrich, J. Wu, N. Ravi, E. J. Doyle, Jr., R. Gregory

Therapy for Chronic Low Back Pain." *Pain* 115, no. 1-2 (2005): 107-17. Williams, K., C. Abildso, L. Steinberg, E. Doyle, B. Epstein, D. Smith, G. Hobbs, R. Gross, G.

Clinical Trials." *Clin Rheumatol* 30, no. 9 (2011): 1257-62.

Controlled Trial." *Ann Intern Med* 143, no. 12 (2005): 849-56.

Chronic Low Back Pain." *Arch Intern Med* (2011).

2005.

no. 10 (2001): 968-77.

(2008): 637-44.

(2011): 13-5.

*Spine* 29, no. 1 (2004): 79-86.

There Causing Bad Karma over Here." *The Independent. (U. K.)* , Sunday, 30 January

Maldonado, D. Duran, A. H. Kaplan, and N. S. Wenger. "A Comparison Study of Multiple Measures of Adherence to Hiv Protease Inhibitors." *Ann Intern Med* 134,

Health Care Expenditures among Individuals with Back Pain in the United States."

"Yoga for Chronic Low Back Pain in a Predominantly Minority Population: A Pilot Randomized Controlled Trial." Altern Ther Health Med 15, no. 6 (2009): 18-27. Sherman, K. J., D. C. Cherkin, J. Erro, D. L. Miglioretti, and R. A. Deyo. "Comparing Yoga,

Exercise, and a Self-Care Book for Chronic Low Back Pain: A Randomized,

Deyo. "A Randomized Trial Comparing Yoga, Stretching, and a Self-Care Book for

Yoga Program on Pain, Functional Disability and Spinal Flexibility in Chronic Low Back Pain: A Randomized Control Study." J Altern Complement Med 14, no. 6

Aplin, A. Semlyen, A. Trewhela, I. Watt, and D. J. Torgerson. "Yoga for Chronic Low Back Pain: A Randomized Trial." *Ann Intern Med* 155, no. 9 (2011): 569-78. Ülger, Ö., and N. V. Yağli. "Effects of Yoga on Balance and Gait Properties in Women with

Musculoskeletal Problems: A Pilot Study." Complement Ther Clin Pract 17, no. 1

Juckett, M. Munoz Kolar, R. Gross, and L. Steinberg. "Effect of Iyengar Yoga

Kelley, and L. Cooper. "Evaluation of the Effectiveness and Efficacy of Iyengar Yoga Therapy on Chronic Low Back Pain." *Spine (Phila Pa 1976)* 34, no. 19 (2009): 2066-76.

### *Edited by Ali Asghar Norasteh*

This book includes two sections. Section one is about basic science, epidemiology, risk factors and evaluation, section two is about clinical science especially different approach in exercise therapy. I envisage that this book will provide helpful information and guidance for all those practitioners involved with managing people with back pain-physiotherapists, osteopaths, chiropractors and doctors of orthopedics, rheumatology, rehabilitation and manual medicine. Likewise for students of movement and those who are involved in re-educating movement-exercise physiologists, Pilates and yoga teachers etc.

Low Back Pain

Low Back Pain

*Edited by Ali Asghar Norasteh*

Photo by Eraxion / iStock


**3. Fibroblast cells**

#### **3.1. Human gingival fibroblasts**

#### *3.1.1. Generality*

Fibroblasts are spindle-shaped connective-tissue cells of mesenchymal origin that secretes proteins and especially molecular collagen from which the extracellular fibrillar matrix of connective tissue forms. They have oval or circular nucleus and a little developed cyto‐ plasm giving rise to long prolongation forms [34]. These cells do not have a basal lamina and their surfaces are often in contact with the fibers of the collagen. Their cytoplasm contains a rough endoplasmic reticulum, an important Golgi apparatus, few mitochon‐ dria and a little bit quantity of cytoplasmic filaments. Fibroblasts synthesize enormous quantities of the extracellular matrix constituents. Indeed, the majority part of the extrac‐ ellular matrix components consists of collagen made in the intracellular space where fi‐ broblasts sustain structural modifications.

