*Article* **An Ultra-High-Q Lithium Niobate Microresonator Integrated with a Silicon Nitride Waveguide in the Vertical Configuration for Evanescent Light Coupling**

**Jianhao Zhang 1,2, Rongbo Wu 1,2, Min Wang 3,4,\*, Youting Liang 3,4, Junxia Zhou 3,4, Miao Wu 3,4, Zhiwei Fang 3,4 , Wei Chu 4,\* and Ya Cheng 1,3,4,5,6,7,8,\***

**Abstract:** We demonstrate the hybrid integration of a lithium niobate microring resonator with a silicon nitride waveguide in the vertical configuration to achieve efficient light coupling. The microring resonator is fabricated on a lithium niobate on insulator (LNOI) substrate using photolithography assisted chemo-mechanical etching (PLACE). A fused silica cladding layer is deposited on the LNOI ring resonator. The silicon nitride waveguide is further produced on the fused silica cladding layer by first fabricating a trench in the fused silica while using focused ion beam (FIB) etching for facilitating the evanescent coupling, followed by the formation of the silicon nitride waveguide on the bottom of the trench. The FIB etching ensures the required high positioning accuracy between the waveguide and ring resonator. We achieve Q-factors as high as 1.4 × 107 with the vertically integrated device.

**Keywords:** lithium niobate microring resonator; silicon nitride waveguide; photolithography assisted chemo-mechanical etching


*4.4. Statistical Design and Analysis*

Measurements of growth dynamics of internodes, petioles and leaflets of each phytomer were used to fit the beta-function (Equation (3)) [51]:

$$L(t) = L\_{\max} \left( 1 + \frac{t\_\ell - t}{t\_\ell - t\_m} \right) \left( \frac{t}{t\_\ell} \right)^{\frac{t\_\ell}{t\_\ell - t\_m}} \ 0 \le t\_m < t\_\ell \tag{3}$$

$$L(t > t\_\ell \ ) \ = L\_{\max}$$

where *L(t)* is the size at day *t*, *Lmax* is the final size, *te* is the day when the final size is reached and *tm* is the day on which the growth rate peaks. The parameters *Lmax*, *te* and *tm* were estimated with the nls-function in the R-package stats [52].

To determine the plants used for the destructive measurements during each experiment, randomizations were performed within two blocks (plant location). The first block comprised the first and last row and the second block the two center rows (Figure 7).

The six light treatments of the experiment were performed with two replicates. Given three LED chambers, three out of the six light treatments could be tested in the same run, i.e., each replicate comprised two runs, resulting in four runs in total (Supplementary Materials, S1). For the arrangement of treatments within the LED chambers over time and space, an α-design with two replicates and a block (time) size of two was used. The effect of BPFD on biomass, morphology, leaf physiology and parameters of the beta-function was tested. The second and mostly third phytomer (hypocotyl counted as first phytomer) were chosen for specific analysis, because they comprised the most comprehensive measurements from beginning to end of growth. According to the experimental design, the following mixed model was used to analyze the data in the SAS® software (SAS Institute, Inc., Cary, NC, USA):

$$y\_{iklmu} = \mu + b\_k + i\_{kl} + p\_{klm} + \tau\_{klmn} + \tau\_i + e\_{iklmn} \tag{4}$$

where µ is the intercept, *bk* is the fixed effect of the *k* th complete replicate, *ikl* is the random effect of the *l* th incomplete block (time) within the *k* th replicate, *pklm* is the random effect of the *m*th chamber within the *l* th run, *rklmn* is the random effect of the *n* th block (plant location) within the *m*th chamber of the *l* th run and the *k* th replicate, τ*i* is the main effect of the *i* th light treatment, and *eiklmn* is the error effect of observation *yiklmn* with homogeneous variance. Residuals were checked graphically for normal distribution and homogeneous variance. After finding significant effects via F-test, a multiple t-test to compare least square means was used to create a letter display [53]. Note that least square means are presented in the results section as data was not balanced, because only three out of six light treatments were tested within each run. Least square means are based on model (Equation (4)) to adjust for block effects.

**Figure 7.** Illustration of the randomization for the plants used for the destructive measurements within the first (light blue square) and second (dark blue square) block (plant location). The numbers exemplarily show the day of the destructive measurements the plants were used for.


**CHRONIC LYMPHOCYTIC LEUKEMIA**

Edited by **Pablo Oppezzo**

**INTECHOPEN.COM**

http://dx.doi.org/10.5772/1156 Edited by Pablo Oppezzo

### **Contributors**

Segundo Gonzalez, Günter Krause, Mirjam Kuckertz, Susan Kerwien, Michaela Patz, Michael Hallek, Juan M Zapata, Gema Perez-Chacon, José-Ángel Hernandez, Marcos González, Jesús-María Hernández, Pablo Oppezzo, Florencia Palacios, Cecilia Abreu, Pilar Moreno, Romina Gamberale, Mirta Giordano, Dariusz Wolowiec, Marcin Wojtowicz, Jozo Delic, Karim Maloum, Florence Nguyen-Khac, Véronique Leblond, Jacques-Louis Binet, Fréderic Davi, Zahia Agui, Sylvie Chevillard, Hélène Merle-Beral, Jean-Brice Marteau, Lidia Karabon, Irena Frydecka, Ida Franiak-Pietryga, Marek Mirowski, Zivadin Bugarcic, Biljana Petrovic, Jovana Bogojeski, Elizabeth Raveche, Siddha Kasar, Yao Yuan, Chingiz Underbayev, Daniel Vollenweider, Matthew Hanlon, Victor Chang, Hina Khan, Joseph Slupsky, John Allen, Farhad Abbasi, Ans Rombout, Jan Philippé, Valerie Pede, Bruno Verhasselt, Lucienne Michaux, Natalie Put, Iwona Wlodarska, Peter Vandenberghe, Lina Reslan, Charles Dumontet, John H. H. Williams, Nina Dempsey-Hibbert, Christine Hoyle, Gustavo H. Marin, Eduardo Mansilla, Alfonso Cayota, Rosa Garcia-Silva, Catalina Guida, Guillermo Dighiero

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First published in Croatia, 2012 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia

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27 Marcia Ochoa observes that glamour is often invoked as a "form of power" that enables "legibility," "affirmation" and "survival." (Ochoa 2014, pp. 89–90).

the field of representations mobilized by Alabanza expands visual examples of gender presentations for subjects to emulate and brings new modes of intersectional identities into being. This work begins to create space for new aesthetics of beauty, not measured against dominant systems, but celebrated for their multiplicity and transgressiveness.28

#### **10. Parting Thoughts**

The long-established art historical hierarchy around portraiture based on the method of creation, venue of the exhibition of the work, and the artist's relative situatedness in the art world is not only outmoded, but such thinking leads to critical blind spots, in need of reassessment in order to keep art discourse relevant. Measuring the success of a portrait or self-portrait is more productive when based on its conceptual underpinnings, intellectual rigor, and intervention into various visual matrixes. So, when we assess photographic representations, I propose we ask such questions as: How does the image challenge various ideological structures? How does it intervene in visual culture? How does it disrupt previous established aesthetics, methods and hierarchies? It behooves us to change our assessment of contemporary portraiture and self-portraiture and move beyond archaic and insufficient methods of analysis. In writing off contemporary works made using social media because they are made in a relatively new and widely accessible means is akin to writing off earlier forms of photography, like vernacular photography, color photography, Polaroids, and slideshows—-all of which have been inducted into the canons of art history and photographic practice.29 The category of photographic portraiture and self-portraiture needs to include works in the emergent form of social media, regardless of previous exclusion based on material hierarchies.

Alabanza's self-images are the very definition of a self-portrait: "portraits of oneself done by oneself," but they facilitate a more nuanced understanding of self-portraiture. Alabanza's Instagram feed creates new representations that radically challenge the creation of stereotypes, and, again, they represent a multi-faceted self-portrait of a subject continually evolving—a self-portrait in a state of perpetual production, or what one may call tranifesting (Green and Ellison 2017). Alabanza's self-images produced in the context of their world, their community, and their perspective insert trans non-binary Black British subjectivity in context into broader

28 For more on creating new aesthetics of trans beauty that are not measured against dominant systems, but celebrated as beautiful and worthy of life in their very transgressiveness, see (Lehner 2020; Wilchins 2017).

29 Consider, for example, the success and canonization of William Eggleston's vernacular style color photos, Warhol's prolific use of Polaroids, and Nan Goldin's now legendary vernacular style slide shows.

culture in a way that is not a stereotype and models otherwise unseen ways of being. Thus, effectively, their Instagram feed constitutes a praxis of worlding, of bringing oneself into existence via permitting oneself to appear in visual culture; a radical intervention from someone who represents a constituency actively erased from visual culture and daily life.

Modelling an interdisciplinary trans visual studies method of analyzing Alabanza's work reveals that there are many hierarchies that could factor into the lack of attention garnered by these important cultural interventions. The scholarly neglect of trans femmes prolifically self-imaging on social media may lie in their being femme, or trans, or not Caucasian, or that the majority of these representations appear as selfies. All of these intersecting factors position these self-image makers and their images as antithetical to what have historically been valued forms of self-representation in the West. Yet, studying these images is integral to understanding visual culture and art today, and many of the self-imaging praxis of trans femmes of color like those of Alabanza radically rework Western discursive framing of the ontology of photography, unseating investment in notions of truth and indexicality, challenging how we understand gender, exploring the intersection of gender and racialization, and creating new forms of self-representation. Thus, I suggest that we view self-images like those of Travis Alabanza as critical and necessary praxis in and of themselves, within the discourse of issues in representation, art history, gender, intersectionality, racialization, contemporary art and self-portraiture.

**Funding:** This research received no external funding.

**Acknowledgments:** I would like to thank Travis Alabanza for being generous in the work that they make and in allowing me to use their images herein. Furthermore, it has been a pleasure to meet them and to write about their work. Their contributions to culture, art, and humanity are invaluable, their spirit indomitable, and their passion for their work inspiring. I would like to acknowledge Derek Conrad Murray, Jennifer A. González, Amelia Jones, and Marcia Ochoa for their ongoing mentorship and encouragement around my work. I would also like to thank the peer reviewers of this work, the publishers at MDPI and the contributors to this volume, all of whom have been invaluable interlocutors to this research.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

Benjamin, Ruha. 2019. *Race after Technology: Abolitionist Tools for the New Jim Code*. Medford: Polity.

