*3.5. Model Calibration and Validation*

The water balance of the basin was estimated based on climatic data analysis from 1980–2016. The base year is considered from January to December 2016, which is taken as a normal hydrological year. The results showed that the total water balance of the basin during the study period was found to be 427 MCM. The values of parameters for the calibration results in the WEAP model is shown (Table 4).

**Table 4.** The calibration parameters' possible ranges used in the WEAP model.

The WEAP model was calibrated and validated with monthly observed streamflow from 1980–1999 and 2000–2014, respectively (Figure 7). In order to simulate the streamflow values in the model, the crop coefficient of (kc) of different land covers, soil water capacity, root zone conductivity, and preferred flow direction were manually calibrated using the default values of the WEAP model (Table 4). Hence, during the calibration period of this study, the values of the coefficient of determination (R2) ranged from 0.73 to 0.91, with an average value of 0.82. On the other hand, the value of the Nash–Sutcliffe efficiency (NSE) was 0.65 and 0.83, the minimum and maximum values, respectively, with an average value of 0.74. However, during validation periods, 0.54 and 0.91 are the minimum and maximum values of the coefficient of determination (R2), with an average value of 0.73. As far as the values of the Nash–Sutcliffe efficiency (NSE), 0.50 and 0.84 were the minimum and maximum values respectively, with an average value of 0.67. From these values, we have observed that in both calibration and validation periods, the values correspond to a perfect match between the observed and the modeled streamflow values. Thus, the performance of the model is acceptable between the trends of observed and simulated streamflow in both calibration and validation of the study basin. This also helps to accurately project the prediction of future discharges based on the future scenario set. The statistical values of Akaki (R2 = 0.73; NSE = 0.65) were less than other gauging stations; Kuntre (R2 = 0.85; NSE = 0.73), Hombole (R2 = 0.89; NSE = 0.83), and Modjo (R2 = 0.91; NSE = 0.85) (Table 5). This may be due to urbanization.

**Table 5.** Statistical values of stations for calibration and validation.

**Figure 7.** Calibration and validation of the four gauging stations and the entire Awash River Basin.


**5.2. Evaluation results**

We evaluate the performance and path solution of search-based algorithms in two scenarios: partially known and unknown 2D grid environment with uniform resolution. The total expanded cells are averaged based on total replanning processes on each simulation instance, with 95% confident. The path solution of each algorithm is counted as the total cells that the robot has traversed from corner to corner of the map. We decrease the suboptimal bound of AD\* for 0.1 per step the robot travels until the suboptimal bound reaches 1.0 (optimal path).

**Figure 16** shows the speedup result throughout the evolution from Replanning A\* to AD\* with different *ε* suboptimal bounds as well as the trade-off of AD\*. The environment is initially generated randomly obstacles that occupy 25% of the map. The initial map is then input to the robot. While the robot is moving, we randomly change the cell states that are 15% of the map, thus forcing the robot to replan its path whenever it detects environmental

Search-Based Planning and Replanning in Robotics and Autonomous Systems

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85

As can be seen, AD\* has the highest performance that has least total expanded cells in replanning process; the higher the suboptimal bound, the better the performance. The reason is AD\* is inflated its heuristic function to make it greedier in expanding cells towards goal. It is interesting that path solution of AD\* is not much longer than optimal path. As the scale of map is increasing, the path between map corner is longer to travel, and thus, the robot is given enough time to improve its solution (path ratio with *ε* = 6.0 is gradually converged to the one

D\*LR slightly improves the performance of D\* Lite; it is because D\*LR relies on computation differences between Replanning A\* and D\* Lite. In fact, the pitfall of D\* Lite rarely happens in scenarios that the robot detects changes near its position. Replanning A\* does not have

The data confirms the fact that AD\*, in average throughout the increasing map scale, improves 125% and 194% performance compared to D\* Lite with *ε* = 3.0 and *ε* = 6.0, respectively. The

**Figure 17.** Comparison between search-based algorithms on unknown environment with increasing map scale in terms

incremental property and thus uses the highest computation.

path produces by AD\* only 1% longer than optimal path in average.

changes.

*ε* = 3.0 at 1.016).

of computation and path solution.

**Figure 15.** Simulated environment on our framework.

**Figure 16.** Comparison between search-based algorithms on partially known environment with increasing map scale in terms of computation and path solution.

**Figure 16** shows the speedup result throughout the evolution from Replanning A\* to AD\* with different *ε* suboptimal bounds as well as the trade-off of AD\*. The environment is initially generated randomly obstacles that occupy 25% of the map. The initial map is then input to the robot. While the robot is moving, we randomly change the cell states that are 15% of the map, thus forcing the robot to replan its path whenever it detects environmental changes.

As can be seen, AD\* has the highest performance that has least total expanded cells in replanning process; the higher the suboptimal bound, the better the performance. The reason is AD\* is inflated its heuristic function to make it greedier in expanding cells towards goal. It is interesting that path solution of AD\* is not much longer than optimal path. As the scale of map is increasing, the path between map corner is longer to travel, and thus, the robot is given enough time to improve its solution (path ratio with *ε* = 6.0 is gradually converged to the one *ε* = 3.0 at 1.016).

D\*LR slightly improves the performance of D\* Lite; it is because D\*LR relies on computation differences between Replanning A\* and D\* Lite. In fact, the pitfall of D\* Lite rarely happens in scenarios that the robot detects changes near its position. Replanning A\* does not have incremental property and thus uses the highest computation.

The data confirms the fact that AD\*, in average throughout the increasing map scale, improves 125% and 194% performance compared to D\* Lite with *ε* = 3.0 and *ε* = 6.0, respectively. The path produces by AD\* only 1% longer than optimal path in average.

**Figure 17.** Comparison between search-based algorithms on unknown environment with increasing map scale in terms of computation and path solution.

**Figure 16.** Comparison between search-based algorithms on partially known environment with increasing map scale in

terms of computation and path solution.

**Figure 15.** Simulated environment on our framework.

84 Advanced Path Planning for Mobile Entities

**Figure 17** describes the evaluation case on unknown environment. The environment is initially generated with random obstacles that occupy 15% of the map. The robot does not know the initial conditions; it will replan its path whenever it detects obstacles that do not exist in its map.

**Author details**

United Kingdom

**References**

<IP_ADDRESS>.1387

\* and Than D. Le2

University, Hồ Chí Minh, Vietnam

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Electrical Engineering and Information Technology, Vietnamese-German

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[1] Lavalle SM, Kuffner JJ Jr. Rapidly-exploring random trees: Progress and prospects. In: Algorithmic and Computational Robotics: New Directions. 2000. pp. 293-308. DOI:

[2] N. Preda, A. Manurung, O. Lambercy, R. Gassert and M. Bonfè. Motion planning for a multi-arm surgical robot using both sampling-based algorithms and motion primitives. In: 2015 IEEE/RSJ International Conference on Intelligent Robots and Systems (IROS);

[3] Ferguson D, Stentz A. Field D\*: An interpolation-based path planner and replanner. Journal of Robotics Research. 2007:239-253. DOI: 10.1007/978-3-540-48113-3\_22

[4] Stentz A. Optimal and efficient path planning for partially-known environments. In: Proceedings of the 1994 IEEE International Conference on Robotics and Automation;

[5] Matthies L, Xiong Y, Hogg R, Zhu D, Rankin A, Kennedy B, Hebert M, Maclaehlan R, Won C, Frost T, Sukhatme G, Mchenry M, Goldberg S. A portable, autonomous, urban reconnaissance robot. In: Proceedings of the International Conference on Intelligent

[6] Doan KN, Le AT, Le TD, Peter N. Swarm robots' communication and cooperation in motion planning. In: Zhang D, Wei B, editors. Mechatronics and Robotics Engineering for Advanced and Intelligent Manufacturing. Cham: Springer; 2016. pp. 191-205. DOI:

[7] Hwang YK, Ahuja N. Gross motion planning—A survey. ACM Computing Survey.

[8] Dechter R, Pearl J. Generalized best-first search strategies and the optimality of a\*.

Journal of the ACM (JACM). 1985;**32**(3):505-536. DOI: 10.1145/3828.3830

2 Faculty of Engineering, Bristol Robotics Laboratory, Bristol University, Bristol,

Hamburg. 2015. pp. 1422-1427. DOI: 10.1109/IROS.2015.7353554

San Diego, CA. 1994. pp. 3310-3317. DOI: 10.1109/ROBOT.1994.351061

Autonomous Systems; 2000. DOI: <IP_ADDRESS>.9353

1992;**24**(3):219-291. DOI: 10.1145/136035.136037

10.1007/978-3-319-33581-0\_15

An T. Le1

For unknown environment scenario, D\*LR performs significantly better than D\* Lite as increasing map scale. The reason is that if the replanned path is much longer than the initial path, which is the common case in unknown environment, the replanning process of D\* Lite is also expensive. AD\* still has the least computation compared to old search-based algorithm; it reduces drastically the computation of D\* Lite with 845% better performance, in the case *ε* = 10.0, while still maintains good path solution.


**Synthesis and Application of Copper Nanowires and Silver Nanosheet-Coated Copper Nanowires as Nanofillers in Several Polymers**

Ningning Zeng, Shuguang Fan, Jingyi Ma, Yujuan Zhang, Shengmao Zhang, Pingyu Zhang and Zhijun Zhang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67782

#### **Abstract**

A large amount of copper (Cu) nanowires was synthesized through the reduction of Cu(OH)2 by hydrazine in an aqueous solution containing NaOH and ethylenediamine. Besides, Cu nanowires coated by silver nanosheet (denoted as Cu@Ag nanowires) were prepared with a facile transmetalation reaction method. In the meantime, the as‐prepared Cu and Cu@Ag nanowires were used as the nanofillers of polyvinyl chloride (PVC), ultra‐ high molecular weight polyethylene (UHMWPE) and epoxy resin (EP), and their effects on the thermal properties and mechanical properties as well as friction and wear behavior of the polymer‐matrix composites nanocomposites were examined. Results indicate that the as‐prepared Cu@Ag nanowires consist of Cu nanowires core and Ag nanosheet shell. The Ag nanosheet shell can well inhibit the oxidation of the Cu nanowires core, thereby providing the as‐prepared Cu@Ag nanowires with good thermal stability even at an ele‐ vated temperature of 230°C. As compared with Cu nanowires, Cu@Ag nanowires could effectively increase the thermal stability of the PVC matrix composites. Moreover, due to the special morphology and microstructure, the as‐prepared Cu@Ag nanowires can effec‐ tively improve the mechanical properties and wear resistance of PVC, UHMWPE, and EP.