#### *3.1.2. Gingival tissue*

It's the tissue that surrounds the necks of teeth and covers the alveolar parts of the jaws; broadly: the alveolar portion of a jaw with its enveloping soft tissues [35]. It consists in a pink connective tissue with fibrous collagen surrounded by an epithelial tissue. Its pink col‐ or changes from one person to another, depending on pigmentation, epithelium thickness, its keratinization level and on the underlying vascularization [36]. Fibroblasts are the basic component of the gingival chorion whose intercellular matrix is essentially formed by colla‐ gen and elastin.

#### **3.2. Cell-Biomaterial: Interface and interactions**

#### *3.2.1. Biocompatibility concept*

While a cell is in contact with a biomaterial, many reactions can occur and a sensing phe‐ nomenon will launch between this cell and the biomaterial [37]. Indeed, the cell has a signal network reached as a result of the surface exploration and sensing made in order to verify whether the new environment (biomaterial) is in accordance with its expected physiological conditions necessary for a normal biological activity [38]. Thus, before putting a new materi‐ al in contact with a cell it's of a great importance to choose the corresponding material in such a way that this material obey the cell's norm by not being toxic or injurious and not causing immunological rejection. In one word, this material must be biocompatible.

The biological tolerance of a biomaterial led scientists to regroup the different parameters and mechanisms controlling the interface biomaterial/cell (or tissue) so that they can deduce a concrete and a common definition for biocompatibility concept. Indeed, biocompatibility includes the understanding of the interactive mechanisms relating the biomaterial with its biological environment. Generally, biocompatibility represents the ability of a material to be accepted by a living organism.

In 1987, Williams D.F suggested the following definition «biocompatibility is the ability of a material to be used with an appropriate and suitable reaction of the host for a spe‐ cific application».

According to Exbrayat [39] « biocompatibility is a set of the different interrelations between a biomaterial and its environment, and their biological local or general consequences, imme‐ diate or delayed, reversible or definitive».

Indeed, biocompatibility is a group of networks that liaises between the biomaterial and its environment and takes into account the possible effect of this biomaterial on its environ‐ ment and vice versa. Interactions existing in the interface biomaterial/biological environ‐ ment differ by their intensity and their duration period depending both on the biomaterial and on the tissue in contact.

Characterizing the surface properties of a biomaterial before putting it in contact with a cell seems to be an obligation. This step allows us to know about different parameters and char‐ acters of this biomaterial (topography, roughness, surface energy etc.) in order to find a cor‐ relation with the cell behavior and therefore we can adjust these physico-chemical properties, when making the biomaterial, so that we have a normal and physiological cell behavior in contact with that biomaterial.

#### *3.2.2. Cell adhesion*

component of the gingival chorion whose intercellular matrix is essentially formed by colla‐

While a cell is in contact with a biomaterial, many reactions can occur and a sensing phe‐ nomenon will launch between this cell and the biomaterial [37]. Indeed, the cell has a signal network reached as a result of the surface exploration and sensing made in order to verify whether the new environment (biomaterial) is in accordance with its expected physiological conditions necessary for a normal biological activity [38]. Thus, before putting a new materi‐ al in contact with a cell it's of a great importance to choose the corresponding material in such a way that this material obey the cell's norm by not being toxic or injurious and not

causing immunological rejection. In one word, this material must be biocompatible.

The biological tolerance of a biomaterial led scientists to regroup the different parameters and mechanisms controlling the interface biomaterial/cell (or tissue) so that they can deduce a concrete and a common definition for biocompatibility concept. Indeed, biocompatibility includes the understanding of the interactive mechanisms relating the biomaterial with its biological environment. Generally, biocompatibility represents the ability of a material to be

In 1987, Williams D.F suggested the following definition «biocompatibility is the ability of a material to be used with an appropriate and suitable reaction of the host for a spe‐

According to Exbrayat [39] « biocompatibility is a set of the different interrelations between a biomaterial and its environment, and their biological local or general consequences, imme‐

Indeed, biocompatibility is a group of networks that liaises between the biomaterial and its environment and takes into account the possible effect of this biomaterial on its environ‐ ment and vice versa. Interactions existing in the interface biomaterial/biological environ‐ ment differ by their intensity and their duration period depending both on the biomaterial

Characterizing the surface properties of a biomaterial before putting it in contact with a cell seems to be an obligation. This step allows us to know about different parameters and char‐ acters of this biomaterial (topography, roughness, surface energy etc.) in order to find a cor‐ relation with the cell behavior and therefore we can adjust these physico-chemical properties, when making the biomaterial, so that we have a normal and physiological cell

gen and elastin.