© 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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2.2.3. Band Selection

Once the spectral library was finalized, the uSZU band selection technique [47] was performed to identify bands that optimize endmember class discrimination. uSZU is an automated band selection technique which attempts to select bands which maximize variation between endmember classes in a spectral library while minimizing the correlation between selected bands. uSZU assigns each band a Stability Index (SI) value based on the interclass variability divided by the intraclass variability for each class [47]:

$$SI\_i = \frac{\Delta inter\_i i}{\Delta intra\_i i} = \frac{\left| \sum\_{j=1}^k R\_{mean,j,i} \right|}{1.96 \times \sum\_{j=1}^k \sigma\_{j,i}} \tag{1}$$

where *k* is the total number endmembers, *Rmean,j,i* is the mean reflectance for endmember class *j* at wavelength *i*, and σ*j,i* is the standard deviation of class *j* for wavelength *i*. The band with the highest SI value is selected. Then a spectral correlation value (*Corr*) is calculated:

$$Corr(X, Y) = \frac{cov(X, Y)}{\sigma\_X \sigma\_Y} \tag{2}$$

where *cov*(*X*,*Y*) is the covariance between the selected waveband, *X* and each remaining waveband, *Y*; and *σ* is the standard deviation. All bands with *corr* values above a predetermined threshold *c* are then discarded. The process is repeated, with the threshold for correlation needed to discard a band decreasing by the value *i* with each iteration. For this study, values of *c* = 0.99 and *I* = 0.001 were used, both of these values were tested in Somers and Asner [47] and found to give acceptable accuracy. MESMA that was based on uSZU band reduced spectral libraries will be noted by including the term "uSZU" in the name.

## 2.2.4. Endmember Selection

Several approaches have been developed for determining the relative value of individual endmembers for representing their endmember class. One method for doing this is a count-based (CoB) approach, where each endmember is selected iteratively, and, using simple SMA, tested to see how many endmembers it can successfully model within the spectral library, using a predetermined Root Mean Square Error (*RMSE*) threshold to define success [52]. When this approach is applied to an endmember within its own endmember class, it is called (In-CoB); a desirable endmember will have a high In-CoB number (indicating a large number of other endmembers within the endmember class are derivative). This technique can also be applied against all the endmember classes which the endmember does not belong (Out-CoB), in this case, a desirable endmember will have a low Out-CoB number, indicating that this endmember would minimize confusion with other endmember classes.

Another approach for selecting endmembers is Endmember Average Root Mean Square Error (*EAR*), which evaluates each endmember's ability to model all other within class endmembers based on a summed RMSE [33]. EAR is calculated using the following formula:

$$EAR\_{A\_i} = \frac{\sum\_{j=1}^{n} RMSE\_{A\_i A\_j}}{n-1} \tag{3}$$

where *A* is an endmember class, *Ai* is the selected single endmember, and *Aj* are each of the other endmembers within the endmember class, and *n* is the total number of spectra in class *A*. A smaller EAR value is more desirable. Another approach, Minimum Average Spectral Angle (MASA, [34]), is similar to EAR but evaluates the summed spectral angle [53] instead of the RMSE fit. Both EAR and MASA evaluate only within an endmember class and do not evaluate interclass confusion.

Two techniques based on EAR, MASA, and CoB were used in this study. The first technique used a combination of EAR, MASA, and CoB to select three endmembers, it will therefore be abbreviated to "EMC". The combined EMC technique selected three endmembers for each endmember class: an endmember which minimizes the EAR value, an endmember which minimizes the MASA value, and an endmember which maximizes the In-CoB value. If multiple endmembers had the same In-CoB value, the endmember with the smallest Out-CoB value was selected. If the same endmember is selected through multiple EMC criteria (for example, the spectra that minimizes MASA also minimizes EAR), then fewer than three endmembers were used for that endmember class. The In-CoB [35] technique was also used in this study. For this technique, the In-CoB value of each endmember in an endmember class was evaluated, and any endmember with a unique In-CoB value was selected. If multiple endmembers have the same In-CoB value, the endmember with the minimum EAR value was selected. Another approach to endmember selection is Iterative Endmember Selection (IES) [31], which first picks two endmembers that maximize the performance of two-endmember SMA classifying the entire spectral library, as determined by using Cohen's kappa [54]. The algorithm then iteratively adds and removes endmembers in order to maximize kappa until the smallest spectral library that maximizes kappa is developed. While the other endmember selection approaches focus on within-class variability, IES explicitly considers confusion of endmembers between classes. In this study, the parameter of 0.025 was used as an RMSE constraint for the two-endmember SMA needed to classify the spectral library [32]. Spectral libraries processed in this way will be referred to as "IES" for the remainder of this study.

IES generally results in relatively large spectral libraries that can make the resultant MESMA analysis computationally expensive. Roberts et al. [55] proposed a method for reducing the size of IES generated spectral libraries through an iterative process, wherein the brightest endmember in each endmember class is initially chosen and all endmembers are modeled as a mixture of that endmember and shade are subsequently removed from the spectral library. Following the first pass, a new set of bright endmembers are selected from the reduced library, targeting endmembers that are most spectrally distinct from the first set. The process is repeated iteratively until either there are no more endmembers within a class in the reduced library, or new endmember selections fail to further reduce the library. The goal of this process is to identify the smallest set of endmembers for each class that also fully characterizes the spectral diversity of that class. The final result is a spectral library that retains the high accuracy of IES, while significantly reducing the number of endmember combinations. Spectral libraries processed in this way will be referred to as "Reduced IES".


**Optical In-Process Measurement Systems**

Editor

**Andreas Fischer**

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**2. Fluids and physiology**

Water is the most important and abundant element of the human body, and the physiology that surrounds it is extensive. The following principles are at best, the foundations toward an informed fluid practice.

#### **2.1. The body fluid compartments**

Water, on average, makes up 60% of the total body weight. The percentage will vary depending on the gender and the fat content in the body. There is an inverse correlation between the water content of the body and the fat content as adipose tissue contains less water than lean tissue. This explains why women have lower percentages of water than men as they have a higher percentage of adipose tissue.

Water in the body is functionally distributed among the two main body fluid compartments, the intracellular fluid (ICF) and the extracellular fluid (ECF) (**Figure 1**). The ICF constitutes approximately two-thirds of the total body water or 40% of the body weight and the ECF the remaining one-third or 20% of the body weight [4]. Water crosses between the ICF and the ECF through aquaporin channels in the cell membrane to attain osmotic equilibrium. The cell

**Figure 1.** The distribution of total body water.

membrane also contains active pumps and transporters that distribute individual solutes, including electrolytes. These electrolytes account for the effective osmolality (tonicity) that governs the water movement. The mechanisms of these electrolyte movements are further defined by the Gibbs-Donnan effect of the nondiffusible large anions like protein. The end results are an ICF compartment with potassium (K+ ) as the predominant ion and an ECF compartment with sodium (Na+ ) and chloride (Cl− ) as the predominant ions [5].

The ECF is further divided into the interstitial fluid and the plasma compartments, the two separated by the capillary wall. Except for plasma proteins and blood cells, the pores on the capillary wall permit the flux of water and small solutes. This contributes to the two compartments having almost similar electrolyte composition with only small differences contributed by the Gibbs-Donnan effect of the plasma proteins. By volume, these plasma proteins constitute 7% of the plasma volume with the remaining 93% plasma water. As a side note, these proteins are solids in the plasma, and the changes in their plasma load will affect the waterbased measurements of plasma electrolyte concentrations [6].

#### **2.2. The body fluid regulation**

Intravenous fluids are drugs, and like other drugs, there are potential complications. In the acute setting where these fluids are commonplace, it is imperative that the practice aims at administering the right patient the right fluids, at the right volume and rate, with the right

Water is the most important and abundant element of the human body, and the physiology that surrounds it is extensive. The following principles are at best, the foundations toward an

Water, on average, makes up 60% of the total body weight. The percentage will vary depending on the gender and the fat content in the body. There is an inverse correlation between the water content of the body and the fat content as adipose tissue contains less water than lean tissue. This explains why women have lower percentages of water than men as they have a

Water in the body is functionally distributed among the two main body fluid compartments, the intracellular fluid (ICF) and the extracellular fluid (ECF) (**Figure 1**). The ICF constitutes approximately two-thirds of the total body water or 40% of the body weight and the ECF the remaining one-third or 20% of the body weight [4]. Water crosses between the ICF and the ECF through aquaporin channels in the cell membrane to attain osmotic equilibrium. The cell

overall fluid balance.

informed fluid practice.

**2. Fluids and physiology**

40 Essentials of Accident and Emergency Medicine

**2.1. The body fluid compartments**

higher percentage of adipose tissue.

**Figure 1.** The distribution of total body water.

A complex interaction of regulatory mechanisms from different organs helps the body to maintain an effective fluid volume in different circumstances. The key pathway that underpins this volume regulation is the hormonally mediated renin-angiotensin II-aldosterone-system (RAAS), with the faster neutrally mediated baroreceptor reflex contributing an indirect role through its interplay of the pressure regulation. In the context of the fluid therapy scope of the chapter, the RAAS will be elaborated below.

The RAAS pathway is activated by a decrease in the renal perfusion pressure, detected by the juxtaglomerular apparatus (JGA) (**Figure 2**). In the JGA, the reduced renal perfusion stimulates the granular cells of the afferent arteriole to secrete the proteolytic enzyme renin through a direct intrarenal baroreceptor activity and detection of reduced sodium chloride concentrations by the macula densa in the wall of the ascending limb of the loop

**Figure 2.** The juxtaglomerular apparatus.

of Henle. Besides these mechanisms, the renin release is also controlled by renal sympathetic nerves and angiotensin II.

Renin as an enzyme will then catalyze the conversion of angiotensinogen, a large protein produced in the liver, to angiotensin I, a decapeptide. This is the rate-limiting step of the RAAS pathway (**Figure 3**). Angiotensin I has little biologic activity apart from being the precursor to angiotensin II. Its conversion to angiotensin II involves the removal of two amino acid moieties by the angiotensin-converting enzyme (ACE). ACE is primarily located in the pulmonary capillaries, but it is also found in the kidney epithelial cells.

