**Energy Data Analytics for Smart Meter Data**

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*Article* **Modulation and Control of Wettability and Hardness of Zr-Based Metallic Glass via Facile Laser Surface Texturing**

**Qinghua Wang 1,2, Yangyang Cheng 3 , Zhixian Zhu 1,2, Nan Xiang 1,2 and Huixin Wang 4,\***

**Abstract:** Bulk metallic glass (BMG) has received consistent attention from the research community owing to its superior physical and mechanical properties. Modulating and controlling the surface functionalities of BMG can be more interesting for the surface engineering community and will render more practical applications. In this work, a facile laser-based surface texturing technique is presented to modulate and control the surface functionalities (i.e., wettability and hardness) of Zr-based BMG. Laser surface texturing was first utilized to create periodic surface structures, and heat treatment was subsequently employed to control the surface chemistry. The experimental results indicate that the laser textured BMG surface became superhydrophilic immediately upon laser texturing, and it turned superhydrophobic after heat treatment. Through surface morphology and chemistry analyses, it was confirmed that the wettability transition could be ascribed to the combined effects of laser-induced periodic surface structure and controllable surface chemistry. In the meantime, the microhardness of the BMG surface has been remarkably increased as a result of laser surface texturing. The facile laser-based technique developed in this work has shown its effectiveness in modification and control of the surface functionalities for BMG, and it is expected to endow more useful applications.

**Keywords:** laser surface texturing; wettability; hardness; Zr-based metallic glass

#### **1. Introduction**

Bulk metallic glass (BMG) has received considerable attention from the research community during the past several decades since its first discovery in the 1990s, mainly owing to its superior mechanical and physical properties [1], including high values of yield strength [2], high hardness [3], relatively low Young's modulus [4], good corrosion and wear resistance [5], as well as excellent magnetic properties [6]. This gives BMG a variety of potential applications in the fields of bioimplants, magnetic materials, structural materials, sensors, microelectromechanical systems (MEMS), and micro/macro devices [7,8].

Besides its intrinsic outstanding physical and mechanical properties, researchers have also attempted to modify the surface functionalities of BMG, which can be achieved by introducing micro/nanostructures into BMG. The typical fabrication methods of surface structuring include magnetron sputtering [9,10], electro-oxidation [11], thermoplastic shaping [12–14], and laser surface texturing [15,16]. Among all the existing surface modification methods, laser surface texturing has demonstrated its strong potential as a highly efficient and cost-effective approach due to several key advantages including process efficiency, flexibility, ease for automation, and environmental friendliness [17].

In recent years, laser-based surface texturing has been proved to be one of the most efficient techniques to modify and control important surface functionalities, including

**Citation:** Wang, Q.; Cheng, Y.; Zhu, Z.; Xiang, N.; Wang, H. Modulation and Control of Wettability and Hardness of Zr-Based Metallic Glass via Facile Laser Surface Texturing. *Micromachines* **2021**, *12*, 1322. https://doi.org/10.3390/mi12111322

Academic Editors: Youmin Rong, Congyi Wu and Yu Huang

Received: 27 September 2021 Accepted: 27 October 2021 Published: 28 October 2021

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**Copyright:** © 2021 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/).

surface wettability [18–22], reflectivity [23,24], anti-icing property [25,26], corrosion resistance [27], etc. For example, wettability transition from superhydrophilicity to superhydrophobicity has been achieved on various materials including aluminum [28], copper [29], stainless steel [30,31], and titanium [32–36] by combining laser surface texturing and lowtemperature annealing. In terms of laser texturing for BMG materials, there have been some recent research efforts on modification of surface properties of BMG via creation of laser-induced surface texture and change of surface chemistry [15,16,37–42]. Huang et al. utilized nanosecond pulsed laser irradiation to fabricate hierarchical micro/nanostructures on the Zr-based metallic BMG substrate in order to increase the effective surface area [16]. The laser-modified BMG surface retained amorphous characteristics, and the elemental distribution on the surface was very uniform. Jiao et al. developed a nanosecond laser texturing technique to fabricate periodic surface structures, including dimples and grooves on Zr-based BMG surfaces [37]. They also investigated the effect of laser surface texturing on the wettability [15] and cytocompatibility [38] of the BMG surfaces. The modification of the surface wettability could be attributed to the laser-induced surface roughness and alteration of surface chemistry, and the enhanced cytocompatibility of the groove-textured BMG resulted from the combined effects of surface chemistry, wettability, and roughness. Du et al. fabricated laser-induced periodic surface structure (LIPSS) and nanoparticle structures on four types of Zr-based BMGs using femtosecond laser irradiation [40]. The experimental results indicated that the femtosecond laser nanostructured Zr-based BMG surface could lead to a distinct decrease in bacterial adhesion compared with the polished surfaces, which was strongly related to the laser-induced surface morphology and wettability. Although the above-mentioned research efforts have effectively modified the surface properties of BMG and achieved improved surface functionalities, none of them has attempted to realize the precise control of the key surface functions of BMG, e.g., control of surface wettability and microhardness, which could be more attractive and challenging, and also help to meet more different applications [43]. Further exploration of a time-efficient and cost-effective laser-based technique to realize the control of surface functionalities on the BMG surface is still of particular interest for the surface engineering community.

Previously, the authors' group has developed a facile nanosecond laser-based surface texturing method to achieve switchable wettability control of titanium alloy [36]. In this work, this laser-based surface texturing technique was further extended to modulate surface wettability and hardness on the BMG substrate. The periodic surface textures were directly created via laser texturing, and the surface chemistry was effectively controlled via heat treatment. The surface wettability was shown to convert from superhydrophilicity immediately upon lase texturing to superhydrophobicity after heat treatment, and the surface microhardness was significantly enhanced on the laser-induced surface texture. The underlying processing mechanisms were elucidated using scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDAX), and X-ray photoelectron spectroscopy (XPS). Compared with the existing research works on laser texturing for functional surfaces, the laser-based surface texturing technique developed in this work proposed a novel and highly efficient approach to modulate and control the surface functionalities, which was achieved by the combination of high-speed UV nanosecond laser surface texturing and subsequent heat treatment. It is expected that the developed technique could provide a viable solution for the surface modification of Zr-based metallic glass, thus rendering a series of applications in the industrial and biomedical fields.

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

#### *2.1. Materials*

Commercially available Zr-based bulk metallic glass Vitreloy 1 with the nominal elemental composition Zr41.2Ti13.8Cu12.5Ni10Be22.5 (at%, purchased from METALLAB, Changzhou, China) was used in this work due to its excellent mechanical properties. The samples were mechanically grinded using grid SiC papers and further machined into thin sheets with a dimension of 30 mm × 30 mm × 2 mm using wire electrical discharge ma-

chining. Then, before laser surface texturing experiments, they were ultrasonically cleaned with acetone, ethanol, and deionized water successively to remove the contaminants.

#### *2.2. Laser-Based Surface Texturing*

The laser-based surface texturing experiment mainly consists of two steps: (1) laser surface texturing and (2) heat treatment. Laser surface texturing experiments employed a laser marking machine (TH-UV200A, Tianhong Laser, Suzhou, China) equipped with a 355 nm UV laser source (AWAVE 355-15W-30K, Advanced Optowave, Ronkonkoma, NY, USA). The laser source emits a laser beam guided by reflective mirrors. The intensity of laser power is controlled by the attenuator, and the diameter of the laser beam can be expanded by the beam expander. The focusing lens of the system provides a focal spot diameter of ~35 µm during the laser texturing experiments. Unidirectional line pattern and cross-hatch pattern were used due to the ease and high efficiency of fabrication, as shown in Figure 1a,b. A series of preliminary experimental trials was attempted, and the optimal laser processing parameters were determined and utilized in this work, which can be found in Table 1. The schematics for the laser surface texturing experiments are shown in Figure 2a.

**Figure 1.** Pattern designs used for laser surface texturing of BMG: (**a**) unidirectional; (**b**) cross-hatch. **Table 1.** Processing parameters for the laser-based surface texturing experiments.

**Figure 2.** Schematics for the laser-based surface texturing experiments of BMG: (**a**) laser surface texturing; (**b**) heat treatment.

Immediately upon laser surface texturing, the laser textured BMG surfaces became superhydrophilic. To achieve the wettability transition, the laser textured BMG surfaces were placed in a conventional furnace for low-temperature annealing treatment, as shown in Figure 2b. An annealing temperature of 150 ◦C and a treatment duration of a maximum of 2 h were used during the heat treatment.

#### *2.3. Surface Characterizations*

θ The surface morphology of the laser textured BMG surfaces was analyzed using field emission scanning electron microscopy (FESEM, Navo Nano SEM450, Hillsboro, OR, USA). The surface chemistry of the laser textured surface was evaluated using energy dispersive Xray analysis (EDAX, FEI Sirion, Hillsboro, OR, USA) and X-ray photoelectron spectroscopy (XPS, PREVAC, Rogów, Poland). To examine the surface wettability of the laser textured surfaces, a contact angle goniometer (SDC-200, Sindin Precision Instrument Co., Ltd., Dongguan, Guangdong, China) equipped with a high-resolution CMOS camera was utilized to measure the static water contact angle value (θw) on each surface. A water droplet with the volume of ~4 µL was dripped onto the sample surface using a digital syringe during each measurement. The optical image of the water contact angle measurement was captured using the CMOS camera, and image analysis software was utilized to determine the θw value for each measurement. For each sample, four θw measurements were performed at various locations, and the averaged θw value was reported. The standard deviation for each averaged θw measurement result was also calculated and added. The microhardness test was carried out using a Vickers hardness machine (HXD-1000TMSC/LCD, Shanghai, China) with a test load of 300 gf and a dwell time of 10 s. Similarly, five microhardness measurements were performed at different locations on the laser textured surface, and the averaged hardness value as well as the standard deviation for each measurement result were reported.