*3.2.1. Biocompatibility concept*

accepted by a living organism.

and on the tissue in contact.

diate or delayed, reversible or definitive».

behavior in contact with that biomaterial.

cific application».

**3.2. Cell-Biomaterial: Interface and interactions**

218 Advances in Biomaterials Science and Biomedical Applications

It is well known that during the contact between a cell and a material, information will be transferred from the material surface to the cell and this contact will induce, in return, an alteration to the material. This situation may cause material remodelling [40,22].

Cells adhere to surfaces through adhesion proteins (i.e. fibronectin, collagen, laminin, vitro‐ nectin) using specific cell receptors, called integrins, attached to the cell membrane. Indeed, when fibroblasts grow on a substrate, most of their cell surface is separated from the sub‐ stratum by a gap of more than 50 nm; but at focal contacts, this gap is reduced to 10 to 15 nm. The main transmembrane linker proteins of focal contacts belong to the integrin family and the cytoplasmic domain of the integrin binds to the protein talin, which in turn binds to vinculin, a protein found also in other actin-containing cell junction. Vinculin associates with α-actinin and is thereby linked to an actin filament [1].

Besides their role as anchors, focal contacts can also relay signals from the extracellular ma‐ trix (ECM) to the cytoskeleton. Several protein kinases are localized to focal contacts and seems to change their activity with the type of the substratum on which the rest. These kin‐ ases can regulate the survival, growth, morphology, movement, and differentiation of cells in response to new environment. Figure 5 shows a possible arrangement of these different proteins during a focal contact.

**Figure 5.** Adhesion proteins involved in focal contacts

The formation of focal contacts occurs when the binding of matrix glycoprotein, such as fi‐ bronectin, on the outside of the cell causes the integrin molecules to cluster at the contact site. Fibronectins are associated together by proteoglycans and constitute thins fibers of the extracellular matrix (ECM).

#### *<IP_ADDRESS>. Extracellular matrix*

The extracellular matrix (ECM) represents an important element in the processes of cell ad‐ hesion. Indeed, at this level, cell adhesion is under the control of a well defined zone in the cytoplasmic membrane called focal contact. At this zone, filaments of actin are linked to fi‐ bronectin through an intracellular complex of proteins, the adherence complex. The extracel‐ lular matrix (ECM) is made of different proteins such as fibronectins, collagen, laminin, vitronectin [41] and represents the mediator of cell adhesion thanks to its integrins.

Although the extracellular matrix generally provides mechanical support to tissues, it serves several other functions as well. Different combinations of ECM components tailor the extrac‐ ellular matrix for specific purposes: strength in a tendon, tooth, or bone; cushioning in carti‐ lage; and adhesion in most tissues. In addition, the composition of the matrix, which can vary, depending on the anatomical site and physiological status of a tissue, can let a cell know where it is and what it should do (environmental cues). Changes in ECM components, which are constantly being remodeled, degraded, and resynthesized locally, can modulate the interactions of a cell with its environment. The matrix also serves as a reservoir for many extracellular signalling molecules that control cell growth and differentiation. In addition, the matrix provides a lattice through or on which cells can move, particularly in the early stages of tissue assembly [42].

Many functions of the matrix require transmembrane adhesion receptors that bind directly to ECM components and that also interact, through adapter proteins, with the cytoskeleton. The principal class of adhesion receptors that mediate cell–matrix adhesion are integrins, a large family of αβ heterodimeric cell surface proteins that mediate both cell–cell and cell– matrix adhesions and inside-out and outside-in signalling in numerous tissues.

#### *<IP_ADDRESS>. Adhesion proteins and receptors in fibroblast cells*

Different proteins and their receptors are involved in fibroblast cells adhesion process. The most important and known are fibronectins and their receptors; integrins:

**•** Fibronectins

Fibronectins are dimers of two similar polypeptides linked at their C-termini by two di‐ sulfide bonds; each chain is about 60–70 nm long and 2–3 nm thick. The combination of different repeats composing the regions, another example of combinatorial diversity, con‐ fers on fibronectin its ability to bind multiple ligands [40].