The ultimate objective of the RAAS, through the activities of angiotensin II and aldosterone as summarized in **Figure 3**, is the preservation of effective fluid volume and pressure. The RAAS demonstrates the strong interconnection between the body fluid and electrolytes in maintaining the fluid homeostasis. In the acute setting, this interconnection is very relevant given the frequent alterations of the electrolyte contents of the body in the acute phase of illness. The assessment of electrolytes in the acute patients should, therefore, be comprehensive and extend beyond the laboratory results. For example, the assessment should also consider the potential electrolyte losses from the gastrointestinal tract, a common organ affected in acute illnesses [7].

in various organs of the body (**Figure 4**). Appreciation of the dynamics of glycocalyx in the microcirculation of the acute cohort of patients will be integral in their fluid resuscitation and

**Figure 4.** The endothelial glycocalyx layer in healthy, equilibrium state (A) and damaged, leaky state (B) that leads to

Approach to Fluid Therapy in the Acute Setting http://dx.doi.org/10.5772/intechopen.74458 43

The history of intravenous fluids began during the cholera pandemic in Europe in the 1830s. The success of Thomas Latta in using a saline solution to resuscitate dying cholera patients paved the way for the widespread use of intravenous fluids and the research to refine their contents [15]. The early milestones in intravenous fluid therapy included the first experiment with albumin in 1834 [16] and the attempt by Sydney Ringer to develop a physiological solution for cardiac contractility with his Ringer's solution in 1876 [17]. Ringer's solution was modified by Alexis Hartmann in 1932 by including lactate to help overcome the acidosis in dehydrated pediatric patients [18]. The gelatins and other solutions with larger molecules only broke into the scene during the Second World War [19], although the first study in humans was performed in 1915 [20]. It is interesting that the history behind the most common type of fluids used, the 0.9% saline, is unclear. The present-day 0.9% saline, often called the "normal saline," has far higher sodium and chloride concentrations than Latta's 1832 saline solution. The only possible connection to 0.9% saline in the history was the in vitro studies of Hamburger in the 1890s that described 0.9%

NaCl as an "indifferent solution" in which erythrocytes were least likely to lyse [21, 22].

From the above breakthroughs, the science of intravenous fluids has grown progressively, especially in the last couple of decades. Whether medicine will find an answer to the ideal intravenous fluid will be debatable, but more evidence has emerged in the comparison

Crystalloids are solutions containing salts in the form of electrolytes and small molecules. The

in future fluid research in such population [13, 14].

between the different types of fluids available.

composition of commonly available crystalloids is given in **Table 1**.

**3.1. Crystalloids**

**3. The types of fluids**

interstitial edema.

#### **2.3. The microcirculation model**

The classic microcirculation model, based on the semipermeability of the capillary and postcapillary venule walls, and the presence of hydrostatic and oncotic pressure gradients across these walls had for long described the flux of fluids and electrolytes between the plasma and the interstitial fluid [8, 9]. The identification of the endothelial glycocalyx layer, a web of membrane-bound glycoproteins and proteoglycans on the luminal side of endothelial cells, has now challenged the classic model [10, 11]. The colloid oncotic pressure from the sub-glycocalyx space is a key determinant of the trans-capillary flow. The disruption to the integrity of the glycocalyx layer, or the "leakiness," in a number of acute situations like sepsis [12], trauma, and postsurgery, has been attributed to the development of interstitial edema

**Figure 3.** The renin-angiotensin II-aldosterone-system (RAAS).

**Figure 4.** The endothelial glycocalyx layer in healthy, equilibrium state (A) and damaged, leaky state (B) that leads to interstitial edema.

in various organs of the body (**Figure 4**). Appreciation of the dynamics of glycocalyx in the microcirculation of the acute cohort of patients will be integral in their fluid resuscitation and in future fluid research in such population [13, 14].


DHD vs CS case:

14 Will-be-set-by-IN-TECH

Healty subjects or SC

 B. Pattern detected for each brain using the mean of the graphics obtained in A.

1 4 7 10 13 16

CS1

Closing size

0 0,02 0,04

0 0,02 0,04

Mean volume Mean volume

0 0,02 0,04

Mean volume

1 4 7 10 13 16

Pattern obtained from a SS

Strabic subjects or SS

Closing size

Fig. 9. Procedure to obtain granulometric patterns. In step A the graphics obtained for one slice of each of the two CS and for one SS case are illustrated. In step B, a common mean pattern is obtained for both CS and one SS. Finally, in step C, a mean pattern is obtained for

1 4 7 10 13 16

Closing size

CS2

Closing size

1 5 9 13 17

Mean pattern obtained from two CS

 C. Mean pattern detected as the mean of the graphics in B.

Mean volume

.

0 0,02 0,04

Closing size

1 4 7 10 13 16

SS

Closing size

1 5 9 13 17

SS

Closing size

1 4 7 10 13 16

CS2

1 4 7 10 13 16

CS2

Closing size

1 4 7 10 13 16

Closing size

CS1

1 5 9 13 17

Closing size

CS1

A. Analysis of dark regions ( ζDark ) per slice.

> 0 0,02 0,04 0,06

0 0,02 0,04

0 0,02 0,04

0 0,02 0,04

ζDark

the two CS.

ζDark

ζDark

ζDark

ζDark

ζDark

0 0,01 0,02 0,03

0 0,05

Fig. 10. Mean volume of clear and dark structures corresponding to SS and SC.

DHD vs CS case:

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

Mean volume Mean volume Mean volume Mean volume

SSAV vs CS case:

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

Fig. 12. WM and GM granulometric patterns for the CS and SS groups.

structures, an index is proposed. Such index is expressed as follows:

*ι* = *vol*(*small structures*)

1 3 5 7 9 11 13 15 17

GM granulometric pattern

WM granulometric pattern

187 Comparison of Granulometric Studies of Brain Slices

from Normal and Dissociated Strabismus Subjects Through Morphological Transformations

Opening size

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

WM granulometric pattern

Closing size

1 3 5 7 9 11 13 15 17

GM granulometric pattern

Opening size

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

In trying to obtain a parameter indicating the absence, thinning, or thickening of WM or GM

*vol*(*medium structures*) + *vol*(*large structures*) (5)

Closing size

CS Mean DHD2 DHD1

> CS mean DHD1 DHD2

> > CS Mean SSAV2 SSAV1

> > > CS Mean SSAV1 SSAV2

Fig. 11. Images illustrating the segmentation of WM and GM.

In order to explain this situation we divide the analysis of WM and GM as follows :

*a) WM analysis.-* In Fig. 13, the terms small, medium and large components are exemplified with some structures. Notice that small components in WM have the appearance of "fingers", and medium components have a characteristic neck that joins them to large structures.

The lack of small components in the SS with respect to CS group, indicates the absence of such components in the WM. Also, the graphs indicate that the existing small components are thin; therefore they are eliminated easily by small structuring elements. On the other hand, the prevalence of medium and large structures is due to absence the circumvolutions, this originates a non-smooth transition mainly between small and medium components; since medium and large components are not to much different.

In Fig. 14 small components are eliminated from slices that belong to DHD, SSAV and a CS. Notice that for DHD cases, WM has thin "fingers" which almost disappear after applying an opening of size 1; whereas for the SSAV case, "fingers" are not so thin and many of them remain after the same transformation is applied.

Note that, for the CS case, WM shows abundant appendages and coral forms. Also, observe that transitions between medium and large sizes are smooth; however for the DHD and SSAV cases, the appendages and coral forms decrease significantly.

*b) GM analysis.-* In the GM there is a prevalence of medium and large components. This is confirmed in the graphs of GM for DHD and SSAV cases ( Fig. 12). Small, medium and large size components in GM are illustrated in Fig. 13.

16 Will-be-set-by-IN-TECH

Fig. 11. Images illustrating the segmentation of WM and GM.

medium and large components are not to much different.

cases, the appendages and coral forms decrease significantly.

remain after the same transformation is applied.

size components in GM are illustrated in Fig. 13.

In order to explain this situation we divide the analysis of WM and GM as follows :

*a) WM analysis.-* In Fig. 13, the terms small, medium and large components are exemplified with some structures. Notice that small components in WM have the appearance of "fingers", and medium components have a characteristic neck that joins them to large structures.

The lack of small components in the SS with respect to CS group, indicates the absence of such components in the WM. Also, the graphs indicate that the existing small components are thin; therefore they are eliminated easily by small structuring elements. On the other hand, the prevalence of medium and large structures is due to absence the circumvolutions, this originates a non-smooth transition mainly between small and medium components; since

In Fig. 14 small components are eliminated from slices that belong to DHD, SSAV and a CS. Notice that for DHD cases, WM has thin "fingers" which almost disappear after applying an opening of size 1; whereas for the SSAV case, "fingers" are not so thin and many of them

Note that, for the CS case, WM shows abundant appendages and coral forms. Also, observe that transitions between medium and large sizes are smooth; however for the DHD and SSAV

*b) GM analysis.-* In the GM there is a prevalence of medium and large components. This is confirmed in the graphs of GM for DHD and SSAV cases ( Fig. 12). Small, medium and large

Fig. 12. WM and GM granulometric patterns for the CS and SS groups.

In trying to obtain a parameter indicating the absence, thinning, or thickening of WM or GM structures, an index is proposed. Such index is expressed as follows:

$$\mu = \frac{vol(small\,\,stnarrow)}{vol(medium\,\,structures) + vol(large\,\,structures)} \tag{5}$$

DHD

189 Comparison of Granulometric Studies of Brain Slices

from Normal and Dissociated Strabismus Subjects Through Morphological Transformations

SSAV

CS

With respect to SS, index *ι* presents the following behavior: a large number of medium components in WM and GM, that yields an inferior value when compared against the index

In other words, index *ι* for WM and GM in SS suggests: a) a lack of small components in the analyzed WM, and whenever present they are characterized by their thinness. Medium structures predominate through the analyzed WM. Given the lack of small components, and the presence of thin small structures in WM, this originates the absence of small structures in

The absence of small components (´´fingers") in the WM may causes a lack of electrical connection among different cortical regions. As a consequence a right electrical

the GM, for which a predominance of medium and large structures is observed.

Fig. 14. Elimination of thin components.

for CS.

In Fig. 15, index *ι* was plotted for WM and GM based on the values obtained for graphs in Fig. 12. When index *ι* for the CS is compared with that for the SS group, significant differences are obtained, since the latter value is much smaller.