#### **3. Results**

#### *3.1. Surface Morphology*

The surface morphology of the laser textured BMG surface using different scanning speeds was examined by SEM, as shown in Figure 3. The SEM micrographs of the laser textured BMG surfaces with unidirectional surface patterns and various scanning speeds can be found in Figure 3a–d, while the SEM micrographs of the laser textured BMG surfaces with cross-hatch surface patterns and various scanning speeds are shown in Figure 3e–h. It is clearly seen that as the laser beam irradiated and ablated the BMG substrates, periodic micro-scale bulge structures were formulated as a result of the strong interaction between the laser beam and the BMG substrates. The SEM micrographs with high magnification indicate that there also have been some sub-micron or nano-scale particles covered on the micro-bulge structures, which can be mainly attributed to the ejection and deposition of nanoparticles during the laser-material interaction. In addition, it can be found that the laser-induced periodic surface structure exhibited distinct differences as the scanning speed increased. When the scanning speed of 20 mm/s was used, solid and defect-free microbulge structures were formed on the laser textured BMG surfaces with both unidirectional and cross-hatch patterns. As the scanning speed further increased, the surface structure gradually changed, which was illustrated by the variation of the micro-bulge height and the appearance of concave sections. Especially when the scanning speeds reached 60 mm/s and 80 mm/s, the concave structure became more distinct along the laser scanned line profiles for both surface patterns. The difference of the surface structure formation when varying the scanning speed should result from the time duration of laser–material interaction. When the lower scanning speed was utilized, the laser beam travelled relatively slowly along the scanned profile, and the number of pulses per irradiation point is higher, which would ensure adequate interaction between the laser beam and the BMG substrate and facilitate the formation of solid micro-bulge structures [44]. However, as the scanning speed increased, the laser beam moved faster and resulted in the decrease in the number of pulses per irradiation point. This would significantly weaken the impact of the laser beam on the BMG substrate. Consequently, due to the insufficient interaction time between the laser beam and the BMG substrate, fewer sub-micron or nano-scale particles and concave structures occurred on the laser textured surface with higher scanning speeds.

The effect of laser power on the surface morphology of the laser textured BMG surface was further examined by SEM, as shown in Figure 4. The SEM micrographs of the laser textured BMG surfaces with unidirectional surface patterns and various laser powers can be found in Figure 4a–e, while the SEM micrographs of the laser textured BMG surfaces with cross-hatch surface patterns and various laser powers are shown in Figure 4e–h. From the SEM micrographs, it is evident that laser power can dramatically affect the formation of laser-induced structures on the BMG surface. By using higher laser powers (15.0 W and 13.5 W), clear concave microgrooves formed on the BMG surface, demonstrating strong vaporization of the material during the laser surface texturing process. As the laser power decreased (12.0 W and 10.5 W), the microgroove structure became less pronounced, only with some holes left on the laser scanned profiles. This indicated that less material evaporated from the substrate, and there appeared to be a restructuring on the laser textured surface. As the laser power kept decreasing (9.0 W), the concave structure almost disappeared, and the convex micro-bulge structure dominated. Similar trends could be observed for the laser textured BMG surfaces with both unidirectional and cross-hatch surface patterns. As clearly pointed out by Feng et al. [45,46], the laser-induced surface structure is a key factor that will affect the surface functionality, i.e., wettability, reflectivity, and hardness, and the laser processing parameters must be carefully chosen to ensure the formation of proper laser-induced structure, thus enabling the realization of desirable surface functionalities.

**Figure 3.** SEM micrographs of the laser textured BMG surfaces using different scanning speeds: (**a**–**d**) unidirectional surface patterns with the scanning speeds of 20 mm/s, 40 mm/s, 60 mm/s, and 80 mm/s; (**e**–**h**) cross-hatch surface patterns with the scanning speeds of 20 mm/s, 40 mm/s, 60 mm/s, and 80 mm/s.

#### *3.2. Surface Wettability*

Surface wettability of the BMG surfaces with different surface patterns and treatment methods were evaluated via contact angle measurements. It can be found that the untreated BMG surface exhibited a θw of 87.4 ± 1.8◦ , as shown in Figure 5a, indicating that the untreated BMG surface is hydrophilic. Immediately upon laser surface texturing using the processing parameters of a laser power of 12 W, a repetition rate of 30 kHz, and a scanning speed of 40 mm/s, the laser textured BMG surfaces with both unidirectional (Figure 5b) and cross-hatch (Figure 5c) surface patterns exhibited a θw of 0◦ . This clearly indicates that the laser textured BMG surfaces are in a saturated Wenzel regime when being treated in the oxygen-containing atmosphere, which agrees well with the experimental results in [47]. After heat treatment for 1 h, the θw measurements indicated that the laser textured BMG surfaces with unidirectional and cross-hatch patterns exhibited θw values of 145.5 ± 1.9◦ and 145.7 ± 2.5◦ , respectively, indicating that a 1 h heat treatment rendered the laser textured BMG with high hydrophobicity. With a 2 h heat treatment, the laser textured BMG surfaces with both unidirectional and cross-hatch patterns became superhydrophobic, with θw values of 154.3 ± 1.9◦ and 153.7 ± 1.1◦ , respectively.

**Figure 4.** SEM micrographs of the laser textured BMG surfaces using different powers: (**a**–**e**) unidirectional surface patterns with the laser powers of 15.0 W, 13.5 W, 12.0 W, 10.5 W, and 9.0 W; (**f**–**j**) cross-hatch surface patterns with the laser powers of 15.0 W, 13.5 W, 12.0 W, 10.5 W, and 9.0 W.

θ θ θ θ Figure 6 shows θw measurement results for the laser textured BMG with varied laser powers and scanning speeds. As shown in Figure 6a, the untreated BMG surface showed a hydrophilic nature with a θw of 84.4 ± 1.2◦ . For the laser texturing experiments, the following laser processing parameters were utilized: a laser power of 10.5 W, a repetition rate of 30 kHz, and a scanning speed of 60 mm/s. The θw measurement results (Figure 6b,c) confirmed the saturated Wenzel regime for the laser textured surfaces with both of the unidirectional and cross-hatch patterns. Subsequently, it can be found that a 1 h heat treatment turned the laser textured surfaces with both patterns highly hydrophobic, with θw values of 144.7 ± 1.1◦ and 146.5 ± 1.7◦ , respectively (Figure 6d,e). A 2 h heat treatment ensured that both of the laser textured surfaces reached superhydrophobicity, with θw values of 155.9 ± 1.2◦ and 154.5 ± 1.9◦ , respectively (Figure 6f,g). As discussed in the previous section, low-temperature heat treatment with an annealing temperature of ~150 ◦C has been proved to be an efficient approach to achieve wettability transition from superhydrophilicity to superhydrophobicity for various metallic materials

such as aluminum, copper, stainless steel, and titanium [28–36]. However, this wetting state transition approach has never been utilized and confirmed for BMG. To the authors' best knowledge, this work represents the first attempt to achieve wettability transition from superhydrophilicity to superhydrophobicity on BMG surfaces using the facile laser surface texturing technique combined with low-temperature annealing. The underlying processing mechanisms associated with the wettability will be explained in detail in the following sections. θ

**Figure 5.** Contact angle measurement results for (**a**) untreated BMG surface; (**b**) laser textured BMG surface using the power of 12 W, the repetition rate of 30 kHz, the scanning speed of 40 mm/s, and a unidirectional surface pattern; (**c**) laser textured BMG surface using the power of 12 W, the repetition rate of 30 kHz, the scanning speed of 40 mm/s, and a cross-hatch surface pattern; (**d**,**e**) laser textured BMG surfaces in (**b**,**c**) after heat treatment for 1 h; (**f**,**g**) laser textured BMG surfaces in (**b**,**c**) after heat treatment for 2 h.

**Figure 6.** Contact angle measurement results for (**a**) untreated BMG surface; (**b**) laser textured BMG surface using the power of 10.5 W, the repetition rate of 30 kHz, the scanning speed of 60 mm/s, and a unidirectional surface pattern; (**c**) laser textured BMG surface using the power of 12 W, the repetition rate of 30 kHz, the scanning speed of 40 mm/s, and a cross-hatch surface pattern; (**d**,**e**) laser textured BMG surfaces in (**b**,**c**) after heat treatment for 1 h; (**f**,**g**) laser textured BMG surfaces in (**b**,**c**) after heat treatment for 2 h.

#### *3.3. Surface Chemistry*

Farasi et al. [48] pointed out that the surface chemical composition of metallic materials can be modified via material vaporization and oxidation during laser surface texturing, and Ngo et al. [31,49] believed that the increase in carbon content during heat treatment indicates the deposition of more hydrophobic functional groups on the laser textured surface, thus leading to the transition of surface wettability. Therefore, to explore and understand the variations of chemical compositions on the BMG surface and to further correlate surface wettability with surface chemistry, the laser textured BMG surfaces before and after heat treatment were analyzed by EDAX, as shown in Figure 7. The laser textured surfaces (with and without heat treatment) considered for the surface chemistry analysis were treated with the average laser power of 12.0 W, repetition rate of 30 kHz, pulse energy of 0.4 mJ, and power intensity of 2.08 GW/cm2 . From Figure 7, it can be clearly seen that all the core elements can be detected on the three different types of surfaces. While compared with the untreated surface, the intensity of the Zr peak for the laser textured surface decreased, which is mainly a result of material vaporization during laser texturing. In the meantime, the O peak shows a notable increase in the laser textured surface compared with that of the untreated surface, indicating the BMG surface has been oxidized along with the formation of a periodic surface structure. As indicated in [50], as the electronic structure of metal oxide facilitates the formation of hydrogen bonds and increases the surface energy, the laser textured surface typically exhibited superhydrophobicity. For the laser textured surface with heat treatment, subtle increases in C and Si peaks can be detected, indicating that hydrophobic functional groups, including –CH2–, –CH3, C=C as well as thin PDMS layer, should have been absorbed and deposited onto the laser textured BMG surface, rendering the heat-treated surface with superhydrophobicity [33,49].