Fibronectins help attach cells to the extracellular matrix by binding to other ECM compo‐ nents, particularly fibrous collagens and heparan sulfate proteoglycans, and to cell sur‐ face adhesion receptors such as integrins. Through their interactions with adhesion receptors (e.g., α5β1 integrin), fibronectins influence the shape and movement of cells and the organization of the cytoskeleton. Conversely, by regulating their receptor-mediated attachments to fibronectin and other ECM components, cells can sculpt the immediate ECM environment to suit their needs.

**•** Integrins

Integrins are the principle adhesion receptors; a large family of αβ heterodimeric cell sur‐ face proteins that mediate both cell–cell and cell–matrix. They are transmembrane pro‐ teins that mediate interactions between adhesion molecules on adjacent cells and/or the extracellular matrix (ECM). They have diverse roles in several biological processes includ‐ ing cell migration during development and wound healing, cell differentiation, and apop‐ tosis. Their activities can also regulate the metastatic and invasive potential of tumor cells. They exist as heterodimers consisting of alpha and beta subunits. Some alpha and beta subunits exhibit specificity for one another, and heterodimers often preferentially bind certain cell adhesion molecules, or constituents of the ECM.

Although they themselves have no catalytic activity, integrins can be part of multimolecu‐ lar signalling complexes known focal adhesions. The two subunits, designated as alpha and beta, both participate in binding.

**Figure 6.** Fibronectin binding to its Integrin receptor (adapted from internet)

Integrins participate in cell-cell adhesion and are of great importance in binding and interac‐ tions of cells with components of the extracellular matrix such as fibronectin. Importantly, integrins facilitate "communication" between the cytoskeleton and extracellular matrix; al‐ low each to influence the orientation and structure of the other. It is clear that interactions of integrins with the extracellular matrix can have profound effects on cell function, and events such as clustering of integrins activates a number of intracellular signally pathways.

#### *3.2.3. Cell adhesion: The physical process*

*<IP_ADDRESS>. Extracellular matrix*

220 Advances in Biomaterials Science and Biomedical Applications

stages of tissue assembly [42].

**•** Fibronectins

**•** Integrins

The extracellular matrix (ECM) represents an important element in the processes of cell ad‐ hesion. Indeed, at this level, cell adhesion is under the control of a well defined zone in the cytoplasmic membrane called focal contact. At this zone, filaments of actin are linked to fi‐ bronectin through an intracellular complex of proteins, the adherence complex. The extracel‐ lular matrix (ECM) is made of different proteins such as fibronectins, collagen, laminin,

Although the extracellular matrix generally provides mechanical support to tissues, it serves several other functions as well. Different combinations of ECM components tailor the extrac‐ ellular matrix for specific purposes: strength in a tendon, tooth, or bone; cushioning in carti‐ lage; and adhesion in most tissues. In addition, the composition of the matrix, which can vary, depending on the anatomical site and physiological status of a tissue, can let a cell know where it is and what it should do (environmental cues). Changes in ECM components, which are constantly being remodeled, degraded, and resynthesized locally, can modulate the interactions of a cell with its environment. The matrix also serves as a reservoir for many extracellular signalling molecules that control cell growth and differentiation. In addition, the matrix provides a lattice through or on which cells can move, particularly in the early

Many functions of the matrix require transmembrane adhesion receptors that bind directly to ECM components and that also interact, through adapter proteins, with the cytoskeleton. The principal class of adhesion receptors that mediate cell–matrix adhesion are integrins, a large family of αβ heterodimeric cell surface proteins that mediate both cell–cell and cell–

Different proteins and their receptors are involved in fibroblast cells adhesion process. The

Fibronectins are dimers of two similar polypeptides linked at their C-termini by two di‐ sulfide bonds; each chain is about 60–70 nm long and 2–3 nm thick. The combination of different repeats composing the regions, another example of combinatorial diversity, con‐

Fibronectins help attach cells to the extracellular matrix by binding to other ECM compo‐ nents, particularly fibrous collagens and heparan sulfate proteoglycans, and to cell sur‐ face adhesion receptors such as integrins. Through their interactions with adhesion receptors (e.g., α5β1 integrin), fibronectins influence the shape and movement of cells and the organization of the cytoskeleton. Conversely, by regulating their receptor-mediated attachments to fibronectin and other ECM components, cells can sculpt the immediate

matrix adhesions and inside-out and outside-in signalling in numerous tissues.

most important and known are fibronectins and their receptors; integrins:

fers on fibronectin its ability to bind multiple ligands [40].