Index *ι*, indicates that in the WM of CS, the volume of small components is more than double that of medium and large structures together; while in GM of CS, index *ι* is almost equal to the unit, that is, vol(small structures) ≈ vol(medium structures) + vol(large structures).

18 Will-be-set-by-IN-TECH

Small size components

In Fig. 15, index *ι* was plotted for WM and GM based on the values obtained for graphs in Fig. 12. When index *ι* for the CS is compared with that for the SS group, significant differences are

Index *ι*, indicates that in the WM of CS, the volume of small components is more than double that of medium and large structures together; while in GM of CS, index *ι* is almost equal to the unit, that is, vol(small structures) ≈ vol(medium structures) + vol(large structures).

Fig. 13. Illustration of small, medium and large structures on WM and GM.

obtained, since the latter value is much smaller.

Small size components

Large size components

Large size components

> Medium size components

Medium size components

DHD

SSAV

CS

Fig. 14. Elimination of thin components.

With respect to SS, index *ι* presents the following behavior: a large number of medium components in WM and GM, that yields an inferior value when compared against the index for CS.

In other words, index *ι* for WM and GM in SS suggests: a) a lack of small components in the analyzed WM, and whenever present they are characterized by their thinness. Medium structures predominate through the analyzed WM. Given the lack of small components, and the presence of thin small structures in WM, this originates the absence of small structures in the GM, for which a predominance of medium and large structures is observed.

The absence of small components (´´fingers") in the WM may causes a lack of electrical connection among different cortical regions. As a consequence a right electrical

method to determine the functional relations among different brain areas. This coherence is altered when some association vias (short and large cortico-corticals, cortico-subcorticals, and short cortico-corticals), are affected structurally; this has been demonstrated in some diseases like Alzheimer's and dementia. Some aspects of the electrical behavior as in the coherence case, depend precisely on the neuro-structural substrate (28; 29). For some authors, a decrease in coherence represents a diagnostic marker. Therefore, further research is necessary on the interrelation between structure and neuronal function, to gain a better understanding into the origin of multiple diseases that have a seat in the cerebral cortex, or at least of those conditions in which the participation of the brain cortex plays a role in their physiopathology.

191 Comparison of Granulometric Studies of Brain Slices

from Normal and Dissociated Strabismus Subjects Through Morphological Transformations

On the other hand, during the development of the cerebral cortex or corticogenesis, there is a cellular migration of neurons that travel from periventricular regions and at the same time start maturing, until they reach more superficial regions of the brain, where their maturation culminates. During this process, interconnection vias are liable to be damaged giving place to different neurological schemes, that can affect the neuronal maturity process as well as the performance of the association routes. In the case of strabismus it is still not known, which structural alterations are the cause of this condition. (30–32). Finally, the granulometric study presented in this work, helps us understand the morphology of WM and GM at macroestructural level; however further studies are necessary to analyze the structure of the

In this work we presented a method for segmenting MRI slices, as well as the computation of two granulometric studies. The first granulometric study, analyzes clear and dark structures in the whole deskulling brain; while the second one, consists in the analysis of WM and GM

Curves obtained for the whole deskulled brains were not appropriated for analyzing clear and

On the other hand, important differences were found in the granulometric plots of WM and GM of the SS when compared to the graphs of CS group. Main differences consist in the lack of small components ("fingers") and predominance of medium and large structures. From this analysis, index *ι* was introduced, which is useful to establish an immaturity degree. In the SS, index *ι* results to be much smaller than that of CS; given the presence of numerous

The information drawn from these results suggests that there is a faulty electrical communication among several cortical areas due to the lack of small components, and also to the thinness of these structures which are present both in WM and GM. Changes in nerve neuroconduction, similar to those observed by means of the DBM in strabismic patients (7; 8; 14; 15), could be related to the microstructural organization of the cerebral mass. Index *ι* values, obtained in this work from healthy and strabismic children, show significant differences in the proportion of granulometric elements in the GM and WM when healthy and SS are compared. Based on the calculated index, strabismic patients with SSAV present a

dark regions, since WM and GM are partly composed by pixels of similar intensities.

This knowledge enable the establishment of better treatments (28).

WM and GM at microestructural level.

medium and large structures.

**5. Conclusions**

separately.

Fig. 15. Table to analyze WM and GM in all subjects through the proposed index *ι*. Volume values(bottom of x-axis) to evaluate the index were obtained from graphs in Fig. 12.

communication between different cortical areas is hindered. Therefore, although there are great areas of GM and WM in the SS, the lack of small components identified as fingers or thin components in the WM and GM, may cause a faulty electrical conduction and communication between these areas.

On the other side, the presence of a large volume formed of medium components in WM can be associated with a certain immaturity process, which is identified by some authors with excessive glial proliferation or abnormalities in myelin maturation or composition (27). This produces a low quality WM characterized by axons presenting certain degree of damage, with the subsequent deficiency in impulse conduction. In our case, index *ι* is also called immaturity index, by means of which the absence and increment of certain structures in WM or GM are measured. In this way, morphometric changes in WM and GM seem to be intimately related to the electrical and neurofunctional behavior of the analyzed SS. The granulometric study carried out in this work suggests that changes in the nerve conduction, as we have observed by means of DBM in strabismic patients (7; 8; 14; 15), could be the consequence of changes in the microstructural organization of the cerebral mass. According to this study, these alterations may be manifested as presence of immaturity of certain brain regions and lack of electrical communication between the structures conforming the WM and GM. We consider that as a result of this immaturity, alternative vias are established affecting the processes of neuronal interconnections; with the subsequent affectation, in different degrees and ways of the grey and white substance; hence different clinical expressions of the dissociated strabismus are originated.

On the other hand, studies of electrical brain function carried out by means of computed neurometry, have revealed the close correlation between the structure and cerebral function. This has been determined from the study of electrical coherence, which is a non invasive

method to determine the functional relations among different brain areas. This coherence is altered when some association vias (short and large cortico-corticals, cortico-subcorticals, and short cortico-corticals), are affected structurally; this has been demonstrated in some diseases like Alzheimer's and dementia. Some aspects of the electrical behavior as in the coherence case, depend precisely on the neuro-structural substrate (28; 29). For some authors, a decrease in coherence represents a diagnostic marker. Therefore, further research is necessary on the interrelation between structure and neuronal function, to gain a better understanding into the origin of multiple diseases that have a seat in the cerebral cortex, or at least of those conditions in which the participation of the brain cortex plays a role in their physiopathology. This knowledge enable the establishment of better treatments (28).

On the other hand, during the development of the cerebral cortex or corticogenesis, there is a cellular migration of neurons that travel from periventricular regions and at the same time start maturing, until they reach more superficial regions of the brain, where their maturation culminates. During this process, interconnection vias are liable to be damaged giving place to different neurological schemes, that can affect the neuronal maturity process as well as the performance of the association routes. In the case of strabismus it is still not known, which structural alterations are the cause of this condition. (30–32). Finally, the granulometric study presented in this work, helps us understand the morphology of WM and GM at macroestructural level; however further studies are necessary to analyze the structure of the WM and GM at microestructural level.

#### **5. Conclusions**

20 Will-be-set-by-IN-TECH

0 0.5 1 1.5 2 2.5 ι

Fig. 15. Table to analyze WM and GM in all subjects through the proposed index *ι*. Volume values(bottom of x-axis) to evaluate the index were obtained from graphs in Fig. 12.

communication between different cortical areas is hindered. Therefore, although there are great areas of GM and WM in the SS, the lack of small components identified as fingers or thin components in the WM and GM, may cause a faulty electrical conduction and communication

On the other side, the presence of a large volume formed of medium components in WM can be associated with a certain immaturity process, which is identified by some authors with excessive glial proliferation or abnormalities in myelin maturation or composition (27). This produces a low quality WM characterized by axons presenting certain degree of damage, with the subsequent deficiency in impulse conduction. In our case, index *ι* is also called immaturity index, by means of which the absence and increment of certain structures in WM or GM are measured. In this way, morphometric changes in WM and GM seem to be intimately related to the electrical and neurofunctional behavior of the analyzed SS. The granulometric study carried out in this work suggests that changes in the nerve conduction, as we have observed by means of DBM in strabismic patients (7; 8; 14; 15), could be the consequence of changes in the microstructural organization of the cerebral mass. According to this study, these alterations may be manifested as presence of immaturity of certain brain regions and lack of electrical communication between the structures conforming the WM and GM. We consider that as a result of this immaturity, alternative vias are established affecting the processes of neuronal interconnections; with the subsequent affectation, in different degrees and ways of the grey and white substance; hence different clinical expressions of the dissociated strabismus are

On the other hand, studies of electrical brain function carried out by means of computed neurometry, have revealed the close correlation between the structure and cerebral function. This has been determined from the study of electrical coherence, which is a non invasive

0 0.5 1 1.5 2 2.5 ι

 **GM SSAV**

**GM DHD**

0.934150077 0.605504587 0.75 CS Mean DHD1 DHD2

0.934150077 0.686619718 0.633587786 CS Mean SSAV1 SSAV2

**WM SSAV**

1.937254902 0.997474747 0.913253012 CS Mean DHD1 DHD2

**WM DHD**

1.937254902 0.890818859 0.831797235 CS Mean SSAV1 SSAV2

0 0.5 1 1.5 2 2.5 ι

between these areas.

originated.

0 0.5 1 1.5 2 2.5 ι

> In this work we presented a method for segmenting MRI slices, as well as the computation of two granulometric studies. The first granulometric study, analyzes clear and dark structures in the whole deskulling brain; while the second one, consists in the analysis of WM and GM separately.

> Curves obtained for the whole deskulled brains were not appropriated for analyzing clear and dark regions, since WM and GM are partly composed by pixels of similar intensities.

> On the other hand, important differences were found in the granulometric plots of WM and GM of the SS when compared to the graphs of CS group. Main differences consist in the lack of small components ("fingers") and predominance of medium and large structures. From this analysis, index *ι* was introduced, which is useful to establish an immaturity degree. In the SS, index *ι* results to be much smaller than that of CS; given the presence of numerous medium and large structures.