The EDAX mapping data were also obtained to reveal the elemental distribution of all the core elements (Zr, Ti, Be, Cu, Ni, Si, C, and O) on each surface, as shown in Figure 8. From EDAX mapping, it can be clearly observed that all the core elements were uniformly distributed on the untreated surface. For the laser textured surfaces, more black areas can be observed, suggesting a reduction of the Zr, Ti, Be, Cu, and Ni elements, corresponding to the material removal during laser surface texturing. Distinct increases for the amount of O can be seen on the EDAX mapping as well, which strongly supports the oxidation during laser surface texturing. For the elemental distribution of C and Si, no clear change can be observed, which will be further examined using XPS.

Given the fact that EDAX is mainly a qualitative analysis method that could not provide conclusive results for the evaluation of the BMG substrate after laser texturing and heat treatment, XPS analysis was further employed to investigate the chemical changes on the surfaces that have been tested by EDAX. The XPS full spectra of the BMG surfaces considered for EDAX analysis and the corresponding atomic percentage of different elements are shown in Figure 9. It can be seen from the XPS full spectra of the untreated BMG surface (Figure 9a) that carbon and oxygen were detected as the major elements, which can be attributed to the contamination and oxidation of the BMG surface. For the constituent elements of the BMG Vitreloy 1 used in this work, small peaks of Zr 3d and Be 1s were observed on the untreated BMG surface, while the content of other elements (Ti 2p, Ni 2p, and Cu 2p) was negligible. Upon laser texturing, the atomic percentage of oxygen increased from 26.88% to 47.85% (Figure 9b). This clearly indicates that the laser textured BMG surface has been oxidized along with the formation of periodic surface structure during laser texturing. As the hydrogen bonds tend to form on the metal oxide, which also increases the surface energy [50], the laser oxidized BMG surface exhibited superhydrophilicity. After heat treatment, a clear atomic percentage increase in carbon from 30.85% to 37.80% can be observed on the laser textured BMG surface with heat treatment (Figure 9c). The increase in carbon content demonstrated that several different types of functional groups that exhibit hydrophobicity could have been absorbed onto the laser textured BMG surface during heat treatment, which rendered the heat-treated BMG surface highly hydrophobic [33,49]. In addition, the element Si was also detected on the heat-treated BMG surface, as shown in

Figure 9c. The appearance of Si might represent the formation of a thin Si-based PDMS layer on the laser textured BMG surface during heat-treatment, which further increased the hydrophobicity of the laser textured BMG surface. The experimental findings in this work agree well with the results of Ti alloy using a similar method [36], and it is believed that the increase in carbon and the appearance of a thin PDMS layer contribute to the superhydrophobicity of the laser textured BMG surface after heat treatment.

**Figure 7.** EDAX spectra for (**a**) untreated BMG surface; (**b**) laser textured BMG surface; (**c**) laser textured BMG surface followed by heat treatment.

**Figure 8.** SEM/EDAX element mapping for (**a**–**i**) untreated BMG surface; (**j**–**r**) laser textured BMG surface; (**s**–**aa**) laser textured BMG surface followed by heat treatment. (**a**,**j**,**s**) are SEM micrographs representing the corresponding analyzed regions; (**b**–**i**,**k**–**r**,**t**–**aa**) are the EDAX maps showing the qualitative elemental distributions of Zr, Ti, Be, Cu, Ni, Si, C, and O.

**Figure 9.** XPS full spectra of (**a**) untreated BMG surface; (**b**) laser textured BMG surface; (**c**) laser textured BMG surface followed by heat treatment.

− − − Core elemental analyses were also performed for C, O, and Si elements, as shown in Figure 10. The core elemental analysis for C element can be found in Figure 10a–c. In Figure 10a, it is clearly shown that C element can be detected, confirming the contamination of the untreated BMG surface. For the laser textured BMG surfaces, it can be observed in Figure 10b and c that after heat treatment, the proportion of C–C and C–H peaks significantly increased, which helped to verify that hydrophobic functional groups with nonpolar C–C or C–H bonds should have been deposited onto the heat-treated BMG surface, inducing hydrophobicity on the surface. The core elemental analysis for O is shown in Figure 10d–f. It is clear that functional groups of (OH)− and O2− were detected on all of the BMG surface, while the proportion of O2− on the laser textured BMG surfaces both with and without heat treatment was higher than that of the untreated BMG surface, proving that oxidation occurred during the laser texturing process. Finally, Figure 10g shows the core elemental analysis for Si element, revealing the appearance of the peaks for three functional groups Si–O, Si–C, and –Si– on the laser textured BMG surface after heat treatment. This should be attributed to the thin PDMS layer deposited on the laser textured BMG surface during heat treatment, which is derived from the silicone component on the furnace used in this work, as indicated in [32]. The underlying mechanism will be explained with details in the following section.

**Figure 10.** The high-resolution core elemental spectra of carbon on (**a**) untreated BMG surface; (**b**) laser textured BMG surface; (**c**) laser textured BMG surface followed by heat treatment. The high-resolution core elemental spectra of oxygen on (**d**) untreated BMG surface; (**e**) laser textured BMG surface; (**f**) laser textured BMG surface followed by heat treatment; and (**g**) the high-resolution core elemental spectra of silicon on the laser textured BMG surface followed by heat treatment.

#### *3.4. Surface Microhardness*

The microhardness measurement results of the untreated BMG surface, the laser textured BMG surface with unidirectional pattern, and the laser textured BMG surface with cross-hatch pattern can be found in Figure 11. The laser textured surfaces considered for the surface microhardness analysis were treated using the same processing parameters as those used for surface chemistry analysis. The experimental results indicated that the microhardness of the untreated BMG surface was 514.4 ± 4.9 HV. By laser surface texturing using the unidirectional surface pattern, the microhardness of the textured surface was increased to 564.9 ± 5.2 HV, representing an increase rate of 9.8% compared with the untreated surface. Meanwhile, the microhardness of the laser textured BMG surface with the cross-hatch pattern was further enhanced up to 596.7 ± 5.8 HV, indicating an increase rate of 16.0% compared with the untreated surface. As evident from the SEM micrographs shown in Figures 3 and 4, the laser surface texturing process created a combination of micro-scale, sub-micron, and nano-scale structures on the BMG substrate, which have generated much finer grains on the surface. As indicated by the Hall–Petch relationship, finer grains will result in higher mechanical strength [51,52]. More grain boundaries can be generated using finer grains, and the resistance to hinder dislocation motion will increase, leading to the increase in surface microhardness. In addition, compared with the laser textured surface with the unidirectional pattern, one more laser scan from the vertical direction would significantly increase the density of surface structures at all scales on the BMG substrate, which would contribute to the further enhancement of microhardness [53]. The microhardness measurement results indicate that the developed laser-based

surface texturing process can effectively enhance the surface mechanical strength of the BMG substrate.

**Figure 11.** Microhardness measurements for untreated surface, laser textured BMG surface with unidirectional pattern, and laser textured BMG surface with cross-hatch pattern.

#### *3.5. Processing Mechanism Analysis*

The underlying mechanism for the wettability transition of BMG surface during laser texturing and heat treatment is schematically depicted in Figure 12. As shown in Figure 12a, nanosecond laser surface texturing not only generated periodic surface structures on the BMG substrate but also oxidized the surface in the ambient air with the existence of water vapor [35,47]. Consequently, formation of oxide and hydroxide layers would occur simultaneously on top of the BMG substrate, which will drastically increase the surface energy and lead to superhydrophilicity [28,54]. During heat treatment, the wettability transition from superhydrophilicity to superhydrophobicity can be ascribed to two aspects: on the one hand, it is proposed that the organic compounds existed in air with nonpolar C–C and C–H bonds that have been absorbed onto the laser-induced micro-bulge or groove structures, and this process has been accelerated as the heat treatment temperature increased. The heat treatment helped to eliminate the hydrophilic –OH functional group from the laser textured BMG surface and created more active sites on the surface for the subsequent absorption of organic compounds [55]. As a result, more hydrophobic functional groups, such as –CH2– and –CH3, have been attracted onto the laser textured BMG surface, leading to the increase in surface hydrophobicity. On the other hand, the XPS analysis confirmed the existence of Si element on the laser textured BMG surface, indicating that a thin PDMS layer could have been deposited on the surface. The source of the Si element was originated from the silicone seal on the furnace, which was partially vaporized and deposited onto the laser textured BMG surface, as shown in Figure 12c. Since the silicon-based organic polymer PDMS is known to be hydrophobic, it could help to further enhance the hydrophobicity of the laser textured BMG surface, leading to superhydrophobicity eventually. It is thus believed that absorption of hydrophobic airborne organic compounds and generation of the thin PDMS layer both contributed to the surface wettability transition on the laser textured BMG surface during heat treatment. Therefore, from an initial contact angle of ~85◦ on the untreated BMG surface, the surface wettability was converted to superhydrophilicity (~0◦ ) after laser texturing, and superhydrophobicity (>150◦ ) after heat treatment, as shown in Figure 12b.

θ

**Figure 12.** Schematic for the mechanism of wettability transition for the laser textured BMG surface after laser texturing and heat treatment: (**a**) alteration of surface chemistry; (**b**) alteration of surface wettability; (**c**) demonstration of the silicone seal on the furnace used in this work; (**d**) SEM micrograph of the untreated BMG surface; (**e**) contact angle measurement results for the untreated BMG surface before and after heat treatment.

θ In the meantime, although surface chemistry plays a key role for the wettability transition, it is proposed that the laser-induced surface structure is equally critical for achieving the target wettability condition. This could be verified by what has been observed on the untreated BMG surface before and after heat treatment. Figure 12d shows the SEM micrograph of the untreated BMG surface, indicating that there are only grinding marks without any distinct periodic surface structures. The contact angle measurement results indicated that the initial θw was 84.4 ± 1.2◦ on the untreated BMG before heat treatment, and the θw was measured to be 88.9 ± 0.8◦ after heat treatment. This clearly indicates that alteration of surface chemistry alone could not render the BMG material with superhydrophobicity. Surface structure and surface chemistry should be modulated and controlled spontaneously to achieve the desired surface functionality.