ECM environment to suit their needs.

*<IP_ADDRESS>. Adhesion proteins and receptors in fibroblast cells*

vitronectin [41] and represents the mediator of cell adhesion thanks to its integrins.

Biological systems exhibit electromagnetic activity in a wide frequency range from the static or quasistatic electric field to optical bands. Fröhlich [43] presumed that biological matter has anomalous polarization properties (e.g. induction of great electric dipole after electric field application). Static charge distribution of dipole and/or multipole nature exists (e.g. in protein molecules). Vibrations in biological molecules, therefore, generate an electromagnet‐ ic field [44]. Pokorny et al.[45], assume that the Fröhlich electromagnetic field can be a fun‐ damental factor of cell adherence.

Surface topography is of an important interest in cell adhesion as well as its chemical com‐ position. Indeed, it has been shown that cells adhere and proliferate depending on the sur‐ face roughness and the more the surface is rough the more cell adhesion and proliferation is better [46]. This effect depends on the cell type. For fibroblasts, they line up along the bioma‐ terial surface microstructures and may adapt their shape with uneven surfaces.

Moreover, recent studies had shown that a weak change in the surface roughness may in‐ duce different cell reactions such as change in their shape and their way of adhesion [47, 48].

#### *<IP_ADDRESS>. Forces involved in cell adhesion*

According to Richards [49], cell adhesion to biomaterials is done thanks to focal adhesion sites which represent strict contact sites with the substrate in a so limited space. For fibro‐ blasts, it has been shown the existence of a force called cohesion force responsible for keep‐ ing contact between cells themselves. However, this force is weaker than the adhesion force involved while a cell adheres to a biomaterial. This difference in force level depends on the cell type and on the nature of the biomaterial used for adhesion, and may explain the differ‐ ent ways of cell adhesion and spreading on different surface structures.

#### *<IP_ADDRESS>. Surface free energy*

Surface free energy is a thermodynamic measurement which contributes to the interpreta‐ tion of the phenomena occurring in interfaces. It has an important effect on cell adhesion in the way that every change in its value induces the modification of the surface wettability, and therefore cell behaviour will be affected too [50, 51, 52].

Cell-biomaterial interface depends on the physico-chemical properties of the biomaterial and every change in the chemical composition or in the electric charge of the surface will affect its surface free energy.

#### *3.2.4. Parameters involved in cell adhesion*

#### *<IP_ADDRESS>. Surface roughness*

Surface roughness has been the subject of many studies as a deciding factor in the process of cell adhesion to biomaterials. Ponsonnet et al.[53] had studied the behaviour of fibroblast cells while adhering to titanium surface with different roughness; they found that cells had adhered to the surface using thin cytoplasmic structures. Indeed, these cells presented a flat‐ tened shape spreading practically over the substrate surface after adhesion to smooth surfa‐ ces. However, on rough surfaces, cell morphology was affected by the surface grooves and they were reoriented by the surface structure.

According to Richards [48], smooth titanium surfaces always increase fibroblasts adhesion and proliferation better than rough surfaces. They suggested that this kind of surfaces should be a better candidate for biological implant thanks to their ability to resist to bacterial infections. Indeed, their weak roughness is unfavourable to the adhesion of bacteria.

#### *<IP_ADDRESS>. The electric charge effect*

ic field [44]. Pokorny et al.[45], assume that the Fröhlich electromagnetic field can be a fun‐

Surface topography is of an important interest in cell adhesion as well as its chemical com‐ position. Indeed, it has been shown that cells adhere and proliferate depending on the sur‐ face roughness and the more the surface is rough the more cell adhesion and proliferation is better [46]. This effect depends on the cell type. For fibroblasts, they line up along the bioma‐

Moreover, recent studies had shown that a weak change in the surface roughness may in‐ duce different cell reactions such as change in their shape and their way of adhesion [47, 48].

According to Richards [49], cell adhesion to biomaterials is done thanks to focal adhesion sites which represent strict contact sites with the substrate in a so limited space. For fibro‐ blasts, it has been shown the existence of a force called cohesion force responsible for keep‐ ing contact between cells themselves. However, this force is weaker than the adhesion force involved while a cell adheres to a biomaterial. This difference in force level depends on the cell type and on the nature of the biomaterial used for adhesion, and may explain the differ‐

Surface free energy is a thermodynamic measurement which contributes to the interpreta‐ tion of the phenomena occurring in interfaces. It has an important effect on cell adhesion in the way that every change in its value induces the modification of the surface wettability,

Cell-biomaterial interface depends on the physico-chemical properties of the biomaterial and every change in the chemical composition or in the electric charge of the surface will

Surface roughness has been the subject of many studies as a deciding factor in the process of cell adhesion to biomaterials. Ponsonnet et al.[53] had studied the behaviour of fibroblast cells while adhering to titanium surface with different roughness; they found that cells had adhered to the surface using thin cytoplasmic structures. Indeed, these cells presented a flat‐ tened shape spreading practically over the substrate surface after adhesion to smooth surfa‐ ces. However, on rough surfaces, cell morphology was affected by the surface grooves and

According to Richards [48], smooth titanium surfaces always increase fibroblasts adhesion and proliferation better than rough surfaces. They suggested that this kind of surfaces

terial surface microstructures and may adapt their shape with uneven surfaces.

ent ways of cell adhesion and spreading on different surface structures.

and therefore cell behaviour will be affected too [50, 51, 52].

damental factor of cell adherence.

222 Advances in Biomaterials Science and Biomedical Applications

*<IP_ADDRESS>. Forces involved in cell adhesion*

*<IP_ADDRESS>. Surface free energy*

affect its surface free energy.

*<IP_ADDRESS>. Surface roughness*

*3.2.4. Parameters involved in cell adhesion*

they were reoriented by the surface structure.

In the majority of the studies carried out about biomaterials made from polyelectrolyte film, as in our case, the electric charge effect is in proportion with the thickness of the film built and depends on the charged functional group of the polyelectrolyte used [54].

For Andrade [25], the notion of the nature of an electric charge is important to be mentioned but its effect is not significant and doesn't induce an efficient change on surface wettability. However, it has been shown that a better adhesion of cells was observed on negatively charged polyelectrolyte [55]. In reality, most of the existed cells and their corresponding ad‐ hesion proteins are negatively charged. Nevertheless, this charge can be without any effect in the case when functional groups become able to control cell adhesion mechanism by their hydrophilic or hydrophobic character as it will be shown later in this text. Dubois [56] pre‐ sumed that an electric charge trapped within an insulating biomaterial, none associated to a particular chemical group, is able to affect its biological environment. Moreover, Maroudas [57] revealed the dependence of cell adhesion and spreading on a solid surface on the sur‐ face charge of the substrate.

#### *<IP_ADDRESS>. Chemical composition*

The different chemical components of a biomaterial must be studied and known before to start investigating cell adhesion to that biomaterial. Therefore, this step is fundamen‐ tal for concluding about the biocompatibility of a given biomaterial and its effect on cell adhesion [58].

The wettability of a surface depends on the chemical composition of the material and each change than can occur at this level will disturb cell adhesion process [59]. Besides the effect of the biomaterial, the adhered cell type plays an important role in adhesion. Indeed, for the same biomaterial surface, different cell reactions were observed for two types of cells [60]; this kind of biomaterial seems to be biocompatible with one cell type but not tolerated by the other cell type.

According to Marmur [61], most of the materials in the nature are rough and heterogeneous and contact angle may change along the contact line with a value depending on the rough‐ ness and heterogeneity level.

#### *<IP_ADDRESS>. Surface hydrophilicity and hydrophobicity*

Contact angle measurement allows us to calculate surface free energy [62]. It also allows knowing about the polar or non polar nature of the interactions at the interface liquid/ solid. Moreover, one can deduce from it the hydrophilic or the hydrophobic character of a surface [63].

A study about polyelectrolyte films found that hydrophobic interactions on a surface in‐ duce the adsorption of proteins and stabilise the complex formed [64]. Indeed, it has been proved that myoglobin or lysozymes are able to adhere to polystyrene sulfonate (PSS) and form many layers. However, this adhesion was not possible when using an‐ other surface having the same electric charge as PSS but with a hydrophilic character. The electrostatic interactions between the protein complex and this hydrophilic surface were easily destructed after water rinsing. Thus, surface hydrophilicity and hydrophobic‐ ity are a determinant parameter for substrate wettability on account of the rearrange‐ ment of the functional groups at the surface of a biomaterial in contact with a cell [65, 66, 67]. Indeed, it has been shown that fibroblast cells adhere and proliferate better on biomaterials with a moderate hydrophilicity [68, 69].

Andrade [66] presumed that, in the case of deformable materials, an elasticity model of 3.5 105 dyn/cm2 is necessary for avoiding contact angle change. A roughness below 0.1 μm has a negligible effect on contact angle. Most of the materials holding over than 20 to 30 % of wa‐ ter present a receding contact angle (θr), in water, near zero because of the hydrophilic char‐ acter which dominates the interface in these conditions. The same author estimated that the majority of polymers have a changeable volume which can be the reason for contact angle change: this change is depending on the duration of the contact with water, on the nature of the liquid and on the temperature of measurement. Non existent contact angle hysteresis may be due to the duration of contact between the material and the liquid which is shorter or longer than the measurement time needed for recording contact angle change. Therefore, surface hydrophilicity and hydrophobicity depends on the volume blowing of the material, on the diffusion phenomenon and on the mobility and reorientation of the molecules on the material surface.

Some materials are able to go out of shape in contact with a liquid depending on their me‐ chanical properties and on their relaxation time and temperature. So, what characterizes a polymer is its chemical composition, roughness, mobility, wettability, surface free energy and its electric charge [70].

#### *<IP_ADDRESS>. Surface wettability: Contact angle hysteresis*

Contact angle hysteresis is the result of contact angle change between the surface we are characterizing and another ideal surface physico-chemically homogeneous. It's the direct re‐ sult of a different sensitivity to the wettability process of heterogeneous surfaces. According to Rupp et al.[71], the receding contact angle value (θr) is under the control of the small hy‐ drophilic particles of the surface which are able to disturb or to delay the non wettability process. Indeed, when the hysteresis remains constant after many immersion and emersion cycles it's called thermodynamic (or true) hysteresis. However, in the opposite case, it's called kinetic hysteresis (see Figure 7).

Thermodynamic hysteresis is due to the surface roughness and heterogeneity. Nevertheless, kinetic hysteresis is caused by the adsorption mechanisms (due to the liquid phase), surface polar group's reorientation and surface deformation [24].

A study about polyelectrolyte films found that hydrophobic interactions on a surface in‐ duce the adsorption of proteins and stabilise the complex formed [64]. Indeed, it has been proved that myoglobin or lysozymes are able to adhere to polystyrene sulfonate (PSS) and form many layers. However, this adhesion was not possible when using an‐ other surface having the same electric charge as PSS but with a hydrophilic character. The electrostatic interactions between the protein complex and this hydrophilic surface were easily destructed after water rinsing. Thus, surface hydrophilicity and hydrophobic‐ ity are a determinant parameter for substrate wettability on account of the rearrange‐ ment of the functional groups at the surface of a biomaterial in contact with a cell [65, 66, 67]. Indeed, it has been shown that fibroblast cells adhere and proliferate better on

Andrade [66] presumed that, in the case of deformable materials, an elasticity model of 3.5

Some materials are able to go out of shape in contact with a liquid depending on their me‐ chanical properties and on their relaxation time and temperature. So, what characterizes a polymer is its chemical composition, roughness, mobility, wettability, surface free energy

Contact angle hysteresis is the result of contact angle change between the surface we are characterizing and another ideal surface physico-chemically homogeneous. It's the direct re‐ sult of a different sensitivity to the wettability process of heterogeneous surfaces. According to Rupp et al.[71], the receding contact angle value (θr) is under the control of the small hy‐ drophilic particles of the surface which are able to disturb or to delay the non wettability process. Indeed, when the hysteresis remains constant after many immersion and emersion cycles it's called thermodynamic (or true) hysteresis. However, in the opposite case, it's

Thermodynamic hysteresis is due to the surface roughness and heterogeneity. Nevertheless, kinetic hysteresis is caused by the adsorption mechanisms (due to the liquid phase), surface

 dyn/cm2 is necessary for avoiding contact angle change. A roughness below 0.1 μm has a negligible effect on contact angle. Most of the materials holding over than 20 to 30 % of wa‐ ter present a receding contact angle (θr), in water, near zero because of the hydrophilic char‐ acter which dominates the interface in these conditions. The same author estimated that the majority of polymers have a changeable volume which can be the reason for contact angle change: this change is depending on the duration of the contact with water, on the nature of the liquid and on the temperature of measurement. Non existent contact angle hysteresis may be due to the duration of contact between the material and the liquid which is shorter or longer than the measurement time needed for recording contact angle change. Therefore, surface hydrophilicity and hydrophobicity depends on the volume blowing of the material, on the diffusion phenomenon and on the mobility and reorientation of the molecules on the

biomaterials with a moderate hydrophilicity [68, 69].