> The information drawn from these results suggests that there is a faulty electrical communication among several cortical areas due to the lack of small components, and also to the thinness of these structures which are present both in WM and GM. Changes in nerve neuroconduction, similar to those observed by means of the DBM in strabismic patients (7; 8; 14; 15), could be related to the microstructural organization of the cerebral mass. Index *ι* values, obtained in this work from healthy and strabismic children, show significant differences in the proportion of granulometric elements in the GM and WM when healthy and SS are compared. Based on the calculated index, strabismic patients with SSAV present a

[6] Gallegos-Duarte M. "Exploratory maneuvers in congenital endotropy ". En: Temas Selectos de Estrabismo. Centro Mexicano de Estrabismo SC. México, Composición

193 Comparison of Granulometric Studies of Brain Slices

from Normal and Dissociated Strabismus Subjects Through Morphological Transformations

[7] Gallegos-Duarte M; Moguel, S.; Rubín de Celis, B.; " Alterations in the cerebral mapping in the variable congenital endotropy ", Rev Mex Oftalmol; 2004; 78 (3): 122-126. [8] Gallegos-Duarte M. " Paradoxical cortical response during the intermittent fotoestimulation in the dissociated strabismus ", Cir y Cir 2005; 73 (3): 161-165. [9] Gallegos-Duarte M, Mendiola-Santibáñez JD, Ortiz-Retana JJ; Rubín de Celis B, Vidal-Pineda R, Sigala-Zamora. " Dissociated desviation. An strabismus of cortical

[10] Olivares, R; Godoy,G; Adaro,L; Aboitiz, F.: "Neuronal density of the visual cortex (area

[11] Horton, J.C., Hocking, D.R., "An adult-like pattern of ocular dominance columns in striate cortex of newborn monkeys prior to visual experience". J. Neuroscience 1996,

[12] Horton, J.C., Hocking, D.R., Timing of the critical period for plasticity of ocular dominance columns in macaque striate cortex. J. Neuroscience 1997; 17:3684-3709,. [13] Tychsen L; Wong AM; Burkhalter A.: Paucity of horizontal connections for binocular vision in V1 of naturally strabismic macaques: Cytochrome oxidase compartment

[14] Gallegos-Duarte, M, Moguel-Ancheita S: "Modifications neurologiques adaptatives après traitement médical et chirurgical du syndrome strabique avec variations des repères angulaires". En: Réunion de printemps, Association française de strabologie. 110 Congres de la société Française d´Ophtalmologie, Paris 2004.

[15] Moguel-Ancheita S, Orozco-Gómez L, Gallegos-Duarte M, Alvarado I, Montes C. " Metabolic changes in the cerebral cortex related to the strabismus treatment. Preliminary

[17] Serra, J. y P. Salembier. "Connected operators and pyramids" ., Proc. of SPIE. Image

[18] Salembier P., and Serra J.,: Flat zones filtering, connected operators and filters by reconstruction. IEEE Transactions on Image Processing, 3(8), (1995) 1153-1160. [19] Vincent L. and Dougherty E. R.:Morphological segmentation for textures and particles. In Digital IMage Processing Methods E.R. Dougherty, editor, . Marcel Dekker, New York,

[20] Mendiola-Santibañez, J.D ,Terol-Villalobos, I.R., Herrera-Ruiz G. , Fernández-Bouzas, A., "Morphological contrast measure and contrast enhancement: One application to the segmentation of brain MRI", *Signal Processing*, vol. 87 , no. 9, pp. 2125-2150, 2007. [21] Soille, P.,: Morphological image analysis: principle and applications, Springer-Verlag,

[23] L. Vincent, "Morphological Grayscale Reconstruction in Image Analysis: Applications and Efficient Algorithms," *IEEE Transactions on Image Processing*, vol. 2, no. 2, pp. 176-201,

[16] Serra, J.: Mathematical Morphology vol. I, Academic Press., London, (1982 ).

[22] Heijmans H., "Morphological Image Operators", Academic Press, USA, 1994.

[24] Matheron G.: Eléments pour une théorie des milieux poreoux. Mason,Paris(1967).

Algebra and Mathematical Morphology'93, San Diego, July , 1993.

17) of two species of wild rodents". Int. J. Morphol 2004, 22 (4): 279-284.

Editorial Láser. México, D.F. 2005, 1-18.

origin". Cir y Cir 2007, 75 (4): (in printt).

specificity. J Com Neurol. 2004, 474 (2): 261-75

http://perso.orange.fr/hoc.lods/demo147.html.

results with SPECT". Cir y Cir 2004; 72:165-170

16:1789-1805.

(1994) 43–102.

2003.

Feb. 1993.

greater degree of immaturity than those patients with DHD; since *ι* index is smaller. This can be more clearly appreciated from the graphs and derived values in Fig. 15.

On the other hand, it is rather convenient that changes consistent with the alterations in the relation volume and granulometric density can be determined in alive subjects; besides this advantage can be complemented with neurofunctional studies such as the DBM. This technique offers a high temporary resolution, though its space resolution is significantly slower. Granulometric studies offer a finest structural analysis available in vivo, that allow the interpretation of neurofunctional studies. On the one hand, granulometry is outlined as an excellent diagnosis marker for several neurologic diseases. The study carried out in this work not only suggests the neuronal immaturity associated with strabismus, but also the lack of small components("fingers") and the predominance of medium size structures mainly in the SS group. These circumstances propitiate alterations in conduction of the neuroelectrical impulses, favoring the presence of strabismus, as well as the alterations encountered in the DBM. Therefore, granulometric studies may help to gain a better insight into the origin, diagnosis and prognosis of this prevalent disease, for which so little is known. Although the amount of brains analyzed here is only six, they provide important information to establish granulometric differences between SC and SS groups. Finally, for our future research, we are considering the following things: I) Improvement of the morphologic operators involved in the obtention of granulometric curves; these operators must be autodual with the purpose of treating clear and dark components of the image separately; and II) Inclusion of a larger sample of healthy children to obtain mean patterns, as well as a larger number of patients with strabismus.

#### **6. Acknowledgements**

The authors wish to thank the Mario Moreno Reyes foundation for the financial support. Jorge D. Mendiola-Santibañez thanks to CONACyT for the financial support.

#### **7. References**

22 Will-be-set-by-IN-TECH

greater degree of immaturity than those patients with DHD; since *ι* index is smaller. This can

On the other hand, it is rather convenient that changes consistent with the alterations in the relation volume and granulometric density can be determined in alive subjects; besides this advantage can be complemented with neurofunctional studies such as the DBM. This technique offers a high temporary resolution, though its space resolution is significantly slower. Granulometric studies offer a finest structural analysis available in vivo, that allow the interpretation of neurofunctional studies. On the one hand, granulometry is outlined as an excellent diagnosis marker for several neurologic diseases. The study carried out in this work not only suggests the neuronal immaturity associated with strabismus, but also the lack of small components("fingers") and the predominance of medium size structures mainly in the SS group. These circumstances propitiate alterations in conduction of the neuroelectrical impulses, favoring the presence of strabismus, as well as the alterations encountered in the DBM. Therefore, granulometric studies may help to gain a better insight into the origin, diagnosis and prognosis of this prevalent disease, for which so little is known. Although the amount of brains analyzed here is only six, they provide important information to establish granulometric differences between SC and SS groups. Finally, for our future research, we are considering the following things: I) Improvement of the morphologic operators involved in the obtention of granulometric curves; these operators must be autodual with the purpose of treating clear and dark components of the image separately; and II) Inclusion of a larger sample of healthy children to obtain mean patterns, as well as a larger number of patients

The authors wish to thank the Mario Moreno Reyes foundation for the financial support. Jorge

[1] Krachmer JH, Marti T.J, Corbett JJ.: Trastornos del quiasma y de las vías visuales retroquiasmáticas, En: Los requisitos en oftalmología: Neuroftalmología. Ed. Harcourt,

[2] Rakic, P; Lombroso, P.J. "Development of the cerebral cortex: I. Forming the cortical

[3] Zeki, SM., Watson, J.D.G, Lueck, C.J., Fristcn, K.J., Kennard, C. And Frackowiat, S.J. "A direct demostration of functional specialization in human visual cortex". J Neurosci 1991,

[4] Mendola JD, Conner IP, Anjali R, Chan ST, Schwartz TL, Odom JV, Kwong KK. "Voxel-based analysis of MRI detects abnormal visual cortex in children and adults with

[5] Suk-tak Ch; Kwok-win T; Kwok-cheung L; Lap-kong Ch; Mendola JD; Kwong KK. Neuroanatomy and adult strabismus: a voxel-based morphometric analisys of magnetic

structure". J. Am Acad Child Adolesc Psychiatry 1998, 37 (1): 116-117.

amblyopia". Human Brain Mapping 2005; 25(2) 222-236

resonance structural scans. Neuroimage 2004 (22) 986-994.

D. Mendiola-Santibañez thanks to CONACyT for the financial support.

be more clearly appreciated from the graphs and derived values in Fig. 15.

with strabismus.

**7. References**

**6. Acknowledgements**

11(3) 641-649.

Madrid, España. 2001; 101-108.

**1. Introduction**

terms of averaged waveforms.

between cortical areas.

Magnetoencephalography (MEG) can monitor the activation of a neuronal population with millisecond temporal resolution, and offer new noninvasive information about basic activities of the human brain. MEG is usable for the description of spontaneous brain activity and the detection of timely events such as stimulus evoked fields. The most recent advances in the field of MEG concerning cortical responses to stimulation are issued from development of multichannel recordings. We can study the temporal order of several cortical areas from averaged waveforms of MEG, e.g., auditory, visual, and somatosensory evoked responses. For three decades, dynamical information contained in MEG has been analyzed mainly in

**Intracerebral Communication Studied by**

*Department of Information Science, Faculty of Engineering, Gifu University*

**Magnetoencephalography**

Kuniharu Kishida

*Yanagido, Gifu,*

*Japan*

**10**

However, lots of brain activities are included in original MEG data. For spontaneous fields there are huge activities in cortical areas. Generally, it is difficult to estimate source locations of current dipole data from MEG data correctly at the present stage, if the number of current dipoles becomes large. The estimation of their locations is an underdetermined problem. In the case of spontaneous fields it is hard to obtain intracerebral communication between active cortical areas. Hence, we consider an overdetermined problem in the case of evoked fields, since their locations of active cortical areas are limited. Furthermore, we should find a possibility to obtain dynamical information by signal processing of fluctuations around concatenate average waveforms, which have been discarded without signal processing in the conventional MEG analysis. The fluctuations may be induced magnetic fields to communicate

In this chapter, we will show this new direction of MEG analysis by way of example of somatosensory evoked field (SEF), and intracerebral communication between active somatosensory cortices can be expressed by system identification of fluctuations. That is, intracerebral communication between the primary somatosensory cortex in the contralateral hemisphere (cSI) and the contralateral secondary somatosensory cortex (cSII) in 2Hz median nerves stimuli will be discussed from correlation functions of SEF fluctuations by the identification method with feedback model (see Section 3.6). In this chapter, intracerebral communication between cSI and cSII will be studied not from averaged waveforms but from

fluctuations around concatenate averaged waveforms by following steps:


**2.10. Prognostic indicators**

The best validated measurement is contralateral lung area assessed by 2D ultrasound through the so-called lung area/head circumference ratio (LHR) [29]. Different methods for measuring were described but the most reproducible and accurate method involves tracing the lung contours.