#### **4. Conclusions**

θ

θ In this work, a facile and efficient laser-based surface texturing method was developed to modulate and control the surface functionalities of Zr-based BMG. The following findings can be summarized:

θ

This method will provide a feasible and highly efficient solution for regulating and controlling the surface functionalities of BMG, which will render more practical and important applications.

**Author Contributions:** Conceptualization, Q.W. and H.W.; Data curation, Q.W., Y.C., Z.Z. and H.W.; Formal analysis, Q.W., Y.C. and N.X.; Funding acquisition, Q.W. and H.W.; Investigation, Q.W. and Z.Z.; Methodology, Q.W.; Project administration, Q.W.; Resources, Q.W. and N.X.; Validation, Q.W.; Writing—original draft, Q.W.; Writing—review and editing, H.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Guangdong Basic and Applied Basic Research Foundation (No. 2019A1515110501), the National Natural Science Foundation of China (No. 52105175), the Natural Science Foundation of Jiangsu Province (No. BK20210235) and the New Faculty Start-up Fund of Southeast University (No.<PHONE_NUMBER>).

**Acknowledgments:** The authors would like to acknowledge the Analysis and Testing Center of Southeast University for the help with SEM, EDAX, and XPS analyses.

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

#### **References**


**Featured Application: In this paper, the dual-view integral imaging 3D display can be used in head-up display, augmented reality and so on.**

**Abstract:** We propose a dual-view integral imaging 3D display based on a multiplexed lens-array holographic optical element (MHOE). A MHOE is a volume holographic optical element obtained by multiplexing technology, which can be used for dual-view integral imaging 3D display due to the angle selectivity of the volume HOE. In the fabrication of the MHOE, two spherical wavefront arrays with different incident angles are recorded using photopolymer material. In the reconstruction, two projectors are used to project the elemental image arrays (EIA) with corresponding angles for two viewing zones. We have developed a prototype of the dual-view integral imaging display. The experimental results demonstrate the correctness of the theory.

**Keywords:** dual-view display; 3D display; integral imaging; lens-array holographic optical element

### **1. Introduction**

Dual-view display provides different images for viewers in different directions. It can meet the needs of different viewers at the same time, and it saves a lot of space and cost. It is increasingly commercialized, especially for car navigation, home entertainment and business applications. For example, when a dual-view display is used in an automobile, it can represent navigation information for the driver and entertainment information for the passenger simultaneously. Some dual-view liquid crystal displays have been proposed [1,2], but they can only display 2D images. For automobile applications, optical see-through capacity is needed because the driver needs to see the road while watching the navigation information.

Recently, 3D displays, including holography display [3–5], integral imaging display [6–9], volumetric display [10,11] and so on, have received extensive attention. Integral imaging is considered to be one of the most promising display technologies. It has the advantages of full parallax [12] and continuous viewpoint [13], and it supplies various physiological depth cues, so viewers can freely change the accommodation and convergence without feeling any visual discomfort [14,15]. Integral imaging has a small viewing angle, and the viewing zone is periodically repeated. Therefore, it is suitable for dual-view or multi-view displays. By using some kinds of multiplexing methods, it can present different 3D images in different directions, and meet the needs of different viewers. In recent years, our team proposed several ways to implement dual-view 3D displays [16–18]. We proposed a dual-view integral imaging 3D display [16], but the viewing angle is only half of the conventional one. In order to increase the viewing angle of the dual-view 3D display, we proposed a dual-view integral imaging 3D display using polarizer parallax barriers [17], but the resolution of each 3D image is reduced to half of the conventional one. In order to solve these issues, Seoul University proposed a projection-type dual-view 3D display based on integral imaging [19], but the display system is bulky. Then we proposed a dual-view integral imaging 3D display using an orthogonal polarizer array and polarization switcher [18], but the brightness is not high.

In this paper, we propose a dual-view integral imaging 3D display system based on a MHOE. The viewing angle and resolution of 3D images in the dual viewing zones are almost same as the conventional one. The dual-view 3D display system can provide different 3D images in different zones via controlling the projection beams. In Section 2, the fabrication and reconstruction principles of the MHOE are analyzed, and the projection part of the dual-view 3D display system is designed. In Section 3, the different 3D images are displayed in different viewing zones, and the imaging characteristics of the MHOE are analyzed. The principle of the proposed system is verified by the display experiments.

### **2. Structure and Principle**

The schematic diagram of the dual-view integral imaging 3D display system by using MHOE is shown in Figure 1, which consists of an MHOE, projector I and II. The MHOE is used as a projection screen, which records the wavefronts of a micro-lens array from two different directions by using two-step fabrication methods. Projection beams I are projected by projector I, which contain elemental image array (EIA) I, and projection beams I are projected on the MHOE with a pair of angles θ*2* and θ*3*, which satisfies the Bragg condition. Then, 3D images I are reconstructed in the left viewing zone. Projection beams II with EIA II are projected by projector II, and projection beams II are incident onto the MHOE with a pair of angles θ*5* and θ*6*. Then the Bragg condition is satisfied and 3D images II are reconstructed in the right viewing zone. Finally, two different 3D images are reconstructed in the dual viewing zones, respectively.

**Figure 1.** Schematic diagram of the dual-view 3D display system by using the MHOE.

#### *2.1. Fabrication and Reconstruction Principles of the MHOE*

Compared with the conventional holographic optical element (HOE), the MHOE is recorded on the volume hologram, which has the characteristics of angular and spatial multiplexing. HOE has angular selectivity; when the incident beam deviates from the Bragg condition, the diffraction efficiency of the reconstructed beam is reduced. When the incident beam greatly deviates from Bragg condition, the diffracted beam cannot be reconstructed. Therefore, multiple optical elements can be recorded on a single holographic plate by angular and spatial multiplexing.

The angular and spatial multiplexing method are used in the fabrication process. Two sets of micro-lens array wavefronts are recorded in a single green sensitive photopolymer material with two different angles. The fabrication process is divided into two steps, which are shown in Figure 2. In the first step, the parallel beam I is incident into the micro-lens array at an oblique angle θ*1*, and then the spherical wavefront array I is generated. The reference beam I is generated by point source I. Reference beam I interferes with spherical wavefront array I with a pair of angles θ*2* and θ*3*, as shown in Figure 2a. In the second step, the parallel beam II is incident into the micro-lens array at an oblique angle θ*4*, and then the spherical wavefront array II is generated. The reference beam II is generated by point source II, and reference beam II interferes with spherical wave array II with a pair of angles θ*5* and θ*6*, as show in Figure 2b. The parallel beams I and II are symmetric to each other. By strictly controlling the exposure time of the first and second steps in the fabrication process. Finally, the required MHOE is obtained.

**Figure 2.** Fabrication principle of the MHOE, (**a**) the first step and (**b**) the second step.

The reconstruction principle of the MHOE is shown in Figure 3. Reference beam I is projected onto the MHOE with a pair of angles θ*2* and θ*3*. The incident angles of the reference beam I are consistent with the angles of fabrication, and the Bragg condition is satisfied. So the spherical wavefront array I can be reconstructed. Reference beam II is also projected onto the MHOE with a pair of angles θ*5* and θ*6*. The incident angles of the reference beam II are the same as the angles of fabrication, and the Bragg condition is also satisfied. Then, the spherical wavefront II is reconstructed. Therefore, the MHOE has the optical characteristics of two sets of micro-lens arrays in different directions. Reference beams I and II are generated by the point source I and II, which have various directional light components. In the reconstruction of 3D images, a Bragg-mismatch occurs when reference beams are produced by projector I and II. So, the angular variation caused by angular deviation of the reconstructed beam should be analyzed.

#### *2.2. MHOE as Imaging Optical Element in the Dual-View 3D Display*

In the fabrication process, reference beams interfere with the spherical wavefront arrays. The spherical wavefront array consists of many small spherical waves, and the interference fringes are recorded on the holographic material. Each elemental lens in the MHOE is generated by the interference of a large spherical wave (reference beam) and a small spherical wave. The grating vectors of different positions on the MHOE are different. The reference beam is considered as a series of plane beams [20], whose wave vectors (**Kr1~rn**) are shown in Figure 4a. The spherical wavefront array can be regarded as a series of small spherical waves, which is shown in Figure 2. Each small spherical

wave in the spherical wavefront array can also be considered as a series of plane beams, whose wave vectors (**Ks1~sn**) are shown in Figure 4a. So the change of the reconstructed beam of the MHOE could be explained by the wave vector relationship of the plane beams.

**Figure 3.** Reconstruction principle of the MHOE.

**Figure 4.** (**a**) Grating vector diagram of the elemental lens in the MHOE, and (**b**) wavefront recording schematic diagram of the elemental lens in the MHOE.

The recorded schematic diagram of the small spherical wave in the MHOE is shown in Figure 4b; the recorded grating vectors **Kg** can be expressed as [20]:

$$\mathbf{K}\_{\mathsf{F}} = \overrightarrow{\mathbf{x}} \left[ |\mathbf{K}\_{\mathsf{S}}| \sin[\theta\_{\mathsf{s}}] - |\mathbf{K}\_{\mathsf{I}}| \sin[\theta\_{\mathsf{I}}] \right] + \overrightarrow{\mathbf{y}} \left[ |\mathbf{K}\_{\mathsf{S}}| \cos[\theta\_{\mathsf{s}}] + |\mathbf{K}\_{\mathsf{I}}| \cos[\theta\_{\mathsf{I}}] \right] \tag{1}$$

where **Ks** and **Kr** are the vectors of the small spherical wave in the spherical wavefront array and reference beam, respectively. In the reconstruction process, the projection beam project onto the MHOE with the vector **Kp**. The angle of the reconstructed beam can be expressed as [20]:

$$\theta\_{\mathcal{E}} = \sin^{-1}\left[\sin\left[\theta\_{\mathcal{P}}\right] + \sin\left[\theta\_{\mathcal{S}}\right] - \sin\left[\theta\_{\mathcal{I}}\right]\right] \tag{2}$$

When the projection angle θ*p* of the projection beam, the incident angle θ*r* of the reference beam and the incident angle θ*s* of the small spherical wave incident into the MHOE are determined, the angle θ*c* of the reconstructed beam changes almost linearly, within the range of the projection angle of the projection beam. Therefore, the MHOE is suitable to be used as the imaging optical element in the dual-view 3D display.

#### *2.3. Design of Projection Part for the Dual-View 3D Display*

The projection part for the MHOE has to satisfy the Bragg conditions. Projection beam I must satisfy the projection angles θ*2* and θ*3* for reconstructed 3D images I in the left viewing zone, and projection beam II also must satisfy the projection angles θ*5* and θ*6* for reconstructed 3D image II in the right viewing zone. In this section, the projection part is realized based on two projectors, so that 3D image I and II are displayed simultaneously, as depicted in Figure 1.

The principle and design of the projection part for the dual-view 3D display are shown in Figure 5. When reference beam I is projected onto the MHOE with a pair of angles θ*2* and θ*3*, spherical wavefront array I is reconstructed with the angle θ*1*. When reference beam II is projected onto the MHOE with a pair of angles θ*5* and θ*6*, spherical wavefront array II is reconstructed with the angle θ*4*. The best view distance of the dual-view 3D display system is *L*, the focal length of the MHOE is *f* and the pitch is *p*. According to the geometric optics, the viewing angle of the left viewing zone can be expressed as:

$$\Omega\_1 = \arctan \frac{2f \tan \theta\_1}{2f - p \tan \theta\_1} - \arctan \frac{2f \tan \theta\_1}{2f + p \tan \theta\_1} \tag{3}$$

and the viewing angle of the right viewing zone can be expressed as:

$$\Omega\_2 = \arctan \frac{2f \tan \theta\_4}{2f - p \tan \theta\_4} - \arctan \frac{2f \tan \theta\_4}{2f + p \tan \theta\_4} \tag{4}$$

The projection part is designed to make sure that the left and right view zone are completely separated, and reconstructed 3D images I and II cannot cause crosstalk, which is shown in Figure 5.

**Figure 5.** Principle and design of the projection part for the dual-view 3D display.

#### **3. Experiments and Results**

#### *3.1. Fabrication of the MHOE*

In the first step, the experimental setup is shown in Figure 6a. The laser's wavelength and power are 532 nm, and 400 mW, respectively. The thickness, resolution, sensitivity and average refractive index of the photopolymer material are 15 um, 12,000 line/mm, 10 mJ/cm2 and 1.47, respectively. The size of the micro-lens array used to fabricate the MHOE is 150 mm × 150 mm, in which the pith of each lens element is 1 mm and the focal length is about 3.3 mm. The laser beam is divided into two beams by a beam splitter (BS). One of the laser beams is expanded by a spatial filter (SF) and a collimating lens (CL), then the laser beam incident into the micro-lens array with the angle θ*1* = 50◦ forms spherical wavefront array I. Another laser beam is reflected by mirror 1 (M1) and mirror 2 (M2), and then passes through the divergent lens (DL) to form spherical wave I. The angles of spherical wave I incident into the holographic plate is controlled to be θ*2* = 61◦, and θ*3* = 81◦. The interference occurs between the spherical waves I and spherical wavefront array I, and the interference fringes are recorded on the holographic plate.

**Figure 6.** Setup for fabrication of the MLAHOE, (**a**) the first step, and (**b**) the second step.

In the second step, the experimental setup is shown in Figure 6b. The laser beam is divided into two beams by a BS. One of the laser beams is expanded by a SF and a CL, then the laser beam incident into the micro-lens array with the angle θ*4* = 50◦ forms spherical wave array II. Another laser beam passes through the DL to form spherical wave II. The angle of spherical wave II incident into the holographic material is controlled to be θ*5* = 61◦, and θ*6* = 81◦. The interference occurs between the spherical waves I and spherical wavefront array I, and the interference fringes are recorded on the holographic plate. The holographic plate is post-processed to obtain the required MHOE. The size of the MHOE is 80 mm × 80 mm, and the diffraction efficiency and transmittance of the MHOE are greater than 85% and 80%, respectively. The exposure time is controlled by the electronic shutters. The detailed parameters of the optical devices are shown in Table 1.

**Table 1.** Parameters of the optical devices in our experiments.

In this paper, the MHOE is used as a projection screen, when the incident projection beam deviates greatly from the Bragg condition, the diffracted beam cannot be reconstructed and the 3D images also cannot be reconstructed. Therefore, the angular selectivity of the MHOE needs to be measured. The maximum deviated angle of the projection beam must be measured but the diffracted beam and 3D images cannot be reconstructed. The diffraction efficiency of the MHOE can be measured by the intensity of the transmitted and diffracted beam. The measurement formula of the diffraction efficiency can be expressed as [21]:

$$\eta = \frac{I\_D}{I\_T + I\_D} \tag{5}$$

where *ID* and *IT* represent the intensity of the diffracted and transmitted beam, respectively. During the measurement, the incident angle of the reference beam is controlled by a rotation table, and the intensity of the diffracted beam of the MHOE is obtained by an optical power meter. The diffraction efficiency of the left and right view zones of the MHOE is measured, as shown in Figure 7. Through observation, when the incident angle of the reference beam is at 13◦, the diffraction efficiency of the MHOE is less than 5%. When the incident angle of the reference beam is between −4.5◦ and 4.5◦, the diffraction efficiency of the MHOE is higher than 50%. As shown in Figure 7, the maximum deviated angle of the MHOE is between −5◦ and 5◦ in the left and right viewing zones.

The angle between the incident angle θ*3* of projector I and the incident angle θ*5* of projector II is much larger than the maximum deviated angle of the MHOE, that is 38◦ > 10◦. The angle between the incident angle θ*2* of projector I and the incident angle θ*6* of projector II is much larger than the maximum deviated angle of the MHOE, that is 38◦ > 10◦. Therefore, there is no crosstalk between the left and right viewing zones.

The projector (BenQ MX518F) is used to ensure the diffraction efficiencies of the MHOE. The horizontal scanning frequency is 102 KHz, and the vertical scanning frequency is 120 Hz. The resolution of the projector is 1600 × 1200. The detailed parameters of the projector are shown in Table 1. Projection beam I is projected onto the MHOE with a pair of angles θ*2* = 61◦ and θ*3* = 81◦, which are the same as in the first step. Then, spherical wavefront array I is reconstructed in the left direction, as shown in Figure 8a. Projection beam II is projected onto the MHOE with a pair of angles θ*5* = 81◦ and θ*6* = 61◦, which are the same as in the second step. Then, spherical wavefront array II is reconstructed in the right direction, as shown in Figure 8b.

**Figure 7.** Diffraction efficiency of the left and right viewing zone.

**Figure 8.** Reconstruction of the wavefronts of the lens array (**a**) in the left viewing zone, and (**b**) in the right viewing zone.

#### *3.2. Experiments for Displaying Images on the Proposed Dual-View 3D Display*

The projector is used in the experiment for displaying images. According to the incident angle requirement of the MHOE, projector I is placed on the left, and projection beam I is incident into the MHOE with a pair of angles θ*2* = 61◦ and θ*3* = 81◦. Projector II is placed on the right, and projection beam II is incident onto the MHOE with a pair of angles θ*5* = 81◦ and θ*6* = 61◦. Therefore, projection beams I and II are symmetrical to each other.

Figure 9 shows the experiment setup for the dual-view 3D display. In order to satisfy the incident angle about the MHOE, the two projectors are placed vertically, and the projection lens is positioned inside. The distance between the projection lenses of the projectors and the MHOE is 31 cm. Projectors I and II are 20 cm apart from each other in the horizontal direction. The projection lenses of projectors I and II are both 21 cm away from the optical table. The height of the MHOE is 19.5 cm from the optical table. The distance between the MHOE and the car model is 3 cm. The projectors are used to project the EIAs onto the MHOE. The projection beams of the projectors that meet the Bragg-match are directly projected onto the MHOE. The MHOE is used as a screen of the dual-view 3D display. Projector I projects EIA I as projection beam I, with a pair of angles θ*2* = 61◦ and θ*3* = 81◦ onto the MHOE. Then, 3D image I is reconstructed in the left viewing zone. Projector II projects EIA II as projection beam II, with a pair of angles θ*5* = 61◦ and θ*6* = 81◦ onto the MHOE. Then, 3D image II is reconstructed in the right viewing zone. In this paper, the pith of the elemental lens in the micro-lens array is 1 mm and the focal length is 3.3 mm. According to Equations (3) and (4), the viewing angles of the left and right viewing zone are Ω1 = 10.2◦ and Ω2 = 10.2◦, respectively. According to the parameters of the micro-lens array, the viewing angle of the conventional integral imaging is about 12◦. Therefore, the viewing angles in the dual viewing zones are almost the same as the conventional one.

**Figure 9.** Experiment setup for the dual-view 3D display.

EIAs I and II are shown in Figure 10a,b, respectively. Each EIA consists of 70 × 70 image elements. EIA I is generated by a computer and consists of the characters "3" and "D", in which "3" is located at −15 mm, and "D" is located at +15 mm. EIA II is also generated by a computer and consists of the characters "A" and "B", in which "A" is located at −15 mm, and "B" is located at +15 mm.

**Figure 10.** (**a**) EIA I and (**b**) EIA II.

3D image I reconstructed in the left view zone taken from the left, right, top and bottom directions are shown in Figure 11. We can see the horizontal parallax of the "3D" images from the left and right directions, and the vertical parallax of the "3D" images from the top and bottom directions. Reconstructed 3D image I has obvious horizontal and vertical parallaxes. Visualizations 1 and 2 clearly show the horizontal and vertical parallaxes between the "3", "D" and the real object. The resolution of reconstructed 3D image I is almost the same as the conventional integral imaging 3D display, and 3D image I in the left view zone has high brightness and definition. The resolution of 3D image I is the same as the number of the image element 70 × 70 in EIA I.

Figure 12 shows 3D image II reconstructed in the right view zone taken from the left, right, top and bottom directions. We can see the horizontal parallax of the "AB" images from the left and right directions, and the vertical parallax of the "AB" images from the top and bottom directions. The reconstructed 3D image II has obvious horizontal and vertical parallaxes. Visualizations 2 and 3 clearly show the horizontal and vertical parallaxes between the "A", "B" and the real object. The resolution of reconstructed 3D image II is also almost the same as the conventional integral imaging 3D display. The resolution of 3D image II is also the same as the number of the image element 70 × 70 in EIA II. The 3D images I and II have the same resolution, according to the parameters of the micro-lens array, the resolution of the conventional integral imaging is 70 × 70. Therefore, the resolution in the dual viewing zones are the same as the conventional one.

It was observed that there is slight image crosstalk in the 3D image reconstructed in the left and right viewing zone. When projection beam I is projected onto the MHOE, the light produces diffuse reflection on the glass substrate of the MHOE, and the diffuse light can produce slight crosstalk with reconstructed 3D image II in the right viewing zone. Reconstructed 3D image I in the left viewing zone has slight crosstalk, which is caused by the same reason as in the right viewing zone.

**Figure 11.** Experimental results in the left viewing zone (see Supplementary Materials visualization 1 and visualization 2), the parallax images in the left, right, top, and bottom four directions.

**Figure 12.** Experimental results in the right viewing zone (see Supplementary Materials visualization 2 and visualization 3), the parallax images in the left, right, top, and bottom four directions.

#### **4. Conclusions**

In this paper, we propose a dual-view 3D display. Two projectors are used to present different 3D images on the MHOE. Viewers can see the two different 3D images in the left and right viewing zones. The viewing angle and resolution of the reconstructed 3D images in the two viewing zones are almost

the same as the conventional integral imaging 3D display. In addition, the fusion of 3D images with real objects is realized. It can be used for the car head up display and medical devices.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3417/9/18/3852/s1.

**Author Contributions:** Conceptualization, Q.W. and D.L.; methodology, H.Z.; software, H.Z.; validation, Q.W., H.D.; formal analysis, H.Z.; investigation, M.H.; writing—original draft preparation, H.Z.; writing—review and editing, H.Z., Q.W., D.H., M.H., D.L.; visualization, H.Z.; funding acquisition, Q.W.

**Funding:** This work is supported by the National Key R&D Program of China (2017YFB1002900) and National Natural Science Foundation of China (61535007 and 61775151).

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

#### **References**

© 2019 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 (http://creativecommons.org/licenses/by/4.0/).


**Abstract**

Farm profitability is the key driver of most livestock enterprises. The productivity and profitability are driven by genetic potential of the animals and the ability to express the superiority in the production environment. In an ideal situation, an animal should produce maximally as dictated by the genetic potential. It is noteworthy that the environment in which an animal lives in impacts on its ability to expose its genetic potential. Studies have shown that it is rarely feasible to provide animals with ideal conditions to express their full genetic potential. The environment in which animals are reared is characterised by many factors that interact in ways that result in different performance even in animals of similar genetic makeup. For instance, thermal environment is critical in poultry production as it affects both the production and reproduction in different ways. The thermal environment affects chicken differently depending on the stage of growth or production phase. This environment has been impacted by the climate change and subsequent increase in climatic variability resulting in thermal challenges in naturally produced chicken thus altering production and reproduction. This implies that there is need to consider thermal resource in the routine poultry management practices. This would result to design of poultry production systems responsive to the thermal environments more so in the light of climate change and the subsequent increase in climatic variability. This chapter explores the impact of heat stress on chicken production, reproduction, health and its dietary amelioration.

**Keywords:** Heat stress, Chicken, Production, Reproduction, Health


**4. Genotype-targeted therapies**

By directly targeting at specific pathogenic mutations causing DCM, genotypetargeted genetic approaches have emerged as promising strategies for effective and individualized therapies to ameliorate disease phenotypes. To carry out genotypebased therapy, many customized genetic strategies were explored. For examples: for loss-of-function mutations that cause reduced or insufficient protein levels, the straightforward gene replacement strategy can be employed. In this scenario, full-length or partial functional cDNA for corresponding mutated gene can be transferred to cardiac tissue to supplement the reduced gene dosage using appropriate gene delivery approach. For mutations that cause dominant-negative effects on a particular gene, exon-skipping or trans-splicing approaches can be considered to remove or modify the mutant transcripts. For pathogenic mutations that cause other protein dysfunction, the highly efficient CRISPRA/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease) system can be explored to directly correct the mutant variants. In addition, other genotypebased therapeutic strategies such as manipulation of the downstream pathways evoked by specific DCM mutations were also explored. Below, we summarize several examples of gene-targeted therapeutic strategies that produced encouraging results in the treatment of DCM (**Table 2**), which hold the opportunities to ultimately improve patient outcome in the future.

#### **4.1 Dystrophin mutations-targeted gene therapies**

Mutations in the dystrophin (*DMD*) gene cause muscular dystrophy patients and X-linked familial DCM in early childhood and patients often die of cardiac and respiratory failure [44]. Currently, there are no effective cures for DMD yet and management

**111**

*Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic…*

of DMD mostly focuses on preserving the limited muscle strength and ameliorating disease symptoms [64]. Mechanistically, dystrophin protein deficiency due to *DMD* gene mutations is the primary cause of DMD and subsequent DCM and heart failure. And the disease severity is mostly correlated with the dystrophin protein level. Thus, a plausible treatment strategy would be to restore the expression level of dystrophin protein [44]. To cure this devastating disease, dystrophin gene-targeted therapeutic strategies such as gene replacement, exon skipping and CRISPRA/Cas9 genome editing techniques are mostly employed, which have shown encouraging results in restoration of dystrophin protein expression and recovery of dystrophin protein function in both

Gene replacement strategy involves delivering a functioning gene to replace or supplement the mutant gene to the target organ and cells to ease the disease phenotype caused by genetic mutation. This approach often utilizes the adeno-associated virus (AAV) as a vector to mediate gene transfection into the skeletal and cardiac muscle cells. However, as the dystrophin gene has 79 exons and its transcript is about 14 Kb, its size is too large to fit in currently available gene construction vector for gene transfection. Alternatively, a mini- or micro- dystrophin gene coding for a functionally similar to dystrophin but smaller in size was thus used. The authors Wang and colleagues firstly defined the minimal functional region of the dystrophin protein, later referred as mini-dystrophin. The authors then packed this min-dystrophin gene into an AAV vector to mediate muscle transfection and demonstrated the effectiveness of gene delivery and restoration of dystrophin gene function [65]. After this study, several other independent groups latter confirmed the effectiveness of a shortened albeit functional dystrophin gene replacement strategy mediated by the AAV gene delivery system in preservation of cardiac and skeletal muscle function and extending the lifespan in the dystrophic mice [66, 67]. Notably, further studies in animal models of muscular dystrophy and human patients detected certain immunologic responses to the shortened dystrophin peptide, which need to be carefully considered

Exon skipping is an RNA-based splice-switching approach that causes cells to "skip" the exon containing the pathogenic variant, resultant in-frame transcripts that produce a shorter peptide, albeit still at least partially functional protein to ameliorate disease phenotypes. This approach was initially developed to mask the pathogenic mutations containing exon 51 using an antisense oligonucleotide (AON), resulting in genetic correction of the open reading frame of the *DMD* gene and partial restoration of dystrophin protein expression and improvement of muscle pathology and function [71–73]. These encouraging studies lead to at least two ongoing clinical trials to evaluate the efficacy and safety of this exon skipping approach targeting to the pathogenic exon 51 or 53 (NCT02255552, NCT02310906). With the revolutionary development of the CRISPRA/Cas9 genome editing technologies that enable precise and efficient genetic modifications from single-nucleotide alternation, insertion of gene of interest to deletion of chromosomal regions, numerous studies have demonstrated the feasibility and high efficiency of this technique in restoration dystrophin expression and function in skeletal and cardiac muscle, both in vitro and in vivo [74, 75]. For example, a recent study carried out by El Refaey and colleagues packaged SaCas9 (Cas9 from *Staphylococcus aureus*)/gRNA constructs into an AAV serotype rh74 and delivered it to the mdx/Utr+/− dystrophic mice through a retro-orbital approach [76]. The authors showed this CRIPRA-mediated genome editing strategy could efficiently excise the mutant exon 23 of dystrophin in the mice model, resulting in the restoration of the dystrophin protein expression and cardiac myofiber architecture, and significantly improved the cardiac contractility in vivo. A follow up study using this AAV-mediated CRISPR genome editing approach found that restored dystrophin expression and improved cardiac function were consistently

*DOI: http://dx.doi.org/10.5772/intechopen.94434*

animal models and clinical trials.

in future clinical application [68–70].

#### *Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94434*

of DMD mostly focuses on preserving the limited muscle strength and ameliorating disease symptoms [64]. Mechanistically, dystrophin protein deficiency due to *DMD* gene mutations is the primary cause of DMD and subsequent DCM and heart failure. And the disease severity is mostly correlated with the dystrophin protein level. Thus, a plausible treatment strategy would be to restore the expression level of dystrophin protein [44]. To cure this devastating disease, dystrophin gene-targeted therapeutic strategies such as gene replacement, exon skipping and CRISPRA/Cas9 genome editing techniques are mostly employed, which have shown encouraging results in restoration of dystrophin protein expression and recovery of dystrophin protein function in both animal models and clinical trials.

Gene replacement strategy involves delivering a functioning gene to replace or supplement the mutant gene to the target organ and cells to ease the disease phenotype caused by genetic mutation. This approach often utilizes the adeno-associated virus (AAV) as a vector to mediate gene transfection into the skeletal and cardiac muscle cells. However, as the dystrophin gene has 79 exons and its transcript is about 14 Kb, its size is too large to fit in currently available gene construction vector for gene transfection. Alternatively, a mini- or micro- dystrophin gene coding for a functionally similar to dystrophin but smaller in size was thus used. The authors Wang and colleagues firstly defined the minimal functional region of the dystrophin protein, later referred as mini-dystrophin. The authors then packed this min-dystrophin gene into an AAV vector to mediate muscle transfection and demonstrated the effectiveness of gene delivery and restoration of dystrophin gene function [65]. After this study, several other independent groups latter confirmed the effectiveness of a shortened albeit functional dystrophin gene replacement strategy mediated by the AAV gene delivery system in preservation of cardiac and skeletal muscle function and extending the lifespan in the dystrophic mice [66, 67]. Notably, further studies in animal models of muscular dystrophy and human patients detected certain immunologic responses to the shortened dystrophin peptide, which need to be carefully considered in future clinical application [68–70].

Exon skipping is an RNA-based splice-switching approach that causes cells to "skip" the exon containing the pathogenic variant, resultant in-frame transcripts that produce a shorter peptide, albeit still at least partially functional protein to ameliorate disease phenotypes. This approach was initially developed to mask the pathogenic mutations containing exon 51 using an antisense oligonucleotide (AON), resulting in genetic correction of the open reading frame of the *DMD* gene and partial restoration of dystrophin protein expression and improvement of muscle pathology and function [71–73]. These encouraging studies lead to at least two ongoing clinical trials to evaluate the efficacy and safety of this exon skipping approach targeting to the pathogenic exon 51 or 53 (NCT02255552, NCT02310906).

With the revolutionary development of the CRISPRA/Cas9 genome editing technologies that enable precise and efficient genetic modifications from single-nucleotide alternation, insertion of gene of interest to deletion of chromosomal regions, numerous studies have demonstrated the feasibility and high efficiency of this technique in restoration dystrophin expression and function in skeletal and cardiac muscle, both in vitro and in vivo [74, 75]. For example, a recent study carried out by El Refaey and colleagues packaged SaCas9 (Cas9 from *Staphylococcus aureus*)/gRNA constructs into an AAV serotype rh74 and delivered it to the mdx/Utr+/− dystrophic mice through a retro-orbital approach [76]. The authors showed this CRIPRA-mediated genome editing strategy could efficiently excise the mutant exon 23 of dystrophin in the mice model, resulting in the restoration of the dystrophin protein expression and cardiac myofiber architecture, and significantly improved the cardiac contractility in vivo. A follow up study using this AAV-mediated CRISPR genome editing approach found that restored dystrophin expression and improved cardiac function were consistently

*Cardiac Diseases - Novel Aspects of Cardiac Risk, Cardiorenal Pathology and Cardiac Interventions*

AV blocker as significant risk factors for disease malignancy [61, 62]. Because of this well-determined genotype–phenotype knowledge, an actionable prognostic genotype–phenotype association, implementation of a lower threshold and earlier implantable cardioverter defibrillator (ICD) therapy than current guidelines recommend in patients with LMNA mutations, was demonstrated to significantly

Based on the Genetic Testing Registry (https://www.ncbi.nlm.nih.gov/gtr/), there are from 40 to 80 genes included in the testing panels for most commercially available genetic testing for DCM. In familial DCM, the yield of genetic testing, resulting in identification of pathogenic mutations, can thus far reach up to 40%, in a comparable level to that of other inherited cardiac disorders such as HCM and long QT syndrome. The sensitivity of genetic testing is compromised partially due to that not all genes implicated in DCM are included in the gene panels for testing. This is especially the case for those identified from sporadic DCM cases or single family. Furthermore, limited by human genetics approaches that heavily rely on pedigree availability and candidate gene approaches, variants for more than half of familial DCM cases have not been identified yet. As technique advances in genetics of cardiomyopathy, identification of the remaining genetic causes in inherited DCM cases and elucidation of the underlying pathogenic mechanisms leading to the phenotype are evolving rapidly. Targeted gene panels for genetic testing are increasing in an unprecedented scale. With further characterization and functional validation, the ever expanding gene panels of genetic testing promise to increase the rate of positive identification and provide individuals and families with a more

By directly targeting at specific pathogenic mutations causing DCM, genotypetargeted genetic approaches have emerged as promising strategies for effective and individualized therapies to ameliorate disease phenotypes. To carry out genotypebased therapy, many customized genetic strategies were explored. For examples: for loss-of-function mutations that cause reduced or insufficient protein levels, the straightforward gene replacement strategy can be employed. In this scenario, full-length or partial functional cDNA for corresponding mutated gene can be transferred to cardiac tissue to supplement the reduced gene dosage using appropriate gene delivery approach. For mutations that cause dominant-negative effects on a particular gene, exon-skipping or trans-splicing approaches can be considered to remove or modify the mutant transcripts. For pathogenic mutations that cause other protein dysfunction, the highly efficient CRISPRA/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease) system can be explored to directly correct the mutant variants. In addition, other genotypebased therapeutic strategies such as manipulation of the downstream pathways evoked by specific DCM mutations were also explored. Below, we summarize several examples of gene-targeted therapeutic strategies that produced encouraging results in the treatment of DCM (**Table 2**), which hold the opportunities to ulti-

Mutations in the dystrophin (*DMD*) gene cause muscular dystrophy patients and X-linked familial DCM in early childhood and patients often die of cardiac and respiratory failure [44]. Currently, there are no effective cures for DMD yet and management

improve patient outcome and survival [63].

comprehensive and conclusive genetic testing.

mately improve patient outcome in the future.

**4.1 Dystrophin mutations-targeted gene therapies**

**4. Genotype-targeted therapies**

**110**

detected at up to 19 months [77]. Similarly, Hakim and colleagues modified the dose of AAV.SaCas9 and AAV-9.gRNA vector that directs Cas9 to introns of 22 and 23 and performed the AAV-9 CRISPRA gene therapy in the mdx mice that carry a nonsense mutation in exon 23 of the dystrophin gene [78, 79]. The authors found significantly increased dystrophin restoration and reduced fibrosis in both skeletal and cardiac muscle and improved muscle function and cardiac hemodynamics at up to 18 months.

### **4.2** *LMNA* **mutations-tailored therapeutic strategy**

A genetic approach referred as trans-splicing approach was initially carried out to correct *LMNA* mutations in the *Lmna* DelK32 knock-in mouse harboring a *LMNA*-related congenital muscular dystrophy mutation in the exon 1 [80, 81]. The *Lmna* DelK32/DelK32 mice exhibited severe muscular and cardiac defects and early premature death. The trans-splicing approach allows converting the targeted endogenous mutated pre-mRNA to a therapeutic pre–trans-spliced molecule containing the wildtype coding sequence, resulting in a processed mRNA transcript devoid of the exon harboring the pathogenic variant [82, 83]. To perform the trans-splicing rescue in the *Lmna* DelK32 knock-in mouse model, 5'-RNA pre-trans-splicing molecules containing the first five exons of *Lmna* and targeting intron 5 of *Lmna* pre-mRNA were firstly developed. After confirmed the induced trans-splicing events on endogenous *Lmna* mRNA in vitro, AAV mediated delivery system was then evaluated in vivo in the newborn mice. Similarly, trans-splicing events were successfully detected in both skeletal and heart muscle of mice up to 50 days after AAV delivery. However, the tans-splicing occurring efficiency needs to be further improved to exert significant rescue effects on cardiac function and premature death.

To target at the *LMNA* pathogenic variants resulting in deficiency of the protein level, another strategy referred as stop codon readthrough was explored through inactivating the molecules participating in nonsense-mediated decay at the RNA level [84]. This approach applies to the scenario when pathogenic variants result in frameshifts and premature stops, subsequently leading to protein deficiency because of increased nonsense-mediated decay of mutant RNAs. PTC124, also known as ataluren, is a chemical compound that can selectively induce ribosomal readthrough of premature but not normal termination codons [85]. PTC124 was tested in iPSC-CMs derived from patients carrying different nonsense and frameshift mutations in the *LMNA* gene [85]. In one of the three frameshift mutants tested, administration of PTC124 significantly increased the translation of full-length LMNA protein and partially restored the protein function, as shown by reduced cardiomyocyte apoptosis and improved excitationcontraction coupling [84].

#### **4.3** *TTN* **truncating variants-targeted gene therapy**

While the pathogenicity of *TTN* missense mutations to DCM remains largely undetermined, *TTN* truncating variants (*TTNtvs*) are the most common cause to DCM. To date, experimental evidence generated largely from rodent animal models, human induced pluripotent stem cell-derived cardiomyocyte, and patient tissues mostly support the mechanism of titin protein haploinsufficiency. Correction of the titin protein deficiency by using traditional viral-mediated gene replacement strategy to increase the expression of titin protein, just as that has been successfully employed to restore the dystrophin deficiency, however, is challenging. Because the *TTN* gene spans 294 Kb of genomic region and its spliced transcript is more than 100 Kb, a size that is way bigger than any currently available cargo capacity for AAV

**113**

capability.

*Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic…*

mediated gene transfection. Alternatively, exon skipping, a genetic approach that had been initially employed to treat the DMD, through using an AON to mask the pathogenic mutations containing exon 51 or 53 and restoration of DMD phenotype [72], as aforementioned, was explored to treat *TTNtvs* related DCM. Notably, this strategy had been also successfully utilized for treating Mybpc3 mutations caused

To employ exon skipping strategy for treating *TTNtvs* caused DCM, Gramlich and colleagues targeted to the Ser14450fsX4 variant located in the exon 326 identified in a DCM patient that caused autosomal-dominant truncating frameshift mutation [87]. They used an AON-mediated exon skipping approach to remove the exon 326 in both patient cardiomyocytes in vitro and mouse heart in vivo. In vitro, skipping of the exon 326 containing the Ser14450fsX4 pathogenic variant restored the impaired myofibril assembly and stabilized its structural integrity and normalized expression of sarcomeric protein in the patient-specific cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) model. Furthermore, in the corresponding Ttn knock-in mice model, skipping of the exon 326 significantly improved sarcomere formation and contractile function and prevented subsequent development of DCM phenotype. These results provide strong evidence to support that the RNA-based exon skipping strategy could be a potential treatment option for DCM caused by gene variants that are otherwise technically difficult to be

Mutations in the δ- sarcoglycan coding gene (*SGCD*) can lead to DCM without

An earlier version of genome editing technique referred as transcription activatoraffected nuclease (TALEN) was explored to directly correct the *PLN* R14del mutation that is associated with high risk for malignant ventricular arrhythmias and end-stage heart failure in DCM patient carriers [89]. The authors Karakikes and colleague firstly derived iPSC-CMs from a DCM patient harboring the *PLN* R14del mutation, followed by detailed phenotypic characterization of this iPSC-CMs model. Next, the authors made the effort to correct the *PLN* R14del mutation in the iPSC-CMs model using TALEN gene editing strategy. This approach successfully corrected the R14del mutation and attenuated the R14del-associated disease phenotypes in the iPSC-CMs model. Furthermore, the authors engineered an AAV6 vector that enabled knock-down the endogenous *PLN*, while simultaneously overexpress a codon-optimized *PLN*, to effectively reverse the disease phenotype in the iPSC-CMs model as well. Thus, this study provides another successful example to using genome editing approach to directly target at the pathogenic mutations associated with DCM

obvious involvement of skeletal muscle disorders [57]. The gene replacement strategy was firstly applied in the treatment of DCM caused by a deletion mutation in the *Sgcd* gene that disrupted the dystrophin-associated glycoprotein complex in a hamster model [88]. The authors Kawada and colleague constructed a recombinant AAV vector containing the full-length cDNA of *Sgcd* gene driven under the cardiac specific expression promoter of CMV and intramurally injected it to the *Sgcd* mutation induced DCM model in the hamster*. They detected* robust expression of the *Sgcd* gene and other types of sarcoglycans as well in the transfected myocardium. The restored expression of *Sgcd* and other sarcoglycans normalized the diameter of transduced cardiomyocytes, improved the contractile function and ultimately prolonged the life span of the animals harboring the *Sgcd* gene mutation. This study provides one of the earliest evidence to employ gene replacement strategy to treat DCM caused by a specific gene mutation with efficient and sustained transfection

*DOI: http://dx.doi.org/10.5772/intechopen.94434*

HCM in a knock-in mouse model [86].

delivered due to size limitation for gene replacement therapy.

**4.4 Genotype-targeted therapies for other DCM genes**

*Genetic Determinant of Familial Dilated Cardiomyopathy and Genotype-Targeted Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94434*

mediated gene transfection. Alternatively, exon skipping, a genetic approach that had been initially employed to treat the DMD, through using an AON to mask the pathogenic mutations containing exon 51 or 53 and restoration of DMD phenotype [72], as aforementioned, was explored to treat *TTNtvs* related DCM. Notably, this strategy had been also successfully utilized for treating Mybpc3 mutations caused HCM in a knock-in mouse model [86].

To employ exon skipping strategy for treating *TTNtvs* caused DCM, Gramlich and colleagues targeted to the Ser14450fsX4 variant located in the exon 326 identified in a DCM patient that caused autosomal-dominant truncating frameshift mutation [87]. They used an AON-mediated exon skipping approach to remove the exon 326 in both patient cardiomyocytes in vitro and mouse heart in vivo. In vitro, skipping of the exon 326 containing the Ser14450fsX4 pathogenic variant restored the impaired myofibril assembly and stabilized its structural integrity and normalized expression of sarcomeric protein in the patient-specific cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) model. Furthermore, in the corresponding Ttn knock-in mice model, skipping of the exon 326 significantly improved sarcomere formation and contractile function and prevented subsequent development of DCM phenotype. These results provide strong evidence to support that the RNA-based exon skipping strategy could be a potential treatment option for DCM caused by gene variants that are otherwise technically difficult to be delivered due to size limitation for gene replacement therapy.

#### **4.4 Genotype-targeted therapies for other DCM genes**

Mutations in the δ- sarcoglycan coding gene (*SGCD*) can lead to DCM without obvious involvement of skeletal muscle disorders [57]. The gene replacement strategy was firstly applied in the treatment of DCM caused by a deletion mutation in the *Sgcd* gene that disrupted the dystrophin-associated glycoprotein complex in a hamster model [88]. The authors Kawada and colleague constructed a recombinant AAV vector containing the full-length cDNA of *Sgcd* gene driven under the cardiac specific expression promoter of CMV and intramurally injected it to the *Sgcd* mutation induced DCM model in the hamster*. They detected* robust expression of the *Sgcd* gene and other types of sarcoglycans as well in the transfected myocardium. The restored expression of *Sgcd* and other sarcoglycans normalized the diameter of transduced cardiomyocytes, improved the contractile function and ultimately prolonged the life span of the animals harboring the *Sgcd* gene mutation. This study provides one of the earliest evidence to employ gene replacement strategy to treat DCM caused by a specific gene mutation with efficient and sustained transfection capability.

An earlier version of genome editing technique referred as transcription activatoraffected nuclease (TALEN) was explored to directly correct the *PLN* R14del mutation that is associated with high risk for malignant ventricular arrhythmias and end-stage heart failure in DCM patient carriers [89]. The authors Karakikes and colleague firstly derived iPSC-CMs from a DCM patient harboring the *PLN* R14del mutation, followed by detailed phenotypic characterization of this iPSC-CMs model. Next, the authors made the effort to correct the *PLN* R14del mutation in the iPSC-CMs model using TALEN gene editing strategy. This approach successfully corrected the R14del mutation and attenuated the R14del-associated disease phenotypes in the iPSC-CMs model. Furthermore, the authors engineered an AAV6 vector that enabled knock-down the endogenous *PLN*, while simultaneously overexpress a codon-optimized *PLN*, to effectively reverse the disease phenotype in the iPSC-CMs model as well. Thus, this study provides another successful example to using genome editing approach to directly target at the pathogenic mutations associated with DCM

*Cardiac Diseases - Novel Aspects of Cardiac Risk, Cardiorenal Pathology and Cardiac Interventions*

detected at up to 19 months [77]. Similarly, Hakim and colleagues modified the dose of AAV.SaCas9 and AAV-9.gRNA vector that directs Cas9 to introns of 22 and 23 and performed the AAV-9 CRISPRA gene therapy in the mdx mice that carry a nonsense mutation in exon 23 of the dystrophin gene [78, 79]. The authors found significantly increased dystrophin restoration and reduced fibrosis in both skeletal and cardiac muscle and improved muscle function and cardiac hemodynamics at up to 18 months.

A genetic approach referred as trans-splicing approach was initially carried out to correct *LMNA* mutations in the *Lmna* DelK32 knock-in mouse harboring a *LMNA*-related congenital muscular dystrophy mutation in the exon 1 [80, 81]. The *Lmna* DelK32/DelK32 mice exhibited severe muscular and cardiac defects and early premature death. The trans-splicing approach allows converting the targeted endogenous mutated pre-mRNA to a therapeutic pre–trans-spliced molecule containing the wildtype coding sequence, resulting in a processed mRNA transcript devoid of the exon harboring the pathogenic variant [82, 83]. To perform the trans-splicing rescue in the *Lmna* DelK32 knock-in mouse model, 5'-RNA pre-trans-splicing molecules containing the first five exons of *Lmna* and targeting intron 5 of *Lmna* pre-mRNA were firstly developed. After confirmed the induced trans-splicing events on endogenous *Lmna* mRNA in vitro, AAV mediated delivery system was then evaluated in vivo in the newborn mice. Similarly, trans-splicing events were successfully detected in both skeletal and heart muscle of mice up to 50 days after AAV delivery. However, the tans-splicing occurring efficiency needs to be further improved to exert significant rescue effects on cardiac function and

To target at the *LMNA* pathogenic variants resulting in deficiency of the protein level, another strategy referred as stop codon readthrough was explored through inactivating the molecules participating in nonsense-mediated decay at the RNA level [84]. This approach applies to the scenario when pathogenic variants result in frameshifts and premature stops, subsequently leading to protein deficiency because of increased nonsense-mediated decay of mutant RNAs. PTC124, also known as ataluren, is a chemical compound that can selectively induce ribosomal readthrough of premature but not normal termination codons [85]. PTC124 was tested in iPSC-CMs derived from patients carrying different nonsense and frameshift mutations in the *LMNA* gene [85]. In one of the three frameshift mutants tested, administration of PTC124 significantly increased the translation of full-length LMNA protein and partially restored the protein function, as shown by reduced cardiomyocyte apoptosis and improved excitation-

While the pathogenicity of *TTN* missense mutations to DCM remains largely undetermined, *TTN* truncating variants (*TTNtvs*) are the most common cause to DCM. To date, experimental evidence generated largely from rodent animal models, human induced pluripotent stem cell-derived cardiomyocyte, and patient tissues mostly support the mechanism of titin protein haploinsufficiency. Correction of the titin protein deficiency by using traditional viral-mediated gene replacement strategy to increase the expression of titin protein, just as that has been successfully employed to restore the dystrophin deficiency, however, is challenging. Because the *TTN* gene spans 294 Kb of genomic region and its spliced transcript is more than 100 Kb, a size that is way bigger than any currently available cargo capacity for AAV

**4.2** *LMNA* **mutations-tailored therapeutic strategy**

**112**

premature death.

contraction coupling [84].

**4.3** *TTN* **truncating variants-targeted gene therapy**

to achieve therapeutic benefit. Notably, while it provides evidence to support the effectiveness of genome editing technique to correct pathogenic variants causing DCM in vitro, germline correction of embryos harboring DCM variants had not been reported yet.