224 Advances in Biomaterials Science and Biomedical Applications

105

material surface.

and its electric charge [70].

*<IP_ADDRESS>. Surface wettability: Contact angle hysteresis*

called kinetic hysteresis (see Figure 7).

polar group's reorientation and surface deformation [24].

**Figure 7.** Immersion and emersion loops showing the two types of hysteresis: (A): thermodynamic hysteresis and (B): kinetic hysteresis. The sample is repeatedly immersed in the liquid leading to typical hysteresis loops. From each loop, wettability parameters (advancing and receding contact angle or wetting tension) can be calculated

Contact angle hysteresis is often assigned to the surface roughness and heterogeneity. Actually, a study made by Lam et al. [26], have shown that hysteresis is related to the molecules' mobility, the liquid diffusion and the surface swelling. These authors had ob‐ served a close dependence between the liquid molecules size and the liquid/material con‐ tact duration. Liquid resorption and retention are the direct causes of hysteresis. However, as the liquid surface free energy is higher that that of the material; therefore the liquid retention into the material will increase the material surface free energy and thus reduces the receding contact angle (θr). Indeed, liquids having smaller molecular chains (or smaller molecular weight) diffuse faster into the polymer surface leading to an important decrease in contact angle.

According to Shananan et al.[72], contact angle hysteresis is related to the polymer polarity. Indeed, when a polymer gets in touch with a polar liquid (water), it orients its mobile polar groups on the surface in order to increase the interfacial water/polymer energy and there‐ fore decreasing the system surface free energy. In the other hand, when the polymer is con‐ tact with a non polar liquid, its functional groups conserve their state and will not reorient. These authors assumed the existence of two parameters behind hysteresis: the intrinsic po‐ larity of the material and the mobility of its polar groups on the surface. Nishioka et al.[73], had observed that the advancing contact angle hysteresis is under the control of surface sites more hydrophobic than those controlling the receding contact angle hysteresis.

The contact angle hysteresis observed on hydrophilic and hydrated polymers is due to the polar groups' orientation on the interfaces polymer/liquid and polymer/air. This reorienta‐ tion represents the polymer reaction to every environmental change (air, liquid). The reced‐ ing contact angle (θr) depends on the contact duration with water, the environment temperature and on the glass transition temperature (Tg) of the material itself. Each material has its own glass transition temperature (Tg) allowing a defined molecular mobility suffi‐ cient for an important rearrangement [74].

#### **3.3. Conclusion**

The concepts of solid surfaces assumed that the surfaces in question were effectively rigid and immobile. Such assumptions allow one to develop certain models and mathematical re‐ lationships useful for estimating and understanding surface energies, surface stresses, and specific interactions, such as adsorption, wetting, and contact angles. It is assumed that the surfaces themselves do not change or respond in any specific way to the presence of a con‐ tacting liquid phase, thereby altering their specific surface energy [75]. Although such as‐ sumptions are (or may be) valid for truly rigid crystalline or amorphous solids, they more often than not do not apply strictly to polymeric surfaces.

In contact with condensed phases, especially liquids, surface relaxations and transitions can become quite important leading to a possible dramatically change in the interfacial charac‐ teristics of a polymer with possibly important consequences in a particular application. And since the processes are time-dependent, the changes may not be evident over the short span of a normal experiment. For critical applications in which a polymer surface will be in con‐ tact with a liquid phase, such as implant device for biomedical application, it is not only im‐ portant to know the surface characteristics (e.g., coefficient of friction, adhesion, adsorption)under normal experimental conditions but also to determine the effects of pro‐ longed (equilibrium) exposure to the liquid medium of interest. It is therefore important for biomedical as well as many other applications that the surface characteristics of a material of interest be determined under conditions that mimic as closely as possible the conditions of use and over extended periods of exposure to those conditions, in addition to the usual characterizations.