Normal lung area develops four times more than the head circumference between week 12 and 32; therefore, the LHR needs to be adjusted according to gestational age. The effect of gestational age on LHR can be minimized by expressing the observed LHR as a ratio to the expected mean LHR for that gestational age.

*2.10.3. Fetal lung volumes*

*2.10.4. Fetal intervention*

maturation [45].

**3. Conclusions**

as fetal echocardiography.

**Conflict of interest**

No conflict of interest to declare.

On fetal MR imaging there are several methods described for estimation of fetal pulmonary hypoplasia. All of these methods include the measuring of total fetal lung volume (TFLV). MR volumetry is considered the most accurate method for measuring ipsilateral lung and, therefore, for best estimation of fetal lung volumes, compared to three-dimensional ultrasound [35].

Congenital Diaphragmatic Hernia

201

http://dx.doi.org/10.5772/intechopen.74500

Because CDH is an anomaly with poor prognosis that can be identified in the prenatal period, it is justified to perform an intervention which improves lung function. Di Fiore and Wilson studied the concept of triggering lung growth by tracheal occlusion [36]. This was observed in an experiment of nature occurring in fetuses with congenital high airway obstruction syndrome (CHAOS), who display impressive lung growth. Through pregnancy, fetal lungs secrete fluid during their development, which creates a positive pressure under the glottis. Fluid secretion of the lung and a cyclical pressure change are essential for development and growth of the lung [37]. Fluid secretion increases the pressure in airways but during fetal breathing movements, the pressure gradient is normalized as glottis opens. This phenomenon creates cyclical periods of tissue stretch, which are important for an optimal balance between growth and differentiation [38]. Intrauterine tracheal occlusion acts like a closed glottis trapping the fluid causing subsequently increased tissue stretch, which triggers lung growth. The tracheal occlusion decreases the number of type II alveolocytes and lowers surfactant expression in occluded lungs [39–42]. This unwanted effect may be improved by either removing the tracheal occlusion before birth and/or by antenatal steroid administration [43, 44]. Experimentally, in-utero reversal of occlusion (clinically translated as a plug-unplug sequence) achieved morphologically better lung

As prenatal detection of fetal CDH improved in recent years, it is important to use standardized prognosis markers around the world. Currently, available markers are not 100% accurately predictive of outcome and their use is much less accurate in the presence of chromosomal anomalies or other malformations. This shows the fact that all fetuses diagnosed with CDH should undergo an ultrasound examination by a well-trained sonographer, as well


**6. Maintenance of remission**

The relapse rate of AAV is high, as demonstrated by the different induction trials, and occur frequently during a drug withdrawal period [32, 39]. Therefore it is important to maintain long-term immunosuppression, while limiting drug toxicity. When a standardized treatment of induction of remission followed by maintenance therapy is applied, the relapse rate in GPA can be reduced from 76.8% (in cohorts treated before 1993) to 50% (in cohorts treated after 1999) over 5 years follow-up [40]. Several studies have provided different drug alternatives for maintenance of remission.

#### **6.1. Azathioprine**

L. More than 90% of patients had GPA. In total, 49 subjects were treated with methotrex‐ ate (15 mg/week orally escalating to a maximum of 20–25 mg/week by 12 weeks), which was then maintained until month 10 and then tapered and discontinued by month 12. A total of 46 subjects received oral cyclophosphamide (2 mg/kg/day (maximum 150 mg/ day) until remission), for a minimum of 3 and a maximum of 6 months. Dose alterations were made for subjects >60 years of age, and the drug was withdrawn if the total white

1.5 mg/kg/day until month 10, when it was tapered and discontinued by month 12. Both treatment groups received oral prednisolone 1 mg/kg/day, tapered to 15 mg/day at 12 weeks and 7.5 mg/day by 6 months, and discontinued by 12 months. The primary end point was induction of remission within 6 months. Between month 12 to 18, patients

At 6 months, 90% of subjects randomized to methotrexate and 94% of subjects random‐ ized to cyclophosphamide achieved remission (p=0.041). The median time to remission was 3 months (range 1–9) in the methotrexate group and 2 months (range 1–5) in the cyclophos‐ phamide group (p=0.19 log rank test). Of the subjects who achieved remission during the treatment period, 70% of the subjects randomized to methotrexate and 47% of subjects randomized to cyclophosphamide had a relapse, with the time to relapse being significant‐ ly longer in the cyclophosphamide group (median 15 months, range 4-17) compared to the methotrexate group (median 13 months, range 2-17) (P =0.023 log rank test). Leukopenia was more common in the cyclophosphamide group and liver dysfunction was more

The long term follow-up of patients treated in the NORAM study was recently reported [39]. Data was obtained on all 95 original subjects with a median duration of follow-up of 6 years. Subjects in the methotrexate group required a longer duration of corticosteroid therapy during the trial period of 18 months (median 15 months, interquartile range (IQR) 12-18) compared to 12 months (IQR 12-15) in the cyclophosphamide group, p=0.005). During subsequent followup, the median duration of corticosteroid therapy during months 19-60 was 3.0 years in the methotrexate group and only 1.5 years in the cyclophosphamide group (p=0.004). After the trial period of 18 months, patients' treatment was left at the discretion of their physicians. Physicians were asked to provide information regarding drugs used to manage disease flare such as cyclophosphamide, methotrexate, azathioprine and mycophenolate mofetil. Exposure to cyclophosphamide and these other agents was also longer in the methotrexate group

Overall, the cumulative relapse-free survival from the time of first remission was 69% after 1 year, 32% after 3 years, and 24% after 5 years of follow-up, demonstrating a trend to being higher in the cyclophosphamide group (p=0.056, logrank test). The cumulative overall survival did not differ between treatment arms (p=0.88, log-rank test) and was 98% after 1 year, 93% after 3 years, and 89% after 5 years. The number of serious infections did not differ between treatment groups. The authors have concluded that methotrexate therapy was associated with

less effective long-term disease control as compared to cyclophosphamide.

/liter. At remission, cyclophosphamide was reduced to

blood cell count fell below 4 x109

198 Updates in the Diagnosis and Treatment of Vasculitis

received no immunosuppressant agents.

common in the methotrexate group.

(p=0.037; and p=0.031, respectively).

An 18 month prospective open label study (CYCAZAREM) compared the use of oral cyclo‐ phosphamide to azathioprine in the maintenance phase, with 155 subjects with GPA, MPA and renal limited vasculitis recruited from 39 hospitals in 11 European countries [41]. All subjects had received the same remission-induction therapy, consisting of daily oral cyclo‐ phosphamide (2 mg/kg) and prednisolone (initially 1 mg/kg/day, with the dose tapered to 0.25 mg/kg/day by 12 weeks). Renal vasculitis was the most common form of organ involvement, occurring in 94 percent of the patients in the study. Patients attaining remission by 3 months, and those attaining remission between 3 and 6 months, were randomly assigned to treatment with azathioprine (2 mg/kg/day) or to continue cyclophosphamide therapy at a lower dose (1.5 mg/kg/day). Both treatment groups continued to receive prednisolone 10 mg daily. At 12 months after study entry, both groups received azathioprine at a dose of 1.5 mg/kg/day and prednisolone 7.5 mg daily. The primary end point was relapse, either major (threatened function of the kidney, lung, brain, eye, motor nerve or gut) or minor (affecting at least three other items in the Birmingham Vasculitis Activity Score (BVAS)) [42].

Of the initial 155 subjects, clinical remission was achieved in 93% overall, with 77% reaching this target by 3 months and 16% between 3 and 6 months. These patients were randomly assigned to cyclophosphamide (73 patients) or azathioprine (71 patients). Azathioprine was demonstrated to be equivalent to cyclophosphamide for maintenance therapy. Sixteen percent in the azathioprine group had relapses, compared to 14% in the cyclophosphamide group (p=0.65). Five patients in each group had a major relapse. The most frequent adverse event was neutropenia (55% of patients, including the remission and maintenance phase), with 52% of infections occurring during an episode of neutropenia. There was no difference in renal outcomes between the groups, with renal failure occurring in only 3% of patients.