**Keywords:** Cu nanowires, Cu@Ag nanowires, nanocomposite, mechanical properties, tribological properties

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Copper nanowires could have promising applications in electronics [1–6], optoelectron‐ ics [7], solar cells [8–10], photonics, magnetics, genetic engineering, chemical sensors, and lubrication [11, 12], due to their unique characteristics such as high electrical conduc‐ tivity and thermal conductivity as well as high aspect ratio and tribological properties. Particularly, copper nanowires often can effectively improve the mechanical properties, thermal properties, and wear resistance of polymers such as polystyrene [13, 14] and poly‐ amide 6 [15, 16].

A variety of methods such as solution‐phase method and complex‐surfactant‐assisted hydrothermal reduction approach are currently available for the synthesis of Cu nanowires [17–35]. Here the solution‐phase method uses hydrazine (N2 H4 ) to reduce Cu2+ ions in the highly basic aqueous solution of copper nitrate (Cu(NO3 )2 ) at 25–100°C in the presence of ethylenediamine (EDA, C2 H8 N2 ) as the surface‐capping agent [32, 33]. The resultant Cu nanowires have a length of >40 μm and a diameter in the range of 60–160 nm, where a high concentration of NaOH (pH = 13–14) is required to prevent copper ions from form‐ ing copper hydroxide precipitates. The so‐called complex‐surfactant‐assisted hydrothermal reduction approach can achieve facile synthesis of metal copper nanowires with an aver‐ age diameter of 85 nm and a length of several tens of micrometers [34], where the copper nanowires are formed through the reduction of CuII−glycerol complex (Cu(C3 H6 O3 )) by phosphite (HPO3 2−) at 120°C in the presence of surfactant sodium dodecyl benzenesulfonate (SDBS).

The synthesis and application of Cu nanowires, however, are still challenging because Cu nanowires are liable to spontaneous oxidation in air. Therefore, it is imperative to adopt carbon materials [36–39], polymers [40], and metals [10, 41–45] as the sources to intro‐ duce nonoxidizable shells on the surface of Cu nanowires, thereby preventing them from oxidation.

Among various surface‐capped Cu nanowires, the metal‐capped ones are of special inter‐ est, because they can be well prevented from oxidation while basic characteristics of met‐ als are retained. We are particularly interested in Ag as a potential surface‐capping shell of Cu nanowires. This is because, with thermal conductivity and electrical conductivity similar to that of Cu, Ag exhibits excellent oxidation resistance [46–51]. A couple of Ag‐capped Cu nanowires have been reported elsewhere, mainly involving Cu nanowires‐Ag nanowires syn‐ thesized in solution phase by the heterogeneous nucleation and growth of Ag nanocrystals on presynthesized Cu nanowires [50] and Cu@Ag nanowires with rough surface obtained by the transmetalation reaction at the room temperature [51]. The Cu nanowire cores of these Ag‐capped Cu nanowires indeed can be well prevented from oxidation. However, the Ag shell of Cu@Ag nanowires consists of Ag nanoparticles, which is unfavorable for achieving full contact between the nanofillers and polymer matrix.

Therefore, in the present research, we prepare Ag nanosheets on Cu nanowires, hoping to achieve a large contact area between the nanofillers and polymer matrix, thereby acquiring improved mechanical properties and wear resistance. This article reports the preparation of Cu@Ag nanowires as well as the evaluation of their oxidation resistance. Besides, it also deals with the effect of Cu@Ag nanowires on the mechanical properties, thermal properties, and wear resistance of polyvinyl chloride (PVC), ultrahigh molecular weight polyethylene (UHMWPE), and epoxy resin (EP).

## **2. Preparation and characterization of Cu nanowires**

**1. Introduction**

2 Nanowires - New Insights

amide 6 [15, 16].

phosphite (HPO3

(SDBS).

oxidation.

ethylenediamine (EDA, C2

Copper nanowires could have promising applications in electronics [1–6], optoelectron‐ ics [7], solar cells [8–10], photonics, magnetics, genetic engineering, chemical sensors, and lubrication [11, 12], due to their unique characteristics such as high electrical conduc‐ tivity and thermal conductivity as well as high aspect ratio and tribological properties. Particularly, copper nanowires often can effectively improve the mechanical properties, thermal properties, and wear resistance of polymers such as polystyrene [13, 14] and poly‐

A variety of methods such as solution‐phase method and complex‐surfactant‐assisted hydrothermal reduction approach are currently available for the synthesis of Cu nanowires

nanowires have a length of >40 μm and a diameter in the range of 60–160 nm, where a high concentration of NaOH (pH = 13–14) is required to prevent copper ions from form‐ ing copper hydroxide precipitates. The so‐called complex‐surfactant‐assisted hydrothermal reduction approach can achieve facile synthesis of metal copper nanowires with an aver‐ age diameter of 85 nm and a length of several tens of micrometers [34], where the copper

The synthesis and application of Cu nanowires, however, are still challenging because Cu nanowires are liable to spontaneous oxidation in air. Therefore, it is imperative to adopt carbon materials [36–39], polymers [40], and metals [10, 41–45] as the sources to intro‐ duce nonoxidizable shells on the surface of Cu nanowires, thereby preventing them from

Among various surface‐capped Cu nanowires, the metal‐capped ones are of special inter‐ est, because they can be well prevented from oxidation while basic characteristics of met‐ als are retained. We are particularly interested in Ag as a potential surface‐capping shell of Cu nanowires. This is because, with thermal conductivity and electrical conductivity similar to that of Cu, Ag exhibits excellent oxidation resistance [46–51]. A couple of Ag‐capped Cu nanowires have been reported elsewhere, mainly involving Cu nanowires‐Ag nanowires syn‐ thesized in solution phase by the heterogeneous nucleation and growth of Ag nanocrystals on presynthesized Cu nanowires [50] and Cu@Ag nanowires with rough surface obtained by the transmetalation reaction at the room temperature [51]. The Cu nanowire cores of these Ag‐capped Cu nanowires indeed can be well prevented from oxidation. However, the Ag shell of Cu@Ag nanowires consists of Ag nanoparticles, which is unfavorable for achieving

nanowires are formed through the reduction of CuII−glycerol complex (Cu(C3

H4

) as the surface‐capping agent [32, 33]. The resultant Cu

)2

2−) at 120°C in the presence of surfactant sodium dodecyl benzenesulfonate

) to reduce Cu2+ ions in the

H6 O3 )) by

) at 25–100°C in the presence of

[17–35]. Here the solution‐phase method uses hydrazine (N2

highly basic aqueous solution of copper nitrate (Cu(NO3

H8 N2

full contact between the nanofillers and polymer matrix.

Cu nanowires were prepared through the reduction of Cu(OH)2 by hydrazine in an aqueous solution containing NaOH and ethylenediamine (EDA), with which the approach developed by Zeng et al. was modified to scale up the reaction by 3900 times (yield of Cu nanowires from 0.006 g to 23.5 g of) for potential large‐scale production [30]. Briefly, NaOH (18 L, 7 M), Cu(OH)2 (0.36 mol), EDA (135 mL), and hydrazine (18 mL, 35 wt.%) were added to a 20 L reactor and heated at 80°C for 30 min to achieve the reduction of Cu2+ to metallic copper at a rate of 100% (**Figure 1**). The as‐prepared Cu nanowire cake is present on the top of the solution, possibly due to the entrapping of nitrogen bubbles among the nanowires (2Cu 2+ + N 2 H 4 + 4OH ⁻   → 2Cu + N2 + 4 H2 O). **Figure 1a** shows the scanning electron microscopic (SEM) images of the as‐pre‐ pared Cu nanowires. They are straight and have a diameter of 150–200 nm and a length of about 10 μm. **Figure 1b** shows the X‐ray diffraction (XRD) pattern of the Cu nanowires. The peaks at 2*θ* = 43.3, 50.4, 74.1, and 89.9° correspond to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) crystal planes of cubic Cu (PDF No. 89‐2838), which indicates that the copper nanowires exhibit face‐ centered cubic (fcc) structure. No XRD signals of other species are detected, which indicate that the as‐prepared Cu nanowires are highly pure. However, when the as‐prepared Cu nanowires are placed in air for 24 h, three XRD peaks emerge at 2*θ* = 36.4, 42.3 and 52.5° (**Figure 1c**), and they correspond to the (1 1 1), (2 0 0), and (2 1 1) crystal planes of Cu2 O (PDF No. 78‐2076). This means that the as‐prepared Cu nanowires are easily oxidized in air environment.

**Figure 1.** (a) SEM image of as‐prepared Cu nanowires in mother liquor and XRD patterns, (b) as‐prepared Cu nanowires, and (c) Cu nanowires after being stored in air for 24 h.

## **3. Preparation and characterization of Cu@Ag nanowires**

The formation of Cu@Ag nanowires by the transmetalation reaction should be closely related to the different reduction potentials of Cu and Ag. First, 1.5 mmol AgNO3 was dispersed in distilled water. Into the AgNO3 solution was dropwise added the ammonium hydroxide (NH4 OH, 28–30%, BDH) aqueous solution under vigorous stirring until the yellow precipitate disappeared and the solution became clear. The as‐obtained Ag‐amine reagent solution was then directly added into 50 mL of Cu nanowires dispersion (with 0.5 g of Cu nanowires) under vigorous magnetic stirring at 40°C. After 2 h of reaction, the as‐obtained product was washed with deionized water and collected by centrifugation, followed by redispersion in deionized water. During the synthesis progress, the Cu atoms on the surface of Cu nanowires quickly react with Ag+ ions to release Cu2+. Simultaneously, Ag+ ions are reduced into Ag atoms and deposited on the surface of the Cu nanowires, during which the color of Cu nanowires dispersion turns grey along with the appearance of metallic Ag. Therefore, the morphology and microstructure of the as‐prepared Cu@Ag nanowires should be highly dependent on the reaction conditions. Through comprehen‐ sive analysis, the optimal reaction condition is that temperature is 40°C; Cu/Ag molar ratio is 5:1; Cu dispersion concentration is 1% and silver ammonia reagent dropping speed is poured directly.

**Figure 2** shows the SEM and TEM images as well as XRD pattern and EDS line map of the Cu@Ag nanowires prepared under the optimal reaction condition. It can be seen that the as‐ prepared Cu@Ag nanowires have a diameter of about 250 nm and a length of about 10 μm (**Figure 2a**). Besides, the major XRD peaks of the as‐prepared Cu@Ag nanowires can be indexed to metallic Ag (PDF No. 87‐0717) and Cu (PDF No. 89‐2838) with fcc structure (**Figure 2b**). The

**Figure 2.** Characterization of Cu@Ag nanowires prepared under the optimal reaction condition: (a) SEM image, (b) XRD pattern, (c) EDS line map, (d) TEM image, and (e) HRTEM image.

small peak at 2*θ* = 35.5° can be indexed to CuO (PDF No. 80‐1917), which is possibly due to the oxidation of original Cu nanowires before coating with Ag nanosheets. In terms of the element line distribution, the copper element has a maximum value at the center part whereas the Ag shows a complementary profile with two maxima on both sides (**Figure 2c**). This indicates that the as‐prepared Cu@Ag nanowires have a core‐shell structure composed of Cu core and Ag shell. Such a core‐shell structure of the as‐prepared Cu@Ag nanowires is also supported by corresponding TEM image shown in **Figure 2d**. Namely, there is a layer of Ag nanosheets on Cu nanowires. Moreover, the high‐resolution TEM (HRTEM) image of Cu@Ag nanowires indicates that the Ag crystal exhibits a fringe lattice spacing of 0.25 nm (**Figure 2e**), and it cor‐ responds to the (200) plane of the Ag crystal.

**3. Preparation and characterization of Cu@Ag nanowires**

dispersed in distilled water. Into the AgNO3

on the surface of Cu nanowires quickly react with Ag+

pattern, (c) EDS line map, (d) TEM image, and (e) HRTEM image.

hydroxide (NH4

4 Nanowires - New Insights

poured directly.

Ag+

The formation of Cu@Ag nanowires by the transmetalation reaction should be closely related to the different reduction potentials of Cu and Ag. First, 1.5 mmol AgNO3

yellow precipitate disappeared and the solution became clear. The as‐obtained Ag‐amine reagent solution was then directly added into 50 mL of Cu nanowires dispersion (with 0.5 g of Cu nanowires) under vigorous magnetic stirring at 40°C. After 2 h of reaction, the as‐obtained product was washed with deionized water and collected by centrifugation, followed by redispersion in deionized water. During the synthesis progress, the Cu atoms

 ions are reduced into Ag atoms and deposited on the surface of the Cu nanowires, during which the color of Cu nanowires dispersion turns grey along with the appearance of metallic Ag. Therefore, the morphology and microstructure of the as‐prepared Cu@Ag nanowires should be highly dependent on the reaction conditions. Through comprehen‐ sive analysis, the optimal reaction condition is that temperature is 40°C; Cu/Ag molar ratio is 5:1; Cu dispersion concentration is 1% and silver ammonia reagent dropping speed is

**Figure 2** shows the SEM and TEM images as well as XRD pattern and EDS line map of the Cu@Ag nanowires prepared under the optimal reaction condition. It can be seen that the as‐ prepared Cu@Ag nanowires have a diameter of about 250 nm and a length of about 10 μm (**Figure 2a**). Besides, the major XRD peaks of the as‐prepared Cu@Ag nanowires can be indexed to metallic Ag (PDF No. 87‐0717) and Cu (PDF No. 89‐2838) with fcc structure (**Figure 2b**). The

**Figure 2.** Characterization of Cu@Ag nanowires prepared under the optimal reaction condition: (a) SEM image, (b) XRD

OH, 28–30%, BDH) aqueous solution under vigorous stirring until the

was

solution was dropwise added the ammonium

ions to release Cu2+. Simultaneously,

The thermal stability of Cu@Ag nanowires with different treatments was measured by XRD. **Figure 3a** shows the XRD pattern of Cu@Ag nanowires prepared under the optimal con‐ dition after 2 month of storage in ambient condition. All the characteristic peaks remain unchanged after 2 month of storage in air, which indicates that the Ag shell layer can well inhibit the oxidation of the Cu nanowires core. **Figure 3b** shows the XRD patterns of the same product after being sintered at different temperatures. It can be seen that the XRD pat‐ terns have no change before 200°C contrasted with the XRD of as‐prepared samples, while after being sintered at 300°C, a distinct peak of Cu2 O emerges and the major peaks of metallic Cu and Ag still remain, which indicates the Ag coating can protect Cu nanowires core from oxidation at high temperature. This means that the Cu@Ag nanowires show better thermal stability than Cu nanowires.

In order to further determine oxidation resistance of Cu@Ag nanowires, TG analysis was con‐ ducted in air flow. **Figure 4** shows the TG curves of the as‐prepared Cu nanowires and Cu@ Ag nanowires in air. The TG curves show Cu nanowires undergo a weight‐gain around 100°C attributed to the oxidation thereat, while the Cu@Ag nanowires began to add the weight at around 230°C indicating that Cu@Ag nanowires have the higher oxidation resistance temper‐ ature, which also demonstrates that Cu@Ag nanowires exhibit much better thermal stability than Cu nanowires.

**Figure 3.** XRD paterns of Cu@Ag nanowires prepared under the optimal reaction condition: (a)after storage for different times in air, (b) after being sintered at different temperatures.

**Figure 4.** TG curves of Cu nanowires and Cu@Ag nanowires in air at a heating rate of 10°C/min.

## **4. Cu/Cu@Ag nanowires‐PVC nanocomposites**

The component and dosage for the preparation of Cu/Cu@Ag nanowires‐PVC composites materials are listed in **Table 1**. The raw materials were mixed with mechanical stirring for 20 min, then melt blended by a laboratory two roll mill with a rotate speed of 50 rpm under 100°C for 10 min. The milled sheets were compressed by a press machine at 175°C and 20 MPa for 5 min. The samples for performance test were cut from the final sheets.

**Table 1.** The component and dosage for the preparation of Cu nanowires‐ and Cu@Ag nanowires‐PVC composites.

## **4.1. Thermal properties of Cu/Cu@Ag nanowires‐PVC nanocomposites**

Thermal properties of the composites were studied by differential scanning calorimetry (DSC). DSC curves were obtained by heating at 10°C/min rate under N2 at a flow rate of 100 mL/min in the range of 20–120°C by using NETZSCH DSC 204HP instrument (Bavarian, Germany). The heated sample was cooled to 20°C and reheated for the second time using the same heating program.

An exemplary DSC heating curve for Cu nanowires‐PVC nanocomposites is presented in **Figure 5**, with the indication of the procedure of glass transitions temperatures (Tg) deter‐ mination as a central point value of the tangent line to the DSC curve, at the inflection point where the characteristic augment of the base line, related to the increase of the specific heat, was used for the determination of the glass transition. For all samples, the run of the DSC curve at the domain of the glass transition was similar.

The Tg of the Cu@Ag nanowires‐PVC nanocomposites is presented in **Figure 6**. It can be seen that, as compared with Cu nanowires, the introduction of Cu@Ag nanowires results in a larger increase in the Tg value. Besides, the Tg of the Cu@Ag nanowires‐PVC nanocom‐ posites tends to rise with the increase of Cu@Ag nanowire content. This is because, with the increase of the Cu@Ag nanowire content, more Cu@Ag nanowires act as physical cross‐ linkers to be intertwined with macromolecular chains, thereby reducing the chain segmen‐ tal mobility of PVC and increasing the Tg. However, when the content of Cu@Ag nanowires

**4. Cu/Cu@Ag nanowires‐PVC nanocomposites**

**PVC Octadecanoic acid**

**Nano‐filler mass fraction**

6 Nanowires - New Insights

**(%)**

for 5 min. The samples for performance test were cut from the final sheets.

0 160 1.28 0.64 0.64 32 0 0.1 160 1.28 0.64 0.64 32 0.195 0.5 160 1.28 0.64 0.64 32 0.978 1.0 160 1.28 0.64 0.64 32 1.97 3.0 160 1.28 0.64 0.64 32 6.02

**Table 1.** The component and dosage for the preparation of Cu nanowires‐ and Cu@Ag nanowires‐PVC composites.

**Figure 4.** TG curves of Cu nanowires and Cu@Ag nanowires in air at a heating rate of 10°C/min.

The component and dosage for the preparation of Cu/Cu@Ag nanowires‐PVC composites materials are listed in **Table 1**. The raw materials were mixed with mechanical stirring for 20 min, then melt blended by a laboratory two roll mill with a rotate speed of 50 rpm under 100°C for 10 min. The milled sheets were compressed by a press machine at 175°C and 20 MPa

**Component (g)**

**stearate**

**Dioctyl phthalate** **Cu/Cu@Ag nanowires**

**Lead stearate Calcium**

**Figure 5.** An exemplary DSC trace for Cu nanowires‐PVC composite (Cu nanowires contain: 1 wt.%) at the domain of the glass transition.

**Figure 6.** The glass transition temperature (Tg) of Cu/Cu@Ag nanowires‐PVC nanocomposites with various mass fractions of the nanofillers.

is too high, the Tg values of the PVC‐matrix nanocomposites declines to some extent. The reason lies in that a too high content of Cu@Ag nanowires would tend to agglomerate in the polymer matrix and weaken the interaction with the macromolecular chains, thereby limiting the movements of the macromolecular chains and reducing the Tg value of the PVC‐matrix nanocomposites. Moreover, the presence of Cu@Ag nanowires in PVC matrix contributes to increasing the elasticity especially in the glass transition region, which cor‐ responds to enhanced thermal stability of the PVC‐matrix nanocomposites.

#### **4.2. Mechanical properties of Cu/Cu@Ag nanowires‐PVC nanocomposites**

Mean values of five factors on mechanical properties were analyzed in this work. The tensile strength and elongation at break of these samples are shown in **Figure 7**. From **Figure 7**, it can be seen that the elongation at break of composite materials all increase to varying degrees by addition of the nanowires. And the effect of Cu@Ag nanowires is much larger than the Cu nanowires. Meanwhile, the tensile strength keeps no change on the whole.

#### **4.3. Tribology properties of Cu/Cu@Ag nanowires‐PVC nanocomposites**

**Figure 8** shows the friction coefficient and wear rate of Cu/Cu@Ag nanowires‐PVC nano‐ composites (load: 20 N; route: 5 mm; frequency: 2 Hz; time: 5 min; room temperature). The friction coefficient of Cu/Cu@Ag nanowires PVC nanocomposites has little change with Synthesis and Application of Copper Nanowires and Silver Nanosheet-Coated Copper Nanowires... http://dx.doi.org/10.5772/67782 9

**Figure 7.** The elongation at break (a) and tensile strength and (b) Cu/Cu@Ag nanowires‐PVC nanocomposites with different nanowires contents.

**Figure 8.** The friction and wear behavior (friction coefficient and wear rate) of PVC nanocomposites with different contents of (a) Cu nanowires and (b) Cu@Ag nanowires.

is too high, the Tg values of the PVC‐matrix nanocomposites declines to some extent. The reason lies in that a too high content of Cu@Ag nanowires would tend to agglomerate in the polymer matrix and weaken the interaction with the macromolecular chains, thereby limiting the movements of the macromolecular chains and reducing the Tg value of the PVC‐matrix nanocomposites. Moreover, the presence of Cu@Ag nanowires in PVC matrix contributes to increasing the elasticity especially in the glass transition region, which cor‐

**Figure 6.** The glass transition temperature (Tg) of Cu/Cu@Ag nanowires‐PVC nanocomposites with various mass

Mean values of five factors on mechanical properties were analyzed in this work. The tensile strength and elongation at break of these samples are shown in **Figure 7**. From **Figure 7**, it can be seen that the elongation at break of composite materials all increase to varying degrees by addition of the nanowires. And the effect of Cu@Ag nanowires is much larger than the Cu

**Figure 8** shows the friction coefficient and wear rate of Cu/Cu@Ag nanowires‐PVC nano‐ composites (load: 20 N; route: 5 mm; frequency: 2 Hz; time: 5 min; room temperature). The friction coefficient of Cu/Cu@Ag nanowires PVC nanocomposites has little change with

responds to enhanced thermal stability of the PVC‐matrix nanocomposites.

fractions of the nanofillers.

8 Nanowires - New Insights

**4.2. Mechanical properties of Cu/Cu@Ag nanowires‐PVC nanocomposites**

nanowires. Meanwhile, the tensile strength keeps no change on the whole.

**4.3. Tribology properties of Cu/Cu@Ag nanowires‐PVC nanocomposites**

increasing amount of copper nanowire, while the wear rate changes greatly therewith. Namely, pure PVC (without Cu nanowires) has a larger wear rate as much as 1.42 × 10−3 mm3 /N m, while PVC composite containing 0.5 wt.% Cu@Ag nanowires has an obviously reduced wear rate of 0.57 × 10−3 mm3 /N m (reduced by nearly 60%). It can be clearly seen that lower or higher concentrations of fillers in the matrix are less effective in reducing the friction coefficient and wear rate. It can be inferred that, at a too low concentration, the nanofillers can hardly play its role on the friction‐reducing and wear resistance in the friction process, thereby leading to relatively not very good friction property. When the additive concentration is too high, the nanofiller would tend to agglomerate and form a large number of weak interfaces in the matrix, which makes the nanofillers easily fall off from the matrix in the friction process causing a higher abrasive wear [52], thus resulting in relatively poor friction property. Therefore, it is suggested to keep the concentration of the nanofillers in polymer matrix as 0.5 wt.% Cu@Ag nanowires in order to effectively reduce the friction coefficient and wear rate. Moreover, the wear rate of Cu nanowires‐ PVC nanocomposites tends to decline with the increasing content of the Cu nanowires, and the minimum wear rate (0.85 × 10−3 mm3 /N m) is obtained at a Cu nanowires content of 0.1 wt.%.

## **5. Cu/Cu@Ag nanowires‐UHMWPE nanocomposites**

The component and dosage for the preparation of Cu@Ag nanowires‐UHMWPE compos‐ ites are listed in **Table 2**. The nanowires and UHMWPE raw material were mixed with ball milling for 1 h. The mixture was heated to 200°C and pressed under 20 MPa for 10–20 min, followed by cooling to room temperature in the mold to afford the Cu@Ag nanowires‐ UHMWPE nanocomposites. All the samples for performance test were cut from the final sheets.

## **5.1. Crystallization and melting behavior of Cu/Cu@Ag nanowires‐UHMWPE nanocomposites**

The crystallization and melting behavior of Cu/Cu@Ag nanowires‐UHMWPE nanocom‐ posites were studied by differential scanning calorimetry (DSC). DSC curves in the range of 20–200**°**C were measured with a NETZSCH DSC 204HP instrument (Bavarian, Germany) at a heating rate of 10**°**C/min under N2 atmosphere (flow rate: 100 mL/min). **Figure 9** shows the crystallization and melting temperature of Cu/Cu@Ag nanowires‐UHMWPE nanocompos‐ ites. It is seen from **Figure 9a** that Cu nanowires have a strong influence on the crystallization and melting temperature of UHMWPE matrix. When the content of Cu nanowires is 0.1 wt.%, the crystallization temperature of nanocomposite rises from 118.10 to 118.32**°**C, which could be because that the heterogeneous nucleation in the presence of copper nanowire served as nucleating agent contributes to improving the crystallinity of UHMWPE matrix. With the increasing of nanowires content, the crystallization temperature significantly decreases, which indicates that Cu nanowires hinder the orientation of PA6 crystals and restrict the movement of the molecular chains of UHMWPE. When the nanowires content is further increased, the effect of Cu nanowires on the crystallization and melting behaviors tends to decline, possi‐ bly due to the aggregation of the nanofillers in the polymer matrix. When the Cu nanowires content reaches 3 wt.%, however, the crystallization temperature decreases again, probably because that the interaction between excess nanowires and molecular chains of UHMWPE is enhanced to restrict the movement of the molecular chains. Correspondingly, a change in melting temperature occurs with the increasing of nanowires content.

Interestingly, the Cu@Ag nanowires have opposite effects on the crystallization and the melting temperature as compared with Cu nanowires, which could be because the Cu@Ag nanowires

**Table 2.** The component and dosage for the preparation of Cu/Cu@Ag nanowires‐UHMWPE composites.

Synthesis and Application of Copper Nanowires and Silver Nanosheet-Coated Copper Nanowires... http://dx.doi.org/10.5772/67782 11

**5. Cu/Cu@Ag nanowires‐UHMWPE nanocomposites**

sheets.

**nanocomposites**

10 Nanowires - New Insights

a heating rate of 10**°**C/min under N2

**Component Mass fraction (%)**

Cu/Cu@Ag nanowires (g)

The component and dosage for the preparation of Cu@Ag nanowires‐UHMWPE compos‐ ites are listed in **Table 2**. The nanowires and UHMWPE raw material were mixed with ball milling for 1 h. The mixture was heated to 200°C and pressed under 20 MPa for 10–20 min, followed by cooling to room temperature in the mold to afford the Cu@Ag nanowires‐ UHMWPE nanocomposites. All the samples for performance test were cut from the final

The crystallization and melting behavior of Cu/Cu@Ag nanowires‐UHMWPE nanocom‐ posites were studied by differential scanning calorimetry (DSC). DSC curves in the range of 20–200**°**C were measured with a NETZSCH DSC 204HP instrument (Bavarian, Germany) at

crystallization and melting temperature of Cu/Cu@Ag nanowires‐UHMWPE nanocompos‐ ites. It is seen from **Figure 9a** that Cu nanowires have a strong influence on the crystallization and melting temperature of UHMWPE matrix. When the content of Cu nanowires is 0.1 wt.%, the crystallization temperature of nanocomposite rises from 118.10 to 118.32**°**C, which could be because that the heterogeneous nucleation in the presence of copper nanowire served as nucleating agent contributes to improving the crystallinity of UHMWPE matrix. With the increasing of nanowires content, the crystallization temperature significantly decreases, which indicates that Cu nanowires hinder the orientation of PA6 crystals and restrict the movement of the molecular chains of UHMWPE. When the nanowires content is further increased, the effect of Cu nanowires on the crystallization and melting behaviors tends to decline, possi‐ bly due to the aggregation of the nanofillers in the polymer matrix. When the Cu nanowires content reaches 3 wt.%, however, the crystallization temperature decreases again, probably because that the interaction between excess nanowires and molecular chains of UHMWPE is enhanced to restrict the movement of the molecular chains. Correspondingly, a change in

Interestingly, the Cu@Ag nanowires have opposite effects on the crystallization and the melting temperature as compared with Cu nanowires, which could be because the Cu@Ag nanowires

**0 0.1 0.3 0.5 0.8 1.0 3.0**

0 0.120 0.361 0.603 0.968 1.21 3.71

UHMWPE (g) 120 120 120 120 120 120 120

**Table 2.** The component and dosage for the preparation of Cu/Cu@Ag nanowires‐UHMWPE composites.

atmosphere (flow rate: 100 mL/min). **Figure 9** shows the

**5.1. Crystallization and melting behavior of Cu/Cu@Ag nanowires‐UHMWPE**

melting temperature occurs with the increasing of nanowires content.

**Figure 9.** The crystallization and melting temperature of Cu/Cu@Ag nanowires‐UHMWPE nanocomposites with different nanowires contents.

with unique morphology and microstructure exhibit significantly increased contact area with the UHMWPE matrix.

#### **5.2. Mechanical properties of Cu/Cu@Ag nanowires‐UHMWPE nanocomposites**

The effect of Cu/Cu@Ag nanowires on the mechanical properties of UHMWPE is shown in **Figure 10**. We find that the elongation at break of composite materials gradually increases with increasing dosage of Cu/Cu@Ag nanowires; and in particular, the UHMWPE‐based nano‐ composites with 0.8 wt.% of Cu nanowires or with 0.3 wt.% of Cu@Ag nanowires possesses the maximum values of elongation at break (168 and 190%, respectively), larger than that of unfilled UHMWPE by 78.5 and 101.9% (**Figure 10a**). Besides, the elongation rate at break of the UHMWPE‐based nanocomposites tends to decline with further increase of nanowires content, possibly due to the aggregation of the nanowires thereat. Moreover, the elongation rate at break of Cu@Ag nanowires‐UHMWPE is higher than that of Cu nanowires‐UHMWPE, which could be closely related to the special morphology and microstructure of the Cu@Ag nanowires. Furthermore, the UHMWPE‐based nanocomposites change slightly with increas‐ ing dosage of Cu/Cu@Ag nanowires. The UHMWPE‐based nanocomposites with 0.3 wt.% of Cu nanowires or with 0.5 wt.% of Cu@Ag nanowires possess the maximum values of tensile

**Figure 10.** The mechanical properties of Cu/Cu@Ag nanowires‐UHMWPE nanocomposites with different nanowires contents.

strength of 26.2 and 22.6 MPa, respectively, showing an increase by 27.1 and 9.7% in compari‐ son with pure UHMWPE (20.6 MPa; **Figure 10b**).

## **5.3. Tribology properties of Cu/Cu@Ag nanowires‐UHMWPE nanocomposites**

**Figure 11** shows the friction coefficient and wear rate of Cu/Cu@Ag nanowires‐UHMWPE nanocomposites (load: 250 N; route: 5 mm; frequency: 2Hz; time: 5 min; room temperature). For Cu nanowires‐UHMWPE nanocomposites (**Figure 11a**), the friction coefficient and wear rate of Cu nanowires‐UHMWPE nanocomposites gradually decrease with increasing dosage of Cu nanowires; and the friction coefficient is rather large when the Cu nanowires content is below 0.3 wt.%. This may be because that when the content of Cu nanowires is too low to play its role on the friction‐reducing and wear resistance in the friction process, thereby leading to relatively not very good friction property. Cu@Ag nanowires‐UHMWPE nanocomposites show similar variation tendency as Cu nanowires‐UHMWPE nanocomposites (**Figure 11b**) except the one with 3 wt.% of Cu@Ag nanowires is somewhat different.

## **6. Cu/Cu@Ag nanowires‐EP nanocomposites**

The Cu/Cu@Ag nanowires‐EP (epoxy resin) nanocomposites were prepared by mixing the raw materials with high‐speed mixers (1500 rpm, 3 min) in association with curing in the mold under ambient conditions. The content of each component is shown in **Table 3**.

## **6.1. The electrical conductivity Cu/Cu@Ag nanowires‐EP nanocomposites**

In general, the electrical conductivity of silver and copper is similar. The special structure of Cu@Ag nanowires makes it feasible to significantly improve the electrical conductivity of Cu nanowires by incorporating Ag shell because of their better oxidation resistance and more conductive network in the polymer matrix nanocomposites. We measured the electrical resistivity of EP‐matrix composites filled with Cu and Cu@Ag nanowires using a Hall effect

**Figure 11.** The friction and wear behavior (friction coefficient and wear rate) of UHMWPE nanocomposites with different contents of (a) Cu nanowires and (b) Cu@Ag nanowires.

strength of 26.2 and 22.6 MPa, respectively, showing an increase by 27.1 and 9.7% in compari‐

**Figure 11** shows the friction coefficient and wear rate of Cu/Cu@Ag nanowires‐UHMWPE nanocomposites (load: 250 N; route: 5 mm; frequency: 2Hz; time: 5 min; room temperature). For Cu nanowires‐UHMWPE nanocomposites (**Figure 11a**), the friction coefficient and wear rate of Cu nanowires‐UHMWPE nanocomposites gradually decrease with increasing dosage of Cu nanowires; and the friction coefficient is rather large when the Cu nanowires content is below 0.3 wt.%. This may be because that when the content of Cu nanowires is too low to play its role on the friction‐reducing and wear resistance in the friction process, thereby leading to relatively not very good friction property. Cu@Ag nanowires‐UHMWPE nanocomposites show similar variation tendency as Cu nanowires‐UHMWPE nanocomposites (**Figure 11b**)

The Cu/Cu@Ag nanowires‐EP (epoxy resin) nanocomposites were prepared by mixing the raw materials with high‐speed mixers (1500 rpm, 3 min) in association with curing in the

In general, the electrical conductivity of silver and copper is similar. The special structure of Cu@Ag nanowires makes it feasible to significantly improve the electrical conductivity of Cu nanowires by incorporating Ag shell because of their better oxidation resistance and more conductive network in the polymer matrix nanocomposites. We measured the electrical resistivity of EP‐matrix composites filled with Cu and Cu@Ag nanowires using a Hall effect

**Figure 11.** The friction and wear behavior (friction coefficient and wear rate) of UHMWPE nanocomposites with different

mold under ambient conditions. The content of each component is shown in **Table 3**.

**6.1. The electrical conductivity Cu/Cu@Ag nanowires‐EP nanocomposites**

**5.3. Tribology properties of Cu/Cu@Ag nanowires‐UHMWPE nanocomposites**

except the one with 3 wt.% of Cu@Ag nanowires is somewhat different.

**6. Cu/Cu@Ag nanowires‐EP nanocomposites**

contents of (a) Cu nanowires and (b) Cu@Ag nanowires.

son with pure UHMWPE (20.6 MPa; **Figure 10b**).

12 Nanowires - New Insights

**Table 3.** The component and dosage for the preparation of Cu nanowires‐ and Cu@Ag nanowires‐EP composites.

measurement system. The electrical resistivity of pure EP is about 1014 Ω cm, while the intro‐ duction of Cu/Cu@Ag nanowires results in greatly reduced electrical resistivity (**Figure 12**). Namely, the electrical resistivity decreases obviously with increasing content of Cu or Cu@ Ag nanowires, and the EP‐matrix nanocomposites filled with Cu@Ag nanowires exhibit lower electrical resistivity than the counterparts filled with Cu nanowires. For example, the resis‐ tivity of the EP composite containing 3 wt.% Cu nanowires is about 1.49 × 105 Ω cm, and it does not satisfy the conductive requirement for electrostatic conductive materials (104 Ω cm). The resistivity of Cu@Ag nanowires‐EP composite (filler mass fraction: 3 wt.%) is reduced to 2.11 × 104 Ω cm, and it satisfies the conductive requirement for electrostatic conductive materials.

**Figure 12.** The electrical resistivity of Cu/Cu@Ag nanowires‐EP nanocomposites with different nanowires contents.

In other words, encapsulating Cu nanowires with Ag nanosheets makes it feasible for the cop‐ per nanowires to be applied as potential electrostatic conductive filler, thereby broadening their application scope.

#### **6.2. Mechanical properties of Cu/Cu@Ag nanowires‐EP nanocomposites**

The tensile strength and the elongation at break of pure EP and its nanocomposites filled with Cu nanowires or Cu@Ag nanowires are shown in **Figure 13**. Both the tensile strength and elongation at break increase after the introduction of Cu nanowires and Cu@Ag nanowires. Particularly, the EP‐matrix nanocomposite with 0.8 wt.% of Cu nanowires exhibits the maxi‐ mum tensile strength of 74.2 MPa, while the one with 1 wt.% of Cu@Ag nanowires exhibits the maximum tensile strength of 67.5 MPa. In addition, compared with the change of the elon‐ gation at break values, the tensile strength has little change with the increase of nanowires content. When the content of the nanowires is too high, the tensile strength and elongation at break are relatively low, which could be due to the aggregation of the nanowires in EP matrix at a high content.

#### **6.3. Tribology properties of Cu/Cu@Ag nanowires‐EP nanocomposites**

**Figure 14** shows the variation in the friction coefficient and wear rate of Cu/Cu@Ag nanow‐ ires‐EP composites with the content of the nanofillers (load: 20 N; amplitude: 5 mm; fre‐ quency: 2Hz; time: 30 min; room temperature). Unfilled EP has a friction coefficient and wear rate of 0.646 and 4.82 × 10−3 mm3 /N m. Namely, the EP‐matrix nanocomposites with less than 0.5 wt.% of Cu nanowires have reduced friction coefficient and wear rate than neat EP; and in particular, the EP‐matrix composite with 0.1% of Cu nanowires exhibits the best friction coefficient and wear rate (0.555, 1.42 × 10−3 mm3 /N m; a reduction by 14.1 and 70.5% as compared with that of neat EP). However, the increase in the content of the Cu nanowires above 0.5 wt.% results in increased friction coefficient and wear rate, due to aggregation of the nanofillers thereat. Similar variation tendency is also observed for Cu@Ag nanowires‐EP nanocomposites.

**Figure 13.** The mechanical properties of Cu/Cu@Ag nanowires‐EP nanocomposites with different nanowires content.

Synthesis and Application of Copper Nanowires and Silver Nanosheet-Coated Copper Nanowires... http://dx.doi.org/10.5772/67782 15

**Figure 14.** The friction and wear behavior (friction coefficient and wear rate) of EP‐matrix nanocomposites with different contents of (a) Cu nanowires and (b) Cu@Ag nanowires.

## **7. Conclusion**

In other words, encapsulating Cu nanowires with Ag nanosheets makes it feasible for the cop‐ per nanowires to be applied as potential electrostatic conductive filler, thereby broadening their

The tensile strength and the elongation at break of pure EP and its nanocomposites filled with Cu nanowires or Cu@Ag nanowires are shown in **Figure 13**. Both the tensile strength and elongation at break increase after the introduction of Cu nanowires and Cu@Ag nanowires. Particularly, the EP‐matrix nanocomposite with 0.8 wt.% of Cu nanowires exhibits the maxi‐ mum tensile strength of 74.2 MPa, while the one with 1 wt.% of Cu@Ag nanowires exhibits the maximum tensile strength of 67.5 MPa. In addition, compared with the change of the elon‐ gation at break values, the tensile strength has little change with the increase of nanowires content. When the content of the nanowires is too high, the tensile strength and elongation at break are relatively low, which could be due to the aggregation of the nanowires in EP matrix

**Figure 14** shows the variation in the friction coefficient and wear rate of Cu/Cu@Ag nanow‐ ires‐EP composites with the content of the nanofillers (load: 20 N; amplitude: 5 mm; fre‐ quency: 2Hz; time: 30 min; room temperature). Unfilled EP has a friction coefficient and

less than 0.5 wt.% of Cu nanowires have reduced friction coefficient and wear rate than neat EP; and in particular, the EP‐matrix composite with 0.1% of Cu nanowires exhibits the best

as compared with that of neat EP). However, the increase in the content of the Cu nanowires above 0.5 wt.% results in increased friction coefficient and wear rate, due to aggregation of the nanofillers thereat. Similar variation tendency is also observed for Cu@Ag nanowires‐EP

**Figure 13.** The mechanical properties of Cu/Cu@Ag nanowires‐EP nanocomposites with different nanowires content.

/N m. Namely, the EP‐matrix nanocomposites with

/N m; a reduction by 14.1 and 70.5%

**6.2. Mechanical properties of Cu/Cu@Ag nanowires‐EP nanocomposites**

**6.3. Tribology properties of Cu/Cu@Ag nanowires‐EP nanocomposites**

application scope.

14 Nanowires - New Insights

at a high content.

nanocomposites.

wear rate of 0.646 and 4.82 × 10−3 mm3

friction coefficient and wear rate (0.555, 1.42 × 10−3 mm3

A large amount of Cu nanowires is prepared by a solution‐reduction approach, while Cu@Ag nanowires are prepared by a simple and efficient transmetalation reaction method. The mor‐ phology and microstructure of the as‐prepared Cu@Ag nanowires can be well manipulated by properly adjusting the reaction condition. Resultant Cu@Ag nanowires consist of Cu nanocores and uniformly distributed Ag nanosheet shell, and they exhibit improved thermal stability and electrical conductivity as compared with Cu nanowires, due to the incorporation of the Ag shell. Besides, the as‐prepared Cu@Ag nanowires can better improve the mechanical properties and wear resistance than the Cu nanowires.

## **Acknowledgements**

We acknowledge the financial support provided by National Natural Science Foundation of China (Grant no. 51275154, 51405132, 21671053), the Plan for Young Scientific Innovation Talent of Henan Province (Grant no.<PHONE_NUMBER>18) and Natural Science Foundation of Henan Province (14A150006).

## **Author details**

Ningning Zeng1 , Shuguang Fan1 , Jingyi Ma1 , Yujuan Zhang1 , Shengmao Zhang1,2\*, Pingyu Zhang1 and Zhijun Zhang1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Engineering Research Center for Nanomaterials, Henan University, Kaifeng, P. R. China

2 Collaborative Innovation Center of Nano Functional Materials and Applications of Henan Province, Henan University, Kaifeng, P. R. China

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**2.1 Stem cells: The perspective has evolved**

Pluripotent embryonic stem cells (ESC) are derived from the inner cell mass of an embryonic blastocyst (primitive ectoderm). The *pluri*potency of ESC defines that they are capable of differentiation into one of the three germ layers: ectoderm, mesoderm, or endoderm; reviewed in (Rossant, 2008; Bolli & Chaudhry, 2010). Embryonic mesoderm cells (identified by the expression of Brachyury T) in the developing primary heart field undergo committed differentiation into a pre-cardiac lineage (expressing Mesp-1) and have the potential to form committed cardiac progenitor cells (CPC, expressing Nkx2.5), which are capable of differentiation into cardiomyocytes, vascular smooth muscle cells, and endothelial cells; reviewed in (Sturzu & Wu, 2011). A cardiac progenitor cell is thus defined as a *multi*potent progenitor cell of the pre-cardiac lineage.

Originally it was thought that only ESC are pluripotent, and they could only be obtained from living embryos. Consequently, studies of human ESC have been impeded by ethical concerns. However, continued research has led to the realization that pluripotency is in fact a plastic multidirectional state. It has now been clearly demonstrated that a pluripotent state can be induced in adult somatic cells from humans, and other species; reviewed in Yamanaka (Yamanaka & Blau, 2010). The biochemical methods through which so-called induced pluripotent cells (iPSC) may be derived from adult cells are numerous and still expanding. Several recent reviews (Rossant, 2008; Yamanaka & Blau, 2010) of both the technology and the biology of iPSC may be consulted for the interested reader; the topic will not be further discussed here.

Myocardial Self-Repair and Congenital Heart Disease 277

(Tsuchihashi et al., 2011). Cai (Cai et al., 2003) reported that Isl1+ cells are mostly localized in the SHF and give rise to the outflow tract. They demonstrated that disruption of Isl1 results in a complete failure to form the outflow tract in the mutant mice. The role of Fgf8 in early and late heart development has also been studied (Sun et al., 1999; Abu-Issa et al., 2002). Fgf8 is required for migration of mesoderm out of the primitive streak (Sun et al., 1999). In addition Fgf8 plays an important role in development of all the pharyngeal arches and in NCC survival (Abu-Issa et al., 2002). Cardiac NCC are multipotent and after migration to the SHF contribute to cardiovascular patterning (Kirby & Waldo, 1995). Ablation of the pre-migratory cardiac NCC causes numerous outflow tract septation defects: persistent truncus arteriosus, doubleoutlet right ventricle, tetralogy of Fallot, double-inlet left ventricle, tricuspid atresia, straddling tricuspid valve, and the absence of a varying combination of aortic arch arteries derived from pharyngeal arches 3, 4 and 6 (Nishibatake et al., 1987; Hutson & Kirby, 2007). Studies of interactions between cells of SHF origin with NCC in the pharyngeal region of mice (Vitelli et al., 2002; Moraes et al., 2005) have shown that mutation in Tbx1 disrupts formation of the pharyngeal arch arteries and causes septation defects due to failure in NCC migration. Bradshaw (Bradshaw et al., 2009) showed that abnormal distribution of Isl1-expressing cells in a neural crest-deficient mutant mouse causes instability of posterior arch arteries and outflow tract septation defects, leading to a double outlet right ventricle. Discovery of the presence and role of CPC in the normal and abnormal development of heart fields and their possible role in postnatal life suggests that targeting these cells is a valuable approach for both understanding

The implications of resident CPC expressing neural markers in the heart are manifold for congenital heart disease repair. First, the secondary heart field of cells creates the structures most affected by congenital disease: the outflow tracts and pulmonary and aortic trunks (Dyer & Kirby, 2009; Jain et al., 2010). Second, the resident CPC-derived from this field (i.e. Isl1+ or Nestin+ CPC) may form the primary cardiac stem cell pool that would potentially be mobilized to participate in the repair (cell replacement) of structures derived from the anterior/secondary heart field such as the OFT. Thirdly, therapeutics aimed at CPC mobilization may act more effectively on the right heart if they target NCC-derived CPC.

Resident CPC have been localized in small groups or clusters in a unique microenvironment known as a "stem cell niche" which provides conditions for stem cells to maintain their multipotency and renewal capacity (Morrison & Spradling, 2008). Niches comprise a specific arrangement of stem cells, supporting cells, and extracellular matrix. The niche environment provides a paracrine signaling influence that maintains stem cells in their quiescent state and also responds to conditions outside the niche to initiate activation of stem cell replication and/or differentiation, for example in response to tissue injury (Morrison & Spradling, 2008). Classically, three well-characterized stem cell niches have been identified: germline, hematopoietic, and epithelial (Lemischka, 1997; Xie & Spradling, 2000; Spradling et al., 2001). However in the past decade, identification of resident stem cells in most mammalian organs suggests local tissue-specific niches are a general rule. Discovery of CPC niches in the adult mouse heart strengthens the growing appreciation that the heart is not a terminally differentiated organ and resident CPC might play a role in the postnatal remodeling and repair of cardiac tissue (Urbanek et al., 2006). The early study of Messina (Messina et al., 2004) demonstrated that cardiac stem cells are present in both atrial and ventricular human samples and in a wide range of ages (1 to 80 years old). According to this

the ontology of CHD and developing therapeutic approaches.

**2.2.3 Postnatal distribution of CPC in the heart**

A variety of techniques have been used to induce the differentiation of ESC into a specific cell type of interest. The directed differentiation of ESC into cardiomyocytes has been achieved by several methods and has enabled the use of this approach to produce a large enough number of cells for repairing the injured heart to restore myocardial function (Gonzales & Pedrazzini, 2009; Laflamme & Murry, 2011). A large number of studies have been conducted and are still being conducted with the goal of restoring the function of infarcted myocardium using stem cell therapy with ESC-derived cardiomyocytes and also other cell types, with the expectation that they would continue to differentiate into cardiomyocytes after being delivered to the injured heart tissue (Murry et al., 2005). As this therapy is less relevant to *congenital* heart disease, it will not be further discussed here.

#### **2.2 How cardiac progenitor cells form the developing heart**

A brief review of cardiac development illustrates how cardiac stem cells are identified and how multiple distinct populations of pre-cardiac cells coalesce to form the distinct subregions of the heart.

#### **2.2.1 Heart fields and cardiac progenitor cells**

The development of the multi-chambered heart is complex and requires a precise regulation of cell migration, proliferation, and differentiation in a highly organized positional and temporal order. Several distinct CPC populations contribute to the formation of different heart fields. The first heart field (FHF) of cardiac progenitor cells is localized in the primitive streak and mostly contributes to formation of the left ventricle and part of the atria (Garcia-Martinez & Schoenwolf, 1993; Tam et al., 1997). A second heart field (SHF) of CPC from the pharyngeal mesoderm migrates into the arterial pole of the heart and is a major source of the cardiac progenitors that form the outflow tract (OFT), the right ventricle and the atria (Abu-Issa & Kirby, 2007). Continued development of the OFT and heart valves is achieved by migration of a population of non-mesodermal neural crest cells (NCC) from the neural fold into the arterial pole and endocardial cushion (Kirby & Waldo, 1995; Hutson & Kirby, 2007). Another population of mesenchymal CPC, the proepicardium, has been demonstrated to be an additional cell source contributing to the formation of coronary arteries and cardiac fibroblasts (Dettman et al., 1998). CPC from both FHF and SHF act in a close collaboration during the formation of the embryonic heart. During heart morphogenesis, CPC directed by different signaling pathways differentiate to mature cardiac cells. However, CPC of the postnatal heart have been shown to express markers common to both FHF and SHF. Whether these postnatal resident CPC contribute to remodeling and growth of the heart is considered likely but is still unsettled.

#### **2.2.2 Second heart field and congenital heart disease**

Studies of heart development in mice have shown that mutation of genes expressed by cells of the SHF causes congenital heart disease (CHD). Mutations in Nkx2.5 cause a spectrum of congenital heart defects including cardiac conduction abnormalities and ventricular- and atrial- septal defects (VSD, ASD) (Basson et al., 1997). Deletion of Tbx1 results in malformations of the cardiac outflow tract and VSDs due to failure in the migration of NCC to the heart (Jerome & Papaioannou, 2001; Merscher et al., 2001). Mutations in GATA4, some of which disrupt its interaction with Tbx5, cause ASDs and VSDs (Garg et al., 2003). Hand2 is essential for survival of second heart field progenitors and loss of Hand*2* function in this cardiac progenitor population can cause a spectrum of congenital heart malformations

A variety of techniques have been used to induce the differentiation of ESC into a specific cell type of interest. The directed differentiation of ESC into cardiomyocytes has been achieved by several methods and has enabled the use of this approach to produce a large enough number of cells for repairing the injured heart to restore myocardial function (Gonzales & Pedrazzini, 2009; Laflamme & Murry, 2011). A large number of studies have been conducted and are still being conducted with the goal of restoring the function of infarcted myocardium using stem cell therapy with ESC-derived cardiomyocytes and also other cell types, with the expectation that they would continue to differentiate into cardiomyocytes after being delivered to the injured heart tissue (Murry et al., 2005). As this therapy is less relevant to *congenital* heart disease, it will not be further discussed here.

A brief review of cardiac development illustrates how cardiac stem cells are identified and how multiple distinct populations of pre-cardiac cells coalesce to form the distinct

The development of the multi-chambered heart is complex and requires a precise regulation of cell migration, proliferation, and differentiation in a highly organized positional and temporal order. Several distinct CPC populations contribute to the formation of different heart fields. The first heart field (FHF) of cardiac progenitor cells is localized in the primitive streak and mostly contributes to formation of the left ventricle and part of the atria (Garcia-Martinez & Schoenwolf, 1993; Tam et al., 1997). A second heart field (SHF) of CPC from the pharyngeal mesoderm migrates into the arterial pole of the heart and is a major source of the cardiac progenitors that form the outflow tract (OFT), the right ventricle and the atria (Abu-Issa & Kirby, 2007). Continued development of the OFT and heart valves is achieved by migration of a population of non-mesodermal neural crest cells (NCC) from the neural fold into the arterial pole and endocardial cushion (Kirby & Waldo, 1995; Hutson & Kirby, 2007). Another population of mesenchymal CPC, the proepicardium, has been demonstrated to be an additional cell source contributing to the formation of coronary arteries and cardiac fibroblasts (Dettman et al., 1998). CPC from both FHF and SHF act in a close collaboration during the formation of the embryonic heart. During heart morphogenesis, CPC directed by different signaling pathways differentiate to mature cardiac cells. However, CPC of the postnatal heart have been shown to express markers common to both FHF and SHF. Whether these postnatal resident CPC contribute to remodeling and growth of the heart is

Studies of heart development in mice have shown that mutation of genes expressed by cells of the SHF causes congenital heart disease (CHD). Mutations in Nkx2.5 cause a spectrum of congenital heart defects including cardiac conduction abnormalities and ventricular- and atrial- septal defects (VSD, ASD) (Basson et al., 1997). Deletion of Tbx1 results in malformations of the cardiac outflow tract and VSDs due to failure in the migration of NCC to the heart (Jerome & Papaioannou, 2001; Merscher et al., 2001). Mutations in GATA4, some of which disrupt its interaction with Tbx5, cause ASDs and VSDs (Garg et al., 2003). Hand2 is essential for survival of second heart field progenitors and loss of Hand*2* function in this cardiac progenitor population can cause a spectrum of congenital heart malformations

**2.2 How cardiac progenitor cells form the developing heart**

**2.2.1 Heart fields and cardiac progenitor cells**

considered likely but is still unsettled.

**2.2.2 Second heart field and congenital heart disease**

subregions of the heart.

(Tsuchihashi et al., 2011). Cai (Cai et al., 2003) reported that Isl1+ cells are mostly localized in the SHF and give rise to the outflow tract. They demonstrated that disruption of Isl1 results in a complete failure to form the outflow tract in the mutant mice. The role of Fgf8 in early and late heart development has also been studied (Sun et al., 1999; Abu-Issa et al., 2002). Fgf8 is required for migration of mesoderm out of the primitive streak (Sun et al., 1999). In addition Fgf8 plays an important role in development of all the pharyngeal arches and in NCC survival (Abu-Issa et al., 2002). Cardiac NCC are multipotent and after migration to the SHF contribute to cardiovascular patterning (Kirby & Waldo, 1995). Ablation of the pre-migratory cardiac NCC causes numerous outflow tract septation defects: persistent truncus arteriosus, doubleoutlet right ventricle, tetralogy of Fallot, double-inlet left ventricle, tricuspid atresia, straddling tricuspid valve, and the absence of a varying combination of aortic arch arteries derived from pharyngeal arches 3, 4 and 6 (Nishibatake et al., 1987; Hutson & Kirby, 2007). Studies of interactions between cells of SHF origin with NCC in the pharyngeal region of mice (Vitelli et al., 2002; Moraes et al., 2005) have shown that mutation in Tbx1 disrupts formation of the pharyngeal arch arteries and causes septation defects due to failure in NCC migration. Bradshaw (Bradshaw et al., 2009) showed that abnormal distribution of Isl1-expressing cells in a neural crest-deficient mutant mouse causes instability of posterior arch arteries and outflow tract septation defects, leading to a double outlet right ventricle. Discovery of the presence and role of CPC in the normal and abnormal development of heart fields and their possible role in postnatal life suggests that targeting these cells is a valuable approach for both understanding the ontology of CHD and developing therapeutic approaches.

The implications of resident CPC expressing neural markers in the heart are manifold for congenital heart disease repair. First, the secondary heart field of cells creates the structures most affected by congenital disease: the outflow tracts and pulmonary and aortic trunks (Dyer & Kirby, 2009; Jain et al., 2010). Second, the resident CPC-derived from this field (i.e. Isl1+ or Nestin+ CPC) may form the primary cardiac stem cell pool that would potentially be mobilized to participate in the repair (cell replacement) of structures derived from the anterior/secondary heart field such as the OFT. Thirdly, therapeutics aimed at CPC mobilization may act more effectively on the right heart if they target NCC-derived CPC.

#### **2.2.3 Postnatal distribution of CPC in the heart**

Resident CPC have been localized in small groups or clusters in a unique microenvironment known as a "stem cell niche" which provides conditions for stem cells to maintain their multipotency and renewal capacity (Morrison & Spradling, 2008). Niches comprise a specific arrangement of stem cells, supporting cells, and extracellular matrix. The niche environment provides a paracrine signaling influence that maintains stem cells in their quiescent state and also responds to conditions outside the niche to initiate activation of stem cell replication and/or differentiation, for example in response to tissue injury (Morrison & Spradling, 2008). Classically, three well-characterized stem cell niches have been identified: germline, hematopoietic, and epithelial (Lemischka, 1997; Xie & Spradling, 2000; Spradling et al., 2001). However in the past decade, identification of resident stem cells in most mammalian organs suggests local tissue-specific niches are a general rule. Discovery of CPC niches in the adult mouse heart strengthens the growing appreciation that the heart is not a terminally differentiated organ and resident CPC might play a role in the postnatal remodeling and repair of cardiac tissue (Urbanek et al., 2006). The early study of Messina (Messina et al., 2004) demonstrated that cardiac stem cells are present in both atrial and ventricular human samples and in a wide range of ages (1 to 80 years old). According to this

Myocardial Self-Repair and Congenital Heart Disease 279

In the postnatal heart, identification of CPC is based primarily on the cardiac lineage markers Nkx2.5, Mef2c, and Isl1. Identification of c-Kit+ cells in adult mammalian heart suggests another postnatal source of CPC, however, the origin of these cells is unsettled. Although expression of the key CPC markers is preserved both in rodents and humans, species-specific markers limit translation of animal studies to human studies. Our studies of human infant RVOFT myocardium showed a mixed population of c-Kit+, Isl1+, and Nkx2.5+ CPC (Amir et al., 2008). The neonatal human heart is a rich source of CPC bearing markers such as SSEA4, Isl1, Nkx2.5, and c-Kit. In addition to our report of myocardial SSEA4 localization(Amir et al., 2008), SSEA4 expression has been previously only reported in adult human kidney (Ward et al., 2011). Isl1+ CPC have also been localized to the atrium (Laugwitz et al., 2005). Stem cell antigen-1 (Sca-1) is a marker for mouse resident CPC. In the mouse and human studies performed by Messina (Messina et al., 2004) and Beltrami (Beltrami et al., 2003) c-Kit+ cells are considered to be CPC, whereas Oh (Oh et al., 2003)

, Sca-1+ cells as a marker for CPC. This apparent controversy may be explained by

the existence of heterogeneous pools of cardiac stem cells in different stages of differentiation rather than multiple populations of distinct resident CPC. In addition, the anatomical location (i.e., heart field of origin) of the heart regions studied may determine the

Fig. 1. Overview of myocardial CPC Markers in relation to their ontology.

SHF, Second heart field; FHF, First heart field.

Pre-cardiac mesoderm cells express Mesp1 until differentiation to cardiogenic mesoderm, marked by the expression of Nkx2.5. The cardiogenic mesoderm differentiates to form two heart fields. Cells in the first heart field (FHF) express GATA4 in addition to Nkx2.5. Cells of the secondary heart field (SHF) express Isl1 and Nkx2.5. The SHF also becomes populated by neural crest cells (NCC) expressing Pax3. Markers associated with the heart field cells but showing greater variation in their expression are indicated in blue type. In addition to cardiogenic mesoderm, cells from the pro-epicardial organ and also of hematopoietic lineage have been identified in the myocardium. Abbreviations: NCC, Neural crest cells;

**<IP_ADDRESS> Markers of CPC in the postnatal heart**

markers expressed by postnatal CPC.

used c-Kit-

study and mouse studies by Beltrami (Beltrami et al., 2003) and Oh (Oh et al., 2003), cardiac stem cells can be isolated from adult myocardium and differentiated into different cardiac cell lineages. Our own studies (Amir et al., 2008) have investigated the number and characteristics of CPC in human neonatal myocardium from patients with congenital heart disease. We demonstrated that CPC comprise a high percentage relative to cardiomyocytes in the human neonate and that their fractional representation declines as the heart grows in size, suggesting a dilutional effect. In other words, an apparently fixed population of CPC resides in the heart postnatally. Pouly (Pouly et al., 2008) reported that CPC concentration in the right atrium is greater than that of the septum of transplanted hearts. Other myocardial niches have been reported. Schenke-Layland (Schenke-Layland et al., 2011) reported that in human and mouse heart endogenous, multipotent Isl1+/Flk1+ CPC reside within niche clusters in the right ventricular free wall, the atria and outflow tracts. They were tightly circumscribed by the basement membrane proteins collagen V and laminin. However, systematic mapping of the complete heart to localize major niches and determine the distribution of these regions of high CPC density has not been reported. A general concept is that cardiac regions protected from greatest mechanical stress (atria, septum, apex) appear to contain the highest density of CPC. A more useful perspective may be consideration of the cardiac developmental sequence in which fields of CPC undergo migration and strategically controlled distribution, since the niches may constitute remnants of specific precursor fields in the primordial heart anlagen.

The role of niche-associated cells in regulating the stability and activation of resident stem cells is still poorly understood, especially in the heart. Recently, it has been shown that Notch-1 of murine CPC interacts with surrounding cells via the Jagged ligand. Through a Jagged-mediated activation signaling process induced in vitro, notch signaling, down regulation of c-Kit and upregulation of Nkx2.5 were found to be associated with increased myocyte proliferation (Boni et al., 2008). These data support the concept that interaction of CPC with supporting cells and matrix regulates their commitment to the myocyte lineage.

#### **2.2.4 Postnatal Characterization of CPC fates**

#### **<IP_ADDRESS> Prenatal formation of the heart from different fields**

During heart development CPC express field-specific markers **(Figure 1.**) for FHF, SHF, and neural crest (Vincent & Buckingham, 2010). Mesp1 is one of the earliest markers for cardiac primordial cells (Saga et al., 1999). Depending on the stage of cell differentiation, genetic markers will be upregulated or down regulated in the CPC, which complicates clear classification of CPC originating from FHF and SHF based on markers. Nkx2.5 is a critical cardiac transcription factor in the first lineage, and Isl1 along with Foxh1, GATA factors, and Hand2 are key regulators in the second heart progenitor field (Moretti et al., 2006). The earliest differentiated cells in the cardiac crescent express GATA4 and Nkx2.5 (Vincent & Buckingham, 2010). The final differentiation to cardiomyocytes is controlled by Tbx (Takeuchi & Bruneau, 2009). Known markers for SHF CPC are Isl1, Tbx1 and Fgf10 (Kelly et al., 2001; Cai et al., 2003; Xu et al., 2004). Pax3 is recognized as a major regulatory gene for NCC, having an important role in their migration (Bradshaw et al., 2009). There is considerable overlap between the heart field-specific genetic markers. This issue is more evident in the case of the proepicardial organ, which gives rise to coronary arteries and epicardium. Both Isl1 and Nkx2.5 are expressed in the CPC from the proepicardial organ (Dettman et al., 1998), the cells of which also express Tbx18 and Wt1 (Martinez-Estrada et al., 2010).

study and mouse studies by Beltrami (Beltrami et al., 2003) and Oh (Oh et al., 2003), cardiac stem cells can be isolated from adult myocardium and differentiated into different cardiac cell lineages. Our own studies (Amir et al., 2008) have investigated the number and characteristics of CPC in human neonatal myocardium from patients with congenital heart disease. We demonstrated that CPC comprise a high percentage relative to cardiomyocytes in the human neonate and that their fractional representation declines as the heart grows in size, suggesting a dilutional effect. In other words, an apparently fixed population of CPC resides in the heart postnatally. Pouly (Pouly et al., 2008) reported that CPC concentration in the right atrium is greater than that of the septum of transplanted hearts. Other myocardial niches have been reported. Schenke-Layland (Schenke-Layland et al., 2011) reported that in human and mouse heart endogenous, multipotent Isl1+/Flk1+ CPC reside within niche clusters in the right ventricular free wall, the atria and outflow tracts. They were tightly circumscribed by the basement membrane proteins collagen V and laminin. However, systematic mapping of the complete heart to localize major niches and determine the distribution of these regions of high CPC density has not been reported. A general concept is that cardiac regions protected from greatest mechanical stress (atria, septum, apex) appear to contain the highest density of CPC. A more useful perspective may be consideration of the cardiac developmental sequence in which fields of CPC undergo migration and strategically controlled distribution, since the niches may constitute remnants of specific

The role of niche-associated cells in regulating the stability and activation of resident stem cells is still poorly understood, especially in the heart. Recently, it has been shown that Notch-1 of murine CPC interacts with surrounding cells via the Jagged ligand. Through a Jagged-mediated activation signaling process induced in vitro, notch signaling, down regulation of c-Kit and upregulation of Nkx2.5 were found to be associated with increased myocyte proliferation (Boni et al., 2008). These data support the concept that interaction of CPC with supporting cells and matrix regulates their commitment to the myocyte lineage.

During heart development CPC express field-specific markers **(Figure 1.**) for FHF, SHF, and neural crest (Vincent & Buckingham, 2010). Mesp1 is one of the earliest markers for cardiac primordial cells (Saga et al., 1999). Depending on the stage of cell differentiation, genetic markers will be upregulated or down regulated in the CPC, which complicates clear classification of CPC originating from FHF and SHF based on markers. Nkx2.5 is a critical cardiac transcription factor in the first lineage, and Isl1 along with Foxh1, GATA factors, and Hand2 are key regulators in the second heart progenitor field (Moretti et al., 2006). The earliest differentiated cells in the cardiac crescent express GATA4 and Nkx2.5 (Vincent & Buckingham, 2010). The final differentiation to cardiomyocytes is controlled by Tbx (Takeuchi & Bruneau, 2009). Known markers for SHF CPC are Isl1, Tbx1 and Fgf10 (Kelly et al., 2001; Cai et al., 2003; Xu et al., 2004). Pax3 is recognized as a major regulatory gene for NCC, having an important role in their migration (Bradshaw et al., 2009). There is considerable overlap between the heart field-specific genetic markers. This issue is more evident in the case of the proepicardial organ, which gives rise to coronary arteries and epicardium. Both Isl1 and Nkx2.5 are expressed in the CPC from the proepicardial organ (Dettman et al., 1998), the cells

precursor fields in the primordial heart anlagen.

**2.2.4 Postnatal Characterization of CPC fates**

**<IP_ADDRESS> Prenatal formation of the heart from different fields**

of which also express Tbx18 and Wt1 (Martinez-Estrada et al., 2010).

#### **<IP_ADDRESS> Markers of CPC in the postnatal heart**

In the postnatal heart, identification of CPC is based primarily on the cardiac lineage markers Nkx2.5, Mef2c, and Isl1. Identification of c-Kit+ cells in adult mammalian heart suggests another postnatal source of CPC, however, the origin of these cells is unsettled. Although expression of the key CPC markers is preserved both in rodents and humans, species-specific markers limit translation of animal studies to human studies. Our studies of human infant RVOFT myocardium showed a mixed population of c-Kit+, Isl1+, and Nkx2.5+ CPC (Amir et al., 2008). The neonatal human heart is a rich source of CPC bearing markers such as SSEA4, Isl1, Nkx2.5, and c-Kit. In addition to our report of myocardial SSEA4 localization(Amir et al., 2008), SSEA4 expression has been previously only reported in adult human kidney (Ward et al., 2011). Isl1+ CPC have also been localized to the atrium (Laugwitz et al., 2005). Stem cell antigen-1 (Sca-1) is a marker for mouse resident CPC. In the mouse and human studies performed by Messina (Messina et al., 2004) and Beltrami (Beltrami et al., 2003) c-Kit+ cells are considered to be CPC, whereas Oh (Oh et al., 2003) used c-Kit- , Sca-1+ cells as a marker for CPC. This apparent controversy may be explained by the existence of heterogeneous pools of cardiac stem cells in different stages of differentiation rather than multiple populations of distinct resident CPC. In addition, the anatomical location (i.e., heart field of origin) of the heart regions studied may determine the markers expressed by postnatal CPC.

Fig. 1. Overview of myocardial CPC Markers in relation to their ontology. Pre-cardiac mesoderm cells express Mesp1 until differentiation to cardiogenic mesoderm, marked by the expression of Nkx2.5. The cardiogenic mesoderm differentiates to form two heart fields. Cells in the first heart field (FHF) express GATA4 in addition to Nkx2.5. Cells of the secondary heart field (SHF) express Isl1 and Nkx2.5. The SHF also becomes populated by neural crest cells (NCC) expressing Pax3. Markers associated with the heart field cells but showing greater variation in their expression are indicated in blue type. In addition to cardiogenic mesoderm, cells from the pro-epicardial organ and also of hematopoietic lineage have been identified in the myocardium. Abbreviations: NCC, Neural crest cells; SHF, Second heart field; FHF, First heart field.

Myocardial Self-Repair and Congenital Heart Disease 281

population of cardiomyocytes presents a potentially vast opportunity for therapeutic

If the heart can add new cells, then the question arises regarding why it doesn't always heal itself? The generally offered explanation for the fact that resident CPC are unable to naturally rescue a moderately infarcted adult heart is that the region of damaged tissue is too great for this mechanism to work rapidly enough to restore function. Although not widely recognized, "silent "repair by CPC has been observed (see section 3.2.1). It is probable that small lesions in the myocardium are actually self-repaired silently through expansion of the resident CPC population. Large ischemic lesions would be expected to also result in the loss of resident CPC in the infarcted region, further impairing self-repair. However, progress has recently been reported. Intra-myocardial injection of autologous bone marrow derived stem cells has been used in adults with chronic ischemic heart disease (i.e. not infarcted) to achieve functional recovery and reverse ventricular remodeling (Williams et al., 2011). Such reports of success strengthen the concept that endogenous stem

cells can provide clinically significant benefits to heart disease patients of all ages.

Presently, many investigators are attempting to use cardiac stem cells to produce engineered myocardial sheet grafts for myoplasty of larger regions of infarct-damaged adult myocardium. Success has been limited to moderate. Major limiting factors to this approach remain. For example, integration of the graft into the existing myocardium so that it provides clinically significant augmented force development has been problematic. Additionally, achieving sufficient revascularization of the grafts to sustain viability has been difficult; see review by Sui (Sui et al., 2011). Although the potential utility of such grafts in congenital heart disease is not expectedly large, marked progress in this approach could conceivably provide alternative therapies for patients with failing Fontan circulations or

One area of potentially beneficial therapy that has previously received little attention in *congenital* heart disease patients is the engineering of vascular and valve grafts that are capable of meeting the rapid growth rate typical of the neonatal heart and great vessels during the first decade of life. The availability of graft materials with the ability to grow along with the young patient is highly desirable, but thus far not available. This is an area of recently increasing research interest, and one in which further research could provide

Given the apparent difficulty in achieving clinically valuable augmentation of cardiac performance through the delivery of cardiomyocytes to a damaged heart, therapeutic

**3.1.2 Therapies using CPC for tissue engineering**

**<IP_ADDRESS> Tissue engineered myocardial grafts**

possibly for those with cardiomyopathies.

enormous benefit to CHD patients.

**3.1.3 Mobilizing resident CPC**

**<IP_ADDRESS> Tissue engineered myocardial vascular and valve grafts**

**<IP_ADDRESS> Alternatives to cell delivery: "Activation" of resident CPC**

**3.1.1 Role of CPC in homeostatic repair and postsurgical healing**

intervention for congenital heart diseases.

**3. CPC and Therapy for CHD 3.1 Cardiac repair using CPC**

Mouse studies have demonstrated homing of bone marrow-derived stem cells into the infarcted myocardium, suggesting a hematopoietic origin for c-Kit+ CPC. However, transdifferentiation of these cells into cardiomyocytes is controversial (Orlic et al., 2001; Murry et al., 2004; Bearzi et al., 2007). On the other hand, Tallini (Tallini et al., 2009) reported that c-Kit is expressed by immature cardiomyocytes as well as endothelial cells during development of mouse heart and that the populations rapidly expanded during the first 2 days of postnatal life. Their study compared the difference between the multipotency potential of CPC derived from the neonatal heart and the adult injured heart. They concluded that the neonatal c-Kit+ cells showed an ability to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells. In contrast, they found that c-Kit+ cells in the adult injured mouse heart had no myogenic capacity. The co-expression of c-Kit and neuronal marker Nestin in neonatal CPC (Tallini et al., 2009) suggests a transitional status of NCC in postnatal life and the potential for NCC contributing to cardiac repair and remodeling (Drapeau et al., 2005).

#### **2.3 Postnatal heart growth**

The infant heart grows rapidly after birth. The pulmonary valve annular diameter doubles in the first 7 years of life and plateaus by age 14 (Sairanen & Louhimo, 1992) while left ventricular volume doubles over approximately the first 10 years of life (Nielsen et al., 2010). Although cardiomyocytes increase in volume (hypertrophic growth), in part by cellular fusion into very large multinucleate myocytes, there is also an overall increase in cellularity (cell number) in the heart during this rapid expansion phase of heart growth. That the number of myocytes continues to increase after birth has only recently been demonstrated. For decades, it was taken as fact that cardiomyocytes were incapable of dividing postnatally, if only because mitosis of a multinucleated cell is considered impossible.

One of the earliest reports of new cardiomyocytes appearing in the postnatal human heart was provided by studies of adult aortic stenosis patients, in which cells co-expressing cardiac and stem cell markers could be localized (Urbanek et al., 2003). Additionally, studies of transplanted adult donor hearts revealed the appearance of new cardiomyocytes in the donor heart by the presence of sex chromosomes opposite to that of the recipient host (Bayes-Genis et al., 2007). Highly compelling further evidence that new myocytes are added to the heart over time arises from carbon isotope data (Bergmann et al., 2009) showing that nearly 50% of myocytes are replaced over a lifetime. Studies of the hearts of dogs with advanced dilated cardiomyopathy performed by Leri (Leri et al., 2001) showed postnatal proliferation of cardiomyocytes, documented by the expression of cell proliferation marker Ki67 and telomerase. Telomerase activity is not only required for cardiac growth and survival but it also suppresses cardiomyocyte apoptosis (Oh & Schneider, 2002). We (Amir et al., 2008) also demonstrated that the neonatal human heart contains cardiomyocytes expressing Ki67. We also determined that the number of these proliferating myocytes declines nearly 6-fold in the first two months of life.

#### **2.4 Summary: CPC in the postnatal myocardium**

Work by a number of investigators has demonstrated that a population of multipotent cardiac lineage-determined cells (i.e., CPC) capable of further differentiation to all cardiac cell types is present in all hearts. Hence, continued cardiomyocyte renewal has slowly been gaining acceptance as both an important aspect of normal myocardial biology and a potential strategy to assist with repair of a diseased heart. The ability to expand the population of cardiomyocytes presents a potentially vast opportunity for therapeutic intervention for congenital heart diseases.