#### **6.2. Methotrexate**

A prospective, open-label, multicenter trial, comparing methotrexate and azathioprine for maintenance of remission in GPA and MPA (WEGENT) was designed to detect treatment tolerance [43]. Three-quarters of the patients had GPA. Sixty-three patients who had achieved remission with intravenous cyclophosphamide and corticosteroids received oral azathioprine (2 mg/kg/day) and 63 received methotrexate (initial 0.3 mg/kg/week, progressively increased to 25 mg per week) for 12 months. At the end of the scheduled maintenance therapy period, azathioprine and methotrexate were withdrawn over a period of 3 months at the discretion of the treating physician. T/S was recommended for 2 additional years for patients with GPA after discontinuation of the maintenance immunosuppressive agents. The primary end point was an adverse event requiring discontinuation of the study drug or causing death.

choice for maintenance of remission in AAV, but could be used in situations of intolerance or

Treatment of ANCA-Associated Vasculitis in Adults

http://dx.doi.org/10.5772/54290

201

Leflunomide is a disease-modifying agent commonly used in the treatment of rheumatoid arthritis as an alternative to methotrexate. A prospective randomized controlled trial of leflunomide compared to methotrexate in patients with generalized GPA for maintenance of remission was conducted in 5 German rheumatology centres [46]. The study was powered to find equivalence between the 2 drugs. Patients achieving complete or partial remission with daily oral cyclophosphamide (2mg/kg) and prednisolone and maintained remission for at least 3 months were enrolled in the study. Partial remission was defined as partial improvement of the disease persisting for at least 3 months represented by a constant disease extent index and BVAS. Complete remission was defined as the absence of pathological findings in clinical, radiological and serological investigations, irrespective of the ANCA titre. Twenty-eight subjects received oral methotrexate starting at a dose of 7.5 mg/week, increased over 9 weeks to 20 mg/week. Folic acid 10 mg weekly was taken the day after methotrexate. Twenty-six patients received leflunomide with a loading dose of 100 mg daily for 3 days, followed by 20 mg daily and then increased to 30 mg daily after 4 weeks. Prednisone was allowed at a dose of 10mg/day or less, and was tapered by 2.5mg/month in the absence of disease activity until a dose of 5 mg was reached, and then by 1 mg/month thereafter. The primary efficacy outcome

In the leflunomide group, 23% of subjects experienced a relapse, compared to 46% of metho‐ trexate subjects (p=0.09), and the incidence of major relapses was significantly higher in the methotrexate group (p=0.037). The study was terminated prematurely in September 2003 after the advisory board had decided that the high rate of major relapses in the methotrexate group

*Safety*: There was no significant difference in the number of adverse events between the groups. Thirty-four adverse events were observed in the leflunomide group and 17 in the methotrexate group (p=0.09). Leflunomide was stopped in two patients with intractable hypertension, one patient with peripheral neuropathy and one patient with leucopenia, whereas no patient stopped due to adverse events in the methotrexate group. Twenty-five infectious episodes, 13 in the leflunomide group and 12 in the methotrexate group were noted, all responding well to

Some patients have disease that proves to be refractory to the therapies used for induction and maintenance of remission as above. Typically, patients with lung and upper airway involve‐ ment, positive PR3-ANCA and severe renal involvement have more resistant disease [47-49]. Relapse is also frequent in AAV, with an overall risk of 38% at 5 years seen in a large cohort of 535 patients from 70 European trial sites between 1995 and 2002 [32]. The presence of positive

contraindication to azathioprine.

was the number of major and minor relapses.

conventional antibiotic treatment on outpatient basis.

**7. Refractory/relapsing disease**

**6.4. Leflunomide**

was not acceptable.

At the censoring date for analysis, the mean follow-up after randomization was 29 months. Adverse events leading to the primary end point (i.e., discontinuation of the study drug or death) occurred in 11% of the azathioprine group and 19% in the methotrexate group (p=0.21). After starting maintenance therapy, 46% of azathioprine recipients had at least one adverse event as compared with 56% of methotrexate recipients (p= 0.29). Thirty-six percent of azathioprine subjects and 33% of methotrexate subjects had a relapse (p=0.71). In 73% of the patients the relapse occurred after discontinuation of the drugs. This study demonstrated that the two agents were equivalent in safety and also efficacy. There was a trend toward a higher risk of adverse events with methotrexate, with a hazard ratio of 1.65 (95%CI, 0.65-4.18).

#### **6.3. Mycophenolate mofetil**

Mycophenolate mofetil is a prodrug of mycophenolic acid, which is a reversible inhibitor of inosine monophosphate dehydrogenase in guanosine nucleotide synthesis, upon which T and B cells are dependent, and has cytostatic effects on lymphocytes [44]. It has been proposed as a less toxic alternative to azathioprine and has been evaluated in one randomized trial. The IMPROVE study was an open-label trial to assess whether mycophenolate mofetil reduces the risk of relapse compared with azathioprine in patients with AAV in remission [45]. All patients received cyclophosphamide (daily oral or intermittent intravenous doses for a maximum of 6 months) and glucocorticoids (up to 3 g of methylprednisolone over 3 days was allowed for severe disease, then 1 mg/kg/day (maximum 80 mg) of oral prednisolone) for induction of remission. Plasma exchange could also be used for severe disease. Oral steroids were reduced according to a standardized schedule to 15 mg/day at the start of the remission regimen, tapered to 5 mg/day after 12 months, and were withdrawn after 24 months. One hundred and fifty six patients with a new diagnosis of GPA and MPA were enrolled after remission was achieved. The azathioprine group (n=80) initially received 2 mg/kg/day (maximum 200 mg), with dose reductions to 1.5 mg/kg/day after 12 months and 1 mg/kg/day after 18 months, with drug withdrawal after 42 months. Seventy-six patients assigned to the mycophenolate mofetil group received 2 g/day, which was reduced to 1500 mg/day after 12 months, 1000 mg/day after 18 months, and withdrawn after 42 months. The primary end point was relapse-free survival, defined as the time from remission to the first relapse (major or minor), withdrawal, death or loss to follow-up, or the end of the follow-up period.

Median follow-up for both treatment groups from start of maintenance therapy was 39 months. Relapses were more common in the mycophenolate mofetil group. In total, 55% of the mycophenolate mofetil recipients experienced relapses (18 major, 24 minor), as compared to 38% of azathioprine recipients (10 major, 20 minor), with an unadjusted hazard ratio for mycophenolate mofetil use of 1.69 (95%CI, 1.06-2.70; p=0.03) at 4 years. The risk of severe adverse events was not significantly different between groups, with a hazard ratio of 0.53 (95%CI, 0.23-1.18, p=0.12). Therefore mycophenolate mofetil should not be considered as a first choice for maintenance of remission in AAV, but could be used in situations of intolerance or contraindication to azathioprine.

#### **6.4. Leflunomide**

azathioprine and methotrexate were withdrawn over a period of 3 months at the discretion of the treating physician. T/S was recommended for 2 additional years for patients with GPA after discontinuation of the maintenance immunosuppressive agents. The primary end point

At the censoring date for analysis, the mean follow-up after randomization was 29 months. Adverse events leading to the primary end point (i.e., discontinuation of the study drug or death) occurred in 11% of the azathioprine group and 19% in the methotrexate group (p=0.21). After starting maintenance therapy, 46% of azathioprine recipients had at least one adverse event as compared with 56% of methotrexate recipients (p= 0.29). Thirty-six percent of azathioprine subjects and 33% of methotrexate subjects had a relapse (p=0.71). In 73% of the patients the relapse occurred after discontinuation of the drugs. This study demonstrated that the two agents were equivalent in safety and also efficacy. There was a trend toward a higher risk of adverse events with methotrexate, with a hazard ratio of 1.65 (95%CI, 0.65-4.18).

Mycophenolate mofetil is a prodrug of mycophenolic acid, which is a reversible inhibitor of inosine monophosphate dehydrogenase in guanosine nucleotide synthesis, upon which T and B cells are dependent, and has cytostatic effects on lymphocytes [44]. It has been proposed as a less toxic alternative to azathioprine and has been evaluated in one randomized trial. The IMPROVE study was an open-label trial to assess whether mycophenolate mofetil reduces the risk of relapse compared with azathioprine in patients with AAV in remission [45]. All patients received cyclophosphamide (daily oral or intermittent intravenous doses for a maximum of 6 months) and glucocorticoids (up to 3 g of methylprednisolone over 3 days was allowed for severe disease, then 1 mg/kg/day (maximum 80 mg) of oral prednisolone) for induction of remission. Plasma exchange could also be used for severe disease. Oral steroids were reduced according to a standardized schedule to 15 mg/day at the start of the remission regimen, tapered to 5 mg/day after 12 months, and were withdrawn after 24 months. One hundred and fifty six patients with a new diagnosis of GPA and MPA were enrolled after remission was achieved. The azathioprine group (n=80) initially received 2 mg/kg/day (maximum 200 mg), with dose reductions to 1.5 mg/kg/day after 12 months and 1 mg/kg/day after 18 months, with drug withdrawal after 42 months. Seventy-six patients assigned to the mycophenolate mofetil group received 2 g/day, which was reduced to 1500 mg/day after 12 months, 1000 mg/day after 18 months, and withdrawn after 42 months. The primary end point was relapse-free survival, defined as the time from remission to the first relapse (major or minor), withdrawal, death or

Median follow-up for both treatment groups from start of maintenance therapy was 39 months. Relapses were more common in the mycophenolate mofetil group. In total, 55% of the mycophenolate mofetil recipients experienced relapses (18 major, 24 minor), as compared to 38% of azathioprine recipients (10 major, 20 minor), with an unadjusted hazard ratio for mycophenolate mofetil use of 1.69 (95%CI, 1.06-2.70; p=0.03) at 4 years. The risk of severe adverse events was not significantly different between groups, with a hazard ratio of 0.53 (95%CI, 0.23-1.18, p=0.12). Therefore mycophenolate mofetil should not be considered as a first

was an adverse event requiring discontinuation of the study drug or causing death.

**6.3. Mycophenolate mofetil**

200 Updates in the Diagnosis and Treatment of Vasculitis

loss to follow-up, or the end of the follow-up period.

Leflunomide is a disease-modifying agent commonly used in the treatment of rheumatoid arthritis as an alternative to methotrexate. A prospective randomized controlled trial of leflunomide compared to methotrexate in patients with generalized GPA for maintenance of remission was conducted in 5 German rheumatology centres [46]. The study was powered to find equivalence between the 2 drugs. Patients achieving complete or partial remission with daily oral cyclophosphamide (2mg/kg) and prednisolone and maintained remission for at least 3 months were enrolled in the study. Partial remission was defined as partial improvement of the disease persisting for at least 3 months represented by a constant disease extent index and BVAS. Complete remission was defined as the absence of pathological findings in clinical, radiological and serological investigations, irrespective of the ANCA titre. Twenty-eight subjects received oral methotrexate starting at a dose of 7.5 mg/week, increased over 9 weeks to 20 mg/week. Folic acid 10 mg weekly was taken the day after methotrexate. Twenty-six patients received leflunomide with a loading dose of 100 mg daily for 3 days, followed by 20 mg daily and then increased to 30 mg daily after 4 weeks. Prednisone was allowed at a dose of 10mg/day or less, and was tapered by 2.5mg/month in the absence of disease activity until a dose of 5 mg was reached, and then by 1 mg/month thereafter. The primary efficacy outcome was the number of major and minor relapses.

In the leflunomide group, 23% of subjects experienced a relapse, compared to 46% of metho‐ trexate subjects (p=0.09), and the incidence of major relapses was significantly higher in the methotrexate group (p=0.037). The study was terminated prematurely in September 2003 after the advisory board had decided that the high rate of major relapses in the methotrexate group was not acceptable.

*Safety*: There was no significant difference in the number of adverse events between the groups. Thirty-four adverse events were observed in the leflunomide group and 17 in the methotrexate group (p=0.09). Leflunomide was stopped in two patients with intractable hypertension, one patient with peripheral neuropathy and one patient with leucopenia, whereas no patient stopped due to adverse events in the methotrexate group. Twenty-five infectious episodes, 13 in the leflunomide group and 12 in the methotrexate group were noted, all responding well to conventional antibiotic treatment on outpatient basis.


**1. Introduction**

Periodontitis is a chronic inflammatory oral disease caused by bacteria infection [1]. Typically, the infection destroys periodontal cells including gingival fibroblast, periodontal ligament fibroblasts, and alveolar bone, which are the supporting tissue and bone that hold the tooth. As the disease progresses, more oral tissues are damaged, causing deep pockets, which eventually lead to teeth loss if left untreated [2].

Tissue engineering has been employed to regenerate the lost periodontal tissues and restore both structure and function. In this regard, three dimensional (3D) porous scaffolds represent important components for tissue engineering as a supporting material for cell proliferation or differentiation before being applied to repair the damaged area [3,4]. Scaffolds provide attachment sites and structural guidance for cells that enable them to synthesize appropriate extracellular matrix (ECM) proteins and ultimately proliferate into functional tissues [5]. In addition, the choice of scaffold can be critical as its chemical and

**Citation:** Phuegyod, S.; Pramual, S.; Wattanavichean, N.; Assawajaruwan, S.; Amornsakchai, T.; Sukho, P.; Svasti, J.; Surarit, R.; Niamsiri, N. Microbial Poly(hydroxybutyrate-cohydroxyvalerate) Scaffold for Periodontal Tissue Engineering. *Polymers* **2023**, *15*, 855. https:// doi.org/10.3390/polym15040855

Academic Editor: Shashi Kant Bhatia

Received: 4 January 2023 Revised: 31 January 2023 Accepted: 2 February 2023 Published: 9 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

physical properties provide guidance cues for the cells to behave appropriately. Scaffold biomaterials for successful tooth regeneration applications should have some requirements such as being biocompatible, biodegradable, and possess mechanical properties that are consistent with the implanted area as well as being used in the appropriate amount and with an accessible volume of porosities for the diffusion of oxygen, cells, and nutrients [6,7]. To date, many polymeric materials have been reported to create biodegradable scaffolds for dental tissue engineering including poly(lactide) (PLA) [8], poly(lactide-co-glycolide) (PLGA) [9,10], and polycaprolactone (PCL) [11–13].

Polyhydroxyalkanoates (PHAs) are aliphatic polyesters synthesized by microorganisms to store excess carbon and energy. Poly(hydroxybutyrate-co-hydroxyvalerate) or P(HB-HV) copolymers are a member of the PHA family [14]. P(HB-HV) has shown great potential for tissue engineering with attractive characteristics of natural origin, biocompatibility, and biodegradability. The properties are adjustable by changing the content of the HV unit. P(HB-HV) are less crystalline, less stiff, and more flexible than the PHB homopolymer due to the incorporation of the HV monomer in the polymer chain [15]. Recently, studies have revealed that different types of scaffolds made with P(HB-HV) demonstrate desirable advantages for tissue engineering. The application of macroporous P(HB-8HV) matrices in the repair of full-thickness cartilage defects in rabbits in vivo was reported by Kose et al. At 8 and 20 weeks after seeding, in vivo results with chondrocyte seeded P(HB-8HV) matrices presented early cartilage formation resembling normal articular cartilage and revealed minimal foreign body reaction. This study also showed that P(HB-8HV) matrices maintained their integrity for 21 days and permitted appropriate gradual degradation and allowed for tissue remodeling to take place [16]. Abazari et al. demonstrated the increased survival rate and insulin-producing cell (IPC) differentiation potential of induced pluripotent stem cells (iPSCs) cultured on a nanofibrous 3D P(HB-5HV) scaffold in comparison with the 2D substrate. iPSCs-P(HB-5HV), as a promising cell-copolymer construct, could potentially be applied in pancreatic tissue engineering applications to diabetic patient treatment [17]. The P(HB-3HV) scaffold was tested for degradation in simulated body fluid (SBF), pH 7.4. After 8-week periods, the P(HB-3HV) scaffolds revealed about 51% weight loss along time due to the high porous structure when compared with the dense and compact films, which showed about a 9% weight loss. Culturing of MC3T3-E1 pre-osteoblast cells on the P(HB-3HV) scaffold samples obtained after 6 weeks of degradation did not lead to the formation of cytotoxic components [18]. In spite of extensive research on P(HB-HV) and their blends as scaffolds for tissue engineering, the HV molar contents of the available commercial P(HB-HV) published are 12 mol% or lower. P(HB-HV) films consisting of various HV content (5–80%) produced by *Paracoccus denitrificans* have been reported to be biocompatible with connective tissue, bone, and dermal fibroblast cells [19]. *Haloferax mediterranei* ES1 produced P(HB-HV) nanofibrous meshes were also shown to be excellent in vitro and to show in vivo biocompatibility with skin tissues [20]. As reported earlier, the more flexible P(HB-HV) with HV contents of 50 mol% can be successfully biosynthesized from bacteria *Cupriavidus necator* H16. This material has already been employed as a drug delivery platform [21–23]. Until now, there have been no studies available in the literature concerning the application of P(HB-50HV) produced by *C. necator* H16 as a scaffold to support cell growth and promote tissue regeneration.

In this study, the 3D porous scaffolds were fabricated from bacterial derived P(HB-50HV) via a particulate leaching method using salt particles as a strategy for the regeneration of periodontal cells. Comparisons were made with scaffold prepared from poly(hydroxybutyrate) (PHB) and poly(hydroxybutyrate-co-12%hydroxyvalerate) (P(HB-12HV)), and the well-established synthetic polycaprolactone (PCL). The scaffolds were characterized with respect to the morphology of the surface and cross section, porosity, mechanical strength, and protein absorption. Subsequently, biological performance of the scaffolds in terms of biocompatibility and cell proliferation was assessed. In this regard, human gingival fibroblasts (HGFs) [10] and periodontal ligament stem cells (PDLSCs) [24]

were used since they have been widely studied for the initial evaluation of biomaterials for periodontal tissue engineering applications.

#### **2. Materials and Methods**

#### *2.1. Materials*

Poly(hydroxybutyrate) (PHB, Mw 3.5 × 105 g/mol), poly(hydroxybutyrate-cohydroxyvalerate) containing HV content 12 mol% (P(HB-12HV), Mw 2.5 × 105 g/mol), polycaprolactone (PCL, Mw 6.5 × 104 g/mol), and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Chloroform, methanol, and ethanol were purchased from RCI Labscan (Bangkok, Thailand). Sodium chloride (NaCl) (Ajax Chemicals Ltd., Sydney, Australia) with the particle size range of 425–500 μm was obtained by sieving through an analytical sieve shaker Octagon digital (Endecotts Ltd., London, UK) using two certified sieve sizes with 425 and 500 μm. Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA were obtained from Invitrogen (Carlsbad, CA, USA). All chemicals and solutions were used as supplied without further purification. Poly(hydroxybutyrate-co-hydroxyvalerate) with 50% HV content (P(HB-50HV) Mw 1.69 × 106 g/mol) was biosynthesized by in house bacterial cultivation according to a previously described protocol [21].

#### *2.2. Characterization of Polymer Films*

Thin polymeric films of PHB, P(HB-12HV), P(HB-50HV), and PCL were prepared by a casting method using 10 mL of 2% (*w*/*v*) polymer stock solution in chloroform on a clean Petri dish. Chloroform was evaporated in a fume hood at room temperature for 24 h. The final thickness of film ranged from 0.05 to 0.10 mm.

The Fourier transform infrared (FTIR) spectra of all PHA and PCL thin films were obtained with a Perkin-Elmer FTIR ATR-FTIR spectrometer (Perkin-Elmer, Spectrum GX FTIR; Shelton, CT, USA). The sample spectra were recorded over 20 scans between 400 and 4000 cm−1 wavenumbers at a resolution of 4 cm−1.

The hydrophilicity of the polymeric surfaces was examined by an optical bench-type contact angle goniometry DM-CE1 (Kyowa Interface Science, Niiza, Japan) using a sessile drop method at room temperature.

#### *2.3. Fabrication and Characterization of Scaffolds*

Salt-leached scaffolds of PHB, P(HB-12HV), P(HB-50HV), and PCL were fabricated following the established procedure [25]. In brief, the polymer was dissolved in chloroform to prepare a 5% (*w*/*v*) stock solution. The 1 mL polymer solution was then poured on a bed of sieved NaCl particles (with size range of 425–500 μm) in a clean glass vial. The weight ratio of porogen (NaCl) to polymer was set at 9:1. The scaffolds were placed in a fume hood at room temperature for the slow evaporation of chloroform over 2 days followed by repeated rinsing with distilled water to remove any residual salt and air-dried. All scaffolds were prepared as a cylindrical shape with 10 mm diameter and 3 mm height.

The fabricated scaffolds were mounted onto an aluminum stub, gold-coated, and then observed by scanning electron microscopy (SEM, JSM-6360; JEOL Techniques, Tokyo, Japan) with an accelerating voltage of 20 kV for the surface topography and cross section images.

The porosity or void volume fraction Vf (%) of the scaffold was calculated using the following equation:

$$\mathbf{V}\_{\mathbf{f}} = (1 - (\rho\_{\mathbf{s}}/\rho\_{\mathbf{m}})) \times 100$$

where ρs is the apparent density of the porous scaffold and ρm is the density of the polymer material [26].

#### *2.4. Compressive Mechanical Testing of Scaffolds*

The scaffolds were subjected to mechanical measurements under compressive mode in order to determine the compressive stress and compressive modulus (E). The tests were performed at room temperature using a Texture analyzer (TA-XT2i, Stable Micro Systems, Ltd., Godalming, UK) with a 50 kN load cell at a crosshead speed of 0.1 mm/s [27]. Cylindrical specimens were tested under both dry and wet conditions. The load deformation curves of the samples obtained were converted into stress–strain curves. The compressive stress (MPa) was used to calculate the secant modulus according to the following equation: