\r\n\tEqually important are the consequences deriving from the extraordinary nature of the present times. The COVID-19 pandemic and the restrictive measures to contain the infection (lockdown and "physical distancing" in primis) have revolutionized the lives, and a distortion/modification of habits, rhythms, arrangements will continue to be necessary. \r\n\tGovernments have implemented a series of actions to mitigate the spread of infections and alleviate the consequent pressure on the hospital system. On the other hand, the Covid-19 pandemic has caused a series of other cascading effects that will probably be much more difficult to mitigate and which expose to complex consequences. The past two years have brought many challenges, particularly for healthcare professionals, students, family members of COVID-19 patients, people with mental disorders, the frail, the elderly, and more generally those in disadvantaged socio-economic conditions, and workers whose livelihoods have been threatened. Indeed, the substantial economic impact of the pandemic may hinder progress towards economic growth as well as progress towards social inclusion and mental well-being.
\r\n
\r\n\t \r\n\tAlthough in all countries the knowledge on the impact of the pandemic on mental health is still limited and mostly derived from experiences only partially comparable to the current epidemic, such as those referring to the SARS or Ebola epidemics, it is likely that the demand for intervention it will increase significantly in the coming months and years. The extraordinary growth of scientific research in the field of neuroscience now offers the possibility of a new perspective on the relationship between mind and brain and generates new scenarios in understanding the long wave of the pandemic and in the prospects for treatment. Moreover, the pandemic also has led to opportunities to implement remote monitoring and management interventions.
\r\n
\r\n\t \r\n\tOverall this volume will address the complex relationship existing between COVID-19, mental health, acquired knowledge, and possible interventions taking a highly multidisciplinary approach; from physiological and psychobiological mechanisms, and neuromodulation through medical treatment, psychosocial interventions, and self-management.
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Devoted researcher of the European Innovation Partnership on Active and Healthy Aging, appointed Assistant Specialty Chief Editor of Frontiers in Psychology -Neuropsychology and Scientific Director of the Italian National Institute of Philanthropy.",coeditorOneBiosketch:"An academic and industrial investigator involved in basal research, drug discovery, and development of potential psychiatric drugs, covering depression, anxiety, OCD, schizophrenia, and sexual dysfunctions.",coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"233998",title:"Ph.D.",name:"Sara",middleName:null,surname:"Palermo",slug:"sara-palermo",fullName:"Sara Palermo",profilePictureURL:"https://mts.intechopen.com/storage/users/233998/images/system/233998.png",biography:"Sara Palermo has an MSc in clinical psychology and a PhD in experimental neuroscience. 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1. Introduction
During the last about 50 years, Si-based integrated circuits (ICs) have been developed with numerous applications in the computer, communication, and consumer electronics industries. There has also been tremendous progress in the manufacturing of ICs over the past 60 years. The minimum feature size has advanced from 10 μm down to 10 nm, the cost per transistor has decreased by seven orders of magnitude, and the maximum number of transistors per chip has increased by at least 10 orders of magnitude [1]. Generally, technology node advances every 2 years with the shrinkage of the feature size by 0.7 times. Hence, the area of IC chip can be approximately reduced by 50%, resulting in doubling of the IC chips produced in a fixed area.
The main purpose of continuous scaling of the device dimensions is to improve the performance of the semiconductor microprocessors and to pack more devices in the same area. However, as the technology node is advanced to 0.25 μm, the back-end-of-line (BEOL) interconnect of ICs becomes the bottleneck in the improvement of IC performance [2]. In other words, as the feature size of ICs is continuously scaling down, the speed of the device increases due to a shorter channel length, although, resistance-capacitance (RC) delay produced by the interconnects limits the chip speed. This RC delay is the product of the dielectric capacitance (C) and the conductor resistance (R), which can be calculated according to Eqs. (1) and (2), respectively.
C=kLTSE1
R=ρLWTE2
where k is dielectric constant and ρ is metal resistivity. L, W, and T are the length, width, and thickness of metal line, respectively. S is the spacing between metal lines.
Table 1 provides the estimated critical dimensions of the BEOL interconnect from 90 to 7 nm technology nodes. As shown, with the advance of the technology node, the smaller line width and pitch result in the increased resistance of the metal lines and the increased capacitance between the neighboring metal lines. This leads to a larger RC delay in the advanced technology nodes, which surpasses the gate delay and becomes a limiting factor in ICs performance [3, 4, 5, 6].
Table 1.
Interconnect dimensions with technology nodes.
In order to slow down the increase of RC delay, the possible solution is to change the materials used in the BEOL interconnects. A dielectric film with the relative dielectric constant (k) lower than 4.0 (called low-k) had replaced a conventional chemical vapor deposition (CVD)-SiO2 film with a k value of 4.0 as an interconnect insulator because it can provide lower capacitance between the neighboring metal lines. The low-k materials currently used in the BEOL interconnects are SiOF (k = 3.5–3.8), SiCOH (k = 2.2–3.2), or air gap (k~1.0) [7, 8, 9, 10, 11]. On the other hand, to reduce the resistance of BEOL interconnects, a metal material with a lower resistivity (ρ) than that of aluminum (Al), which is the traditional conductor used in 3.0–0.25 μm technology nodes, is considered to be a candidate to replace Al conductor. Table 2 lists the electrical resistivity for different metals. Among all metals in the world, three kinds of metal have lower resistivity than Al with a resistivity of 2.65 Ωμ-cm: Gold (Au; 2.214 Ωμ cm), copper (Cu; 1.678 Ωμ cm), and silver (Ag: 1.587 Ωμ cm). Compared with these three metals, Cu has been recognized to be a candidate as a conductor in the BEOL interconnects for integration consideration. Additionally, higher electromigration reliability than Al by at least 10 times is another advantage for Cu as a conductor because Cu has a lower diffusivity than Al. Based on these reasons, semiconductor industries are fully transitioning toward using Cu instead of Al in future IC applications [12, 13, 14, 15].
Table 2.
Melting point and resistivity of different metals.
In this connection, this chapter is an attempt to provide an overview of Cu conductor used in the BEOL interconnects of ICs in the past, present, and future. This chapter is organized as follows: in Section 2, we describe the process flow of Cu damascene metallization. Then, in Section 3, the deposition methods of Cu metal are introduced and compared. Next, the integration and reliability issues of Cu metallization are discussed in Sections 4 and 5, respectively. Finally, short conclusion and future trend for conductors used in the BEOL interconnects are provided in Section 6.
2. Copper damascene metallization
Unlike Al metallization, Cu cannot be easily patterned by reactive ion etching (RIE) due to the low volatility of Cu etching by-products, such as Cu chlorides and Cu fluorides [16, 17]. Hence, to fabricate Cu interconnects, a different process flow which is called “damascene” process has been developed, including “single damascene” and “dual damascene” processes [18, 19, 20, 21, 22, 23]. In the “single damascene” process, only trench or via is fabricated after completing the process. While in the dual damascene process, both via and trench can be fabricated simultaneously, in which both via and trench can be performed with the same metallization step. Thus, Cu interconnects are usually fabricated by cost-effective dual damascene technology.
In order to fabricate Cu dual damascene interconnects, various process flows were developed. “Via first” and “Trench first” dual damascene processes are commonly used, as plotted in Figure 1.
Figure 1.
Via first (B1–D1) and Trench first (B2–D2) approaches for dual damascene patterning. (A) Dielectrics (SiN/SiCN, SiCOH, SiO2) deposition; (B1) Via-1 lithography and RIE; (B2) M-2 trench lithography and RIE; (C1) ARC plug; (C2) Via-1 lithography; (D1) M-2 trench lithography and RIE and etching stop layer opening; (D2) Via-1 RIE; (E) metal barrier and Cu seed deposition; (F) electroplating Cu deposition; and (G) Cu CMP and dielectric barrier deposition.
The process flow of Cu dual damascene metallization is described as below: After processing of Metal-1 (M-1), the etching stop layer (Cu barrier dielectric layer) and the Via-1 (V-1)/Metal-2 (M-2) dielectric layer (e.g., SiCOH low-k) are subsequently deposited. For the etching stop layer, also called Cu barrier dielectric layer, SiN or SiCN can be used, providing functions to protect Cu from oxidation and protect Cu from diffusion into the low-k dielectric during processing or device operation. These materials have much higher dielectric constants than that of the low-k dielectric. The dielectric constant of SiN film ranges from 6.8 to 7.3 and that of SiCN layer from 4.0 to 5.0, depending on the process conditions [24, 25, 26]. Sometimes, a sandwich dielectric stack film (SiCOH/Si(C)N/SiCOH) is used in order to control the depths of the via and metal precisely. These steps will increase the effective dielectric constant, raising the capacitance. The dense SiO2 layer can be capped onto the SiCOH low-k dielectric to mitigate damage on the low-k dielectric caused by the subsequent process steps, such as photoresist, and Cu chemical mechanical polishing (CMP). This layer will not appear in the final structure of the fabricated Cu interconnects because it can be removed by Cu CMP process.
For “Via first” process, Via-1 is patterned first, stopping on the SiN (or SiCN) layer that protects Cu from oxidation. Then, the Metal-2 trench is patterned and the final step is the removal of the SiN (or SiCN) etch stop from the bottom of the via. For the “Trench first” process, the “via patterning” and “trench pattering” steps are reversed.
The metal deposition in the dual damascene structure consists of three steps: Cu barrier layer, Cu seed layer, and bulk Cu layer. Currently, the first two steps are performed by sputtering and the last step uses Cu electroplating (ECP) method. The used material for Cu barrier layer is a TaN/Ta barrier layer, which prevents Cu from diffusing into the dielectric, A Cu seed layer helps to the growth of electroplated Cu film. Cu electroplating provides to fill in the via and trench. After completing the metal deposition, Cu chemical mechanical polishing (CMP) process is used to remove the excess metal over the field regions. Thus, a layer of Cu dual damascene structure (via and trench) is finished. To construct multiple metal levels, these steps are repeated for each metal level. After the last metal layer is fabricated, thick dielectric passivation layer (e.g., SiO2/SiN bi-layer) is deposited and via is opened to the bond pads.
Further reduction of the capacitance between the meal conductors is required as the device dimensions are continuously scaled down. The porous low-k material with a dielectric constant as low as 2.2 is adopted as an interconnect insulator [9]. The porous low-k material can be produced by adding pores (<2 nm diameter) to the SiCOH film. The obtained dielectric constant depends on the porosity. A higher porosity results in a lower dielectric constant; however, open pore are formed (high pore connectivity). The open pore in the porous low-k film allows water and other contaminations to diffuse into the dielectric. Moreover, during the interconnect fabrication, the porous low-k material is exposed to oxygen plasma environments in the conventional resist strip step. Ions and radicals produced from oxygen plasmas can severely damage the porous low-k material. These issues result in an increased dielectric constant and degraded dielectric breakdown reliability for the porous low-k material. To minimize the damage on the porous low-k material, low-k material optimization and resist strip condition are chosen, and the process integration modification has been provided. The integration approach for dual damascene patterning is transformed to “metal hardmask” method from 32 nm technology node as shown in Figure 2 [27]. In the metal hardmask method, the resist strip damage on the porous low-k material can be minimized because the resist is stripped prior to the trench and via etching. However, this method requires the extra steps and good process control to avoid the integration issues.
Figure 2.
Metal hardmask approach for dual damascene patterning. (A) TiN, ARC, and resist deposition; (B) M-2 metal hardmask RIE; (C) M-2 trench lithography; (D) Via-1 lithography; (E) Via-1 RIE; (F) M-2 oxide hardmask RIE; (G) M-2/Via-1 RIE and M-1 capping layer RIE; and (H) M-2/Via-1 Cu metallization.
3. Copper deposition method
In addition to the need of lower resistivity, the other requirement for Cu film is to fill the high aspect ratio vias and trenches without voids in the dual damascene structure. After continuous research and development for many years, Cu film can now be deposited by various technologies, such as physical vapor deposition (PVD), chemical vapor deposition (CVD), laser reflow, atomic layer deposition (ALD), and plating (electrolytic and electroless) [28, 29, 30, 31, 32, 33]. Table 3 lists the properties of Cu films obtained by different deposition technologies. Evaporation and sputtering methods belong to PVD technology, which can provide a lower resistivity as compared to other technologies. The latter method is widely used in the semiconductor industry.
Table 3.
Comparison of various Cu deposition technologies.
In current Cu metallization, electroplating method is used to fill the high aspect ratio via and trench in the dual damascene structure. However, in order to successfully deposit Cu film during ECP process, a Cu seed layer is needed. Sputtering deposition is the preferred method to deposit the Cu seed layer because it can produce high-purity films. In the sputtering process to deposit Cu film, Ar plasma is used to sputter Cu target and then the sputtering Cu material is deposition on the wafer. The biggest challenge for Cu sputtering process is to achieve good step coverage in the high aspect ratio via and trench. With the reduction of interconnect dimensions in the advanced technology nodes, this problem is becoming thrilling. To achieve adequate conformity in high aspect ratio via and trench in the dual damascene structure for advanced technology nodes, ionized PVD [34] or atomic layer deposition (ALD) [35] technologies have been developed for Cu seed layer deposition with demonstrated good step-coverage.
After depositing a Cu seed layer, ECP process is used to fill-in via and trench dual damascene structure. ECP process is performed by immersing the wafers in a solution containing cupric ions, sulfuric acid, and trace organic additives [36]. By applying an electric current, Cu ion (Cu+2) is reduced to Cu, which deposit onto the seed layer. To achieve void-free filling in the high aspect ratio feature for ECP process, the “bottom-up” or “super-filling” strategy is adopted. By means of the bottom-up (or super-filling) method, the deposition of Cu film is growing from the bottom to the top, so no void is formed in the via and trench. To meet this goal, the additives in the ECP solution play an important role. The additives must consist of both suppressors and accelerators. The former is polymers, such as polyethylene glycol, which reduce the plating rate at the top of features by blocking of growth sites on the Cu surface. While the latter is dimercaptopropane sulfonic acid (SPS) with sulfide and thiol-like functional groups, which enhance the plating rate at the bottom of features because the functional groups strongly absorb on Cu surfaces [36, 37]. Consequently, ECP process can provide a void-free filling process for via and trench dual damascene structure under an adequate combination of suppressor and accelerator additives.
4. Integration issues of copper metal
In the advance technology nodes, the critical dimensions of BEOL interconnects are continuously scaled down. Additionally, new materials (Cu and low-k) and a dual damascene process have been introduced. Furthermore, new technologies, such as electroplating and Cu CMP, have been used in the semiconductor fabrication line. Thus, more integration challenges are raised, as described below:
4.1. Width effect on resistivity of Cu
As the dimensions of Cu interconnects are reduced, the resistivity increases dramatically due to grain boundary scattering, surface scattering, and an increasing fraction of refractory metal liner in the trench (Figure 3) [38, 39].
Figure 3.
Resistivity of metal line and thickness of Cu barrier layer with technology nodes [40].
The first factor that increases the resistivity of the metal line is grain boundary scattering. The smaller gain size in Cu lines results in more grain boundaries, leading to an increased resistivity. Unfortunately, Cu gain size scales as the critical dimensions of the Cu line in dual damascene interconnect. It is difficult to achieve large grain size in narrow lines because grain growth of Cu in trenches is inhibited at small dimensions. Therefore, subtractive Cu method is the possible solution to increase the Cu grain size [41]. That is, Cu film is patterned by etching process. In such a case, Cu grain size would be much larger because Cu deposition is not restricted in the narrow lines. However, there remain many challenges to solve for etching of Cu including etching chemistry, hardmask, and hardware. Moreover, encapsulation of Cu line with barrier/liner materials is another issue to solve.
Surface scattering increases as the critical dimensions of the Cu line becomes smaller than the bulk mean free path of the electrons. To solve this issue, new material such as tungsten (W), silicides, carbon nanotube, or collective excitations could be an alternative to Cu as interconnects [42, 43]. Even though the bulk resistivity of W and silicide films is much larger than that of Cu film, the shorter mean free path of the electrons will lower the surface scattering effect. Another advantage of W interconnects is no-barrier process because the diffusivity of W metal is very low. The latter two materials (carbon nanotube and collective excitations) can provide a different conductance mechanism, but they are still in the research and development phase. Therefore, for these new conductor materials to successfully integrate into the semiconductor industry is a long way off.
As the dimensions of Cu interconnects are continuously scaled down for the advanced technology nodes, a larger fraction of the metal line cross-section is occupied by the refractory metal barrier film. The resistivity of the refractory metal barrier film is far larger than that of Cu metal. Therefore, the overall resistivity of the metal line is significantly increased. The direct strategy to reduce the resistance rise is to decrease the thickness of the metal barrier film. However, the accompanied problems are poor step coverage and Cu diffusion into the dielectric. The improved sputtering method or atomic layer deposition method can be used to deposit a thinner liner layer [44, 45, 46]. The most promising method is to adopt a self-forming barrier process by depositing Mn-based film. The deposited Mn film can react with silicon-based dielectrics to form a self-forming dielectric barrier by annealing. A smooth MnOx layer can be formed at Cu/dielectric film interface. This layer also provides a good adhesion for Cu film deposition. Furthermore, if this self-forming barrier process is controlled precisely, there is no barrier at the via bottom connecting the underlying metal line because of high diffusivity of Mn in Cu [47, 48]. This results in a lower via resistance and a better reliability for Cu interconnects.
4.2. Cu diffusion into the dielectric
Cu is easily diffused into the dielectric under a thermal and/or electric stress, causing a dielectric failure. For this reason, Cu film must be surrounded by a good diffusion barrier layer. Generally, the barrier layer in the sides and bottom of the Cu line is metal barrier film and is typically a TaN/Ta bilayer [49], while that on the top is dielectric barrier film, such as silicon nitride (SiN), silicon carbide (SiC), silicon carbonitride (SiCN), and silicon oxynitride (SiON) [24, 25, 26, 50, 51].
Both Ta and TaN are good Cu diffusion barrier layers. Besides, TaN can provide good adhesion to the dielectric, and Ta can provide a surface with good wettability of the Cu seed layer. Based on these characteristics, a TaN/Ta bilayer instead of a Ta/TaN bilayer is the best choice for a Cu diffusion barrier layer. Moreover, Ti-based and Ru-based barrier layers are alternatives to act as a Cu barrier layer for cost and resistivity consideration [52, 53]. However, the Cu barrier efficiency of Ti and Ru is not as good as that of Ta-based films. Therefore, a multilayer film of Ti/TiN/Ti is used as a Cu diffusion barrier layer; TiN can prevent excessive reaction between Ti and Cu, which can increase the resistivity of the wire. The top Ti layer can provide good wetting of Cu film because Cu wetting on TiN is very poor. Ru-based layer can provide a lower resistivity and a better Cu wettability than Ta layer; however, its Cu diffusion barrier is very poor. So, a TaN/Ru or Ti/Ru bilayer is used for Cu diffusion barrier [54, 55, 56].
The diffusion barrier layer on the top of Cu wires is typically a dielectric barrier film. However, its Cu barrier efficiency and adhesion ability with Cu film are poorer than those of a metal barrier layer. To address these issues, an extra metal layer (Ta/TaN or CoWP) is capped on the top surface of Cu wires before a dielectric barrier film deposition [57]. This extra process to deposit a metal layer is very challenging because of selectivity deposition on the Cu lines. However, control of this process can make a significant improvement on reliability for Cu interconnects if it is controlled precisely.
4.3. Cu oxidation
The other disadvantage for Cu interconnects is that Cu film can be oxidized during the water rinse and exposure to air. In addition to increasing the resistance of Cu wires, the formed Cu oxides cause reliability degradation due to the weakened adhesion at the Cu interfaces. During the fabrication of Cu dual damascene structure, there are two stages in which Cu film could be exposed to air. One stage is via-opening before Cu metallization deposition. The other stage is the completion of Cu CMP before a dielectric barrier layer deposition. Hence, to remove Cu oxides and avoid Cu re-oxidation, an in situ clean is required. The mechanism to remove Cu oxides in clean process can be achieved by either physical removal or chemical reaction [58].
Ar sputtering clean to physically remove Cu oxides is a typical physical method. However, during Ar sputtering process, the corners of vias and trenches are chamfered and re-sputtering Cu atoms are trapped onto the sidewalls of the via [58]. The former phenomenon leads to an increased leakage current between the neighboring wires. The latter phenomenon results in strong degradation in dielectric reliability. Hence, the energy and time in the Ar sputtering clean process must be carefully controlled in order to alleviate these two phenomena. Moreover, a “barrier-first” process was provided to minimize the detrimental effects caused by Ar sputtering clean [59]. In this barrier-first process, a TaN layer is deposited first and Ar sputtering clean is then performed to etch through the TaN layer and the contamination at the bottom of the via. Finally, a Ta layer is deposited. Due to the presence of the TaN layer, the chamfering at the top corner of vias and trenches and the re-sputtered Cu atoms and contaminations into the dielectric can be effectively reduced during Ar sputtering clean process.
The chemical clean to remove Cu oxides can also minimize the detrimental effects caused by the Ar sputtering clean. The mechanism of chemical clean is based on the oxidation-reduction reaction. Hydrogen (H) atom is typically the reducing agent. Hence, H2 or NH3 is widely used reduction gas [60, 61, 62]. The chemical clean is processed under a plasma process, which increases the activity of the reaction. During chemical plasma clean process, Cu oxides can be reduced; however, the dielectric (e.g. low-k) is also exposure to a plasma environment. The dielectric is damaged by plasma irradiation. This leads to an increased dielectric constant and a reduced dielectric breakdown filed. Therefore, the drawback of using chemical plasma cleaning to remove Cu oxides is the plasma-induced damage on the dielectric (e.g. low-k damage). To minimize the damage on the dielectric and keep Cu oxide removal efficiency, a remote plasma technology has been proposed [63].
4.4. Cu chemical mechanical polishing
The chemical mechanical polishing (CMP) process has been used to polish oxide dielectric film and W plug in Al metallization since 0.35 μm technology node. As the BEOL interconnect was transferred to Cu metallization, due to the adoption of damascene structure, dielectrics do not needed to be polished by the CMP process. Instead, the excess Cu films in the damascene structure are necessary to be removed by CMP process. Cu CMP process can be regarded as a new technology and has a different consideration from oxide CMP process, hence, it is very challenging. During CMP process, the wafers are placed face-down on a rotating pad on which the slurry is dispensed, resulting in the removal of the film by chemical reaction and mechanical force. In Cu CMP process, the excess Cu film and metal barrier layer must be removed to fabricate Cu metallization. Typically, there are three main steps in Cu CMP process [64]. The first step is Cu film removal, stopping on the barrier layer. In this step, removal selectivity is not considered because only Cu film is polished. The second step is the barrier layer removal, stopping on the dielectric. During this step, both barrier layer and Cu film are polished. The last step is over-polishing to ensure that all metals are removed from the field regions in all parts of the wafer. Cu film, barrier layer, and the dielectric are polished simultaneously. In the last two steps, the selectivity should be considered because it is of importance to reach high-degreed planarization.
Cu dishing and oxide erosion as shown in Figure 4 are the main problems associated with Cu CMP process [65, 66, 67]. As porous low-k dielectric films are used as the BEOL insulators to further reduce the capacitance between the metal lines, these two issues also become more severe. Therefore, the Cu CMP process is needed to be optimized. Since the formation mechanism of these two problems is due to the faster polish rate and lower selectivity in the slurry, reducing the down-force during Cu CMP process and/or optimizing the used slurry are feasible methods to minimize these effects.
Figure 4.
Side view schematic of Cu dishing and oxide erosion induced by the Cu CMP process.
Additionally, the pattern density of the Cu line also influences the performance of Cu CMP process. Generally, in the region of high Cu pattern density, the polishing rate is high and the thinning of the Cu line is observed due to a high polishing rate, resulting in a large variation in the resistance of the metal line. Thus, design rules to restrict the local Cu pattern density are provided for IC designers based on Cu CMP process [68]. Therefore, to reach high IC performance, inserting the dummy Cu lines to increase Cu pattern density is a general method to minimize the pattern effect of the Cu CMP process. Moreover, low downforce during the over-polish step in Cu CMP process is required to minimize this effect from the perspective of the process [69].
Moreover, cracks, delamination, scratching and contamination are the problems accompanied with Cu CMP process because the Cu CMP process is basically a frictional process. These problems can be solved through: (i) reducing the down-force during Cu CMP process; (ii) improving the adhesion between layers in the interconnect; (iii) optimizing the used slurry; (iv) depositing a relatively dense material, such as SiO2 or nonporous SiCOH films on the top of the porous low-k dielectric film; and (v) performing an optimized wetting clean after the CMP process [70, 71, 72].
5. Reliability of copper metal
In the Cu interconnects, there are three main reliability items: electromigration (EM), stress-induced voiding (SIV), and time-dependent dielectric breakdown (TDDB) [18]. The first two items are used to assess metal reliability, while the last item is to evaluate dielectric reliability. However, all reliability items are related to each component of Cu interconnects.
5.1. Electromigration (EM)
The failure of interconnects through electromigration (EM) has been a long-standing concern for the development of highly reliable ICs. The first EM-related failure of Al-interconnect based circuit was observed in 1966 [73]. For the past 60 years, intense efforts have been made on either Al interconnects or the newly introduced Cu interconnects to enhance the resistance against EM.
The phenomenon of EM involves metal atoms migration in a metal conductor due to a stress with a high electrical current density (~105 A/cm) [74]. As an electric current is applied on a metal lead, the momentum transfer is occurred from the electrons to the metallic atoms, resulting in the migration of the metallic atoms. Therefore, the depletion and accumulation of the metallic atoms in a metal lead would be observed, which occur in the cathode and anode sides of a metal lead, respectively. As a lead is depleted at the cathode side, voids will form and the resistance will increase. If the voids grow large enough to spans the whole line, open line will be observed. At the anode end of the wire, metal atoms will accumulate, resulting in a hydrostatic stress. If the stress is high enough and the dielectrics are weak, metal extrusions may form, causing leakage between the neighboring metal lines [18, 75]. In Cu interconnects, the Ta/TaN barrier layers at the bottom of the via can act as blocking boundaries, which provide a higher EM resistance than Cu. Hence, during an EM stress, the depletion and accumulation phenomenon occur in the Cu line.
This stress produces a back flux of atoms that is opposite in the direction to the flux from electromigration, which is called the “Blech effect” or Short-length effect” [76, 77, 78, 79]. This buildup stress causes a reverse migration process, which reduces or compensates for the effective metal material flow toward the anode side during EM. Thus, the EM failure time can be effectively improved. Moreover, this back-stress force becomes obvious as the length of the wire decreases. Therefore, the short wires that have a length below a critical threshold length (typically on the order of 5–50 μm), the back flux of atoms prevents killer voids from forming, and the wires are immortal.
To accelerate the fails and save the test time, the EM test is performed under a high-current density and a high-temperature condition. The failure time (t) of Cu line is widely described by using Black’s equation [80, 81]:
t=Aj−nexpEakTE3
where j is the current density, Ea is the activation energy for diffusion, k is the Boltzmann constant, T is the temperature, A is a constant, and n is the current exponent, which value is typically between 1 and 2. If n value is close to 1, the EM kinetics is dominated by void growth, whereas n = 2 corresponds to kinetics limited by void nucleation [82].
The activation energy for diffusion is varied by the different diffusional mechanisms as listed in Table 4 [83]. Diffusion process caused by EM can be divided into bulk diffusion, grain boundary diffusion, surface diffusion, and interface diffusion. In Al and Cu interconnects, the activation energies for diffusion in different diffusion paths are different. In Cu interconnects, interface diffusion has the lowest activation energy, presenting the major path for EM. Whereas in Al interconnects, grain boundary diffusion is a fast EM path due to a lower activation energy [83, 84, 85, 86].
Table 4.
Activation energy for different diffusion paths for Al, Al/Cu, and Cu metal.
In Cu metallization, the “line-via” structure is widely used for EM characterization. Two typical EM test structures: “downstream stressing” and “upstreaming stressing,” as shown in Figure 5. In order to minimize the Blech effect on EM results, the length of the tested Cu line must be sufficiently long. Generally, the length is about 200–250 μm. During an EM test, the resistance is monitored with the stressing time. As the monitored resistance is increased by a certain value or a certain percentage, this time is defined as the EM failure time. Generally, 20–30 samples are tested for an EM test. The measured failure times are usually plotted using a log-normal distribution and analyzed [23].
Figure 5.
Side view schematic of electromigration test structures and void formation locations. (A) Downstream stressing structure and (B) upstream stressing structure.
In a “downstream stressing” test structure, electron flow is from metal-2 to metal-1 through Via-1. The EM-induced void will form under the via (early failure) and in the wire far from the via (late failure). In an “upstream stressing” test structure, electron flow is from metal-1 to metal-2 through Via-1. The EM-induced void will form inside the via (early failure) and in the wire (late failure). The early failure occurred in both test structures is related to via process or metal barrier deposition. The late failure is directly linked to Cu/dielectric interface or Cu line property. Therefore, to mitigate Cu EM phenomenon, these related processes are needed to be optimized.
Many factors, such as design related, process related, and environmental related factors, can significantly affect Cu EM reliability, as summarized below:
5.1.1. Scaling effect
As device and wire dimensions are reduced in the advanced technology nodes, it is desirable to increase the maximum required current density in Cu lines, thus a longer EM lifetime of Cu lines should be achieved [40]. Figure 6 plots the maximum required current density at 105°C for Cu lines. The reason for this increase is that the drive current in the devices increases and the switching speed increases as the dimension of the device is scaling. Simultaneously, the dimension of the metal line is minimized. Hence, the metal line should sustain a higher current density.
Figure 6.
Maximum required current density at 105°C for M-1 Cu lines with technology nodes [40].
The EM performance, however, could not be improved as the dimensions of Cu lines decrease. Actually, the EM lifetime decreases as shown in Figure 7. Two reasons can explain this result. First, as the dimensions of via and trench decreases, the void size required to cause a EM fail decreases accordingly [86]. This leads to a short time to form a “killer” void. The other reason is due to the grain size in Cu lines. Experimental results indicated that the grain size decreases with line width as the width of Cu lines is less than 0.2 μm. In Cu lines with the smaller grain size, grain boundary diffusion can be significant during an EM stress, resulting in a lower EM lifetime [87].
Figure 7.
Experiment and model results of electromigration lifetime scaling with the reduction of interconnect dimension. Reproduced with permission from Ref. [86].
For the advanced technology nodes, EM reliability is becoming a critical challenge due to a high EM requirement and a low EM performance. Therefore, a number of Cu interconnect fabrication technologies or ways to improve the EM performance for narrow Cu lines are necessary. Moreover, from the perspective of stressing method, alternative current (AC) stressing can enlarge EM lifetime of Cu lines as compared to the conventional direct current stressing [88, 89]. The improvement in EM lifetime is attributed to the effect of damage healing. Under AC conditions, the partial Cu atoms migrating in one direction at one polarity stress would migrate back to its original location at the reversing polarity stress. Consequently, the Cu line suffers less damage from EM for a given time, resulting in a long EM lifetime. Additionally, the effect of damage healing from AC stress depends on the operation frequency. As the operation frequency is above 10 Hz, the effect of self-healing becomes significant and increases with the operation frequency. When the operation frequency is up to about 10 kHz, this effect is saturated. Thus, no further EM lifetime improvement is observed at the operation frequencies above this point [88].
5.1.2. Cu interface effect
The interface between Cu line and the capping layer is the dominating EM transport path for Cu damascene interconnects due to the lowest activation energy for diffusion [83]. Therefore, to obtain a long EM lifetime, the improvement of Cu interface is the most effective method by increasing the adhesion between these layers [90]. A typical dielectric capping process consists of two main steps: plasma clean to remove Cu oxides and a Cu barrier dielectric deposition (either SiN or SiCN) [91, 92].
A plasma clean has a pronounced effect on the EM improvement as compared to a barrier dielectric deposition. This is attributed to the enhanced adhesion between Cu line and barrier dielectric layer. H2 or NH3 plasma clean is typically used, which can remove the Cu oxide from the top surface of Cu metallization through chemical reaction. The obtained results were contradictory [93, 94, 95] since some authors reported H2-based plasma clean is better. These apparent contradictions may result from the wide variety of plasma chambers and the plasma conditions. Nevertheless, it is clear that both the H2 and NH3 plasma clean can enhance EM lifetime. Additionally, in order to strengthen adhesion, a SiH4 exposure process is inserted between a plasma clean and a dielectric deposition processes to form a thin Cu silicide layer. This way, the EM lifetime was enhanced due to the improved adhesion [96, 97].
The effect of Cu dielectric capping layer on EM is not as obvious as compared to that of a plasma clean although it is concluded that the improvement in the adhesion between Cu line and dielectric capping layer can enhance EM. SiN and SiCN capping layers have similar EM lifetime, but have longer EM lifetime as compared to SiC capping layer [98]. The formation of Cu compound (Cu3N) at the interface for providing a better interface is a possible mechanism.
Based on these results, an alternative to improve Cu interface is through the use of a metal capping layer in replace of a dielectric capping layer. Due to the reduction in interface diffusion, EM lifetime was found to have a huge improvement. The used metal capping layer can be Ta/TaN or CoWP [57], the latter capping layer reported to provide a larger EM improvement than the former. Moreover, in Cu damascene lines with bamboo-like grain structure (i.e., no grain boundary diffusion), the activation energies for diffusion were 1.0 eV for an SiN or SiCN capping layer, 1.4 eV for an Ta/TaN capping layer, and 2.4 eV for a CoWP capping layer [99]. This suggests that the diffusion mechanism is changed from interface diffusion to bulk diffusion for CoWP capping layer. In the cast of Ta/TaN capping layer, although the interface diffusion mechanism is still dominating, the interface bonding between the Cu and the capping layer is enhanced.
Figure 8 compares various technologies for EM improvement in terms of EM improvement efficiency (EM lifetime improvement ratio and the resistance increase ratio). CoWP capping layer is shown to be the best approach with a higher EM life-time improvement and a lower resistance increase.
Figure 8.
Comparison of electromigration lifetime improvement versus the resistance increase for various electromigration improvement technologies [40].
5.1.3. Microstructure effect
The microstructure of Cu interconnects also plays an important role in EM performance. The important microstructure parameters include grain size (with respect to line width), grain distribution, and grain orientation. Each of these parameters influences EM performance and is impacted by Cu metallization steps. Generally, large grain size or bamboo grain structure, tight grain distribution, and (111) grain orientation are helpful for EM improvement. Experimental results indicated that electroplated Cu line has relatively large grain size and tight grain distribution as compared to CVD Cu line, resulting in longer EM lifetime [100]. Furthermore, an annealing (<400°C) step after electroplating and before Cu CMP step can increase the grain size of Cu lines due to gain growth and recrystallization, resulting in increased EM lifetime [101].
5.1.4. Dielectric effect
A lower EM lifetime was found when a low-k dielectric is used as an insulator in Cu interconnects [102, 103]. This reduction is amplified with decreasing the dielectric constant of low-k dielectrics. There are several reasons to explain the lower EM life-time for low-k dielectrics. First, the modulus of the low-k dielectrics is lower than that of SiO2 film and decreases with the reduction of the dielectric constant. Because of the lower modulus, the Blech effect and the critical length for line immortality will be reduced [103]. Second, the barrier layers often have weak adhesion to low-k materials; the weak adhesion can result in extrusion fails during an EM stress [104]. Finally, low-k materials have lower thermal conductivity than does SiO2. Hence, more joule heating is generated for a given current density [105], resulting in a higher temperature in the Cu wire, and therefore a faster diffusion rate of EM.
5.1.5. Cu seed layer doping effect
Doping impurities such as Al [106, 107], Ag [108], Mn [109, 110, 111], Magnesium (Mg) [112, 113], Zirconium (Zr) [114], and Tin (Sn) [115] into the Cu layer is an effective method to improve the EM lifetime. The main disadvantage of this approach is that the impurities increase the resistivity of Cu line. To avoid a huge increase in the resistivity, the dopant concentration is kept relatively low and the dopant is usually introduced in the Cu seed layer deposition process. Additionally, an extra annealing process is needed after completing Cu metallization. The purpose is that the dopant impurities segregate at grain boundaries and interfaces between the Cu line and the capping layer by an annealing. Thus, Cu migration rate of EM is retarded due to the reduction in the grain boundary and interface diffusions [106, 110]. The measured EM lifetime was found to be enhanced by at least one order of magnitude and is positively proportional to the doping concentration.
Among the dopants used, Al and Mn have received more attention because they have shown to increase EM lifetime significantly. Furthermore, Mn is the promising candidate for providing some advantages. Its low solubility in Cu lines allows minimum increase in resistivity by optimizing the post-metal annealing [109]. Moreover, Mn has high affinity for oxygen, resulting in the formation of MnOx layer with the dielectric film by annealing. The formed MnOx layer can act as a Cu barrier layer, thus avoiding depositing a metal barrier layer [47, 48].
5.2. Stress-induced voiding (SIV)
Like EM, voids will form in the metal line for stress-induced voiding. But these two reliability terms have different mechanisms. Whereas EM is induced by electron wind force under an electric field, stress-induced voiding (SIV) is due to stress migration. As a passivated Cu interconnect is annealed at moderate temperatures (200–250°C), tensile stress in the metal is established. If this built-up tensile stress is above the critical stress, voids will form in the Cu line, leading to a resistance increase or an open line. The built-up stress in the metal line is caused by two mechanisms: One is thermal stress due to thermal expansion mismatch between the metal line and the dielectric insulator; and the other is growth stress due to grain growth in the metal line [116, 117, 118].
The unique characteristic of stress-induced void is that the maximum rate of void growth in Cu line does not occur at a high temperature, as shown in Figure 9. To achieve large enough voids to fail the circuit, the stress built-up (void nucleation) and Cu atom migration (void accumulation) must occur in sequence. However, the temperature-dependence effect of these two mechanisms is totally different. If stress-induced void is originated from thermal expansion mismatch during the dielectric capping layer deposition, a “stress-free” temperature can be obtained. This stress-free temperature is related to the deposition temperature of the dielectric capping layer and subsequent processes. The stress-free temperature is close to the inter-level dielectric deposition temperature, generally being 300–450°C. As the stress temperatures is close to the stress-free temperature, the tensile stress (σ) in the metal line is low, so that the void growth rate is low. On the other hand, the Cu diffusivity is increased with increasing the temperature, leading to a high void growth rate at high temperatures. These two different mechanisms result in a significant void growth at intermediate temperatures (150–250°C) [116, 119, 120]. Therefore, for a newly developed process for Cu interconnects, the stress temperature for the maximum rate of void growth in Cu line should be characterized in advance.
Figure 9.
Void growth rate of stress-induced void as a function of temperature [116].
The test structure of stress-induced void is simple via-chain structures. The resistance is monitored as a function of time at the stress temperature [121]. As the resistance is increased by a certain value (5–10%), this time is defined as the lifetime for stress-induced void.
The main affecting factors for stress-induced voids in Cu lines can be categorized as follows.
5.2.1. Scaling effect
The main failure mode of stress-induced void is void formation under vias due to the stress gradient in the underlying Cu line and the presence of the via-metal interface [116]. As the formation void is spanned the whole via, which is called “killer void,” the electric current is stopped, leading to a failure of circuits. Therefore, the failure rate for stress-induced void in Cu line increases with decreasing via size (Figure 10).
Figure 10.
Failure rate of stress-induced void versus M2 line width and V2 via size after annealing stress at 225°C for 1000 h. Reproduced with permission from Ref. [122].
On the other hand, the failure rate for stress-induced void in Cu line increases with increasing line width (Figure 10) opposite to what is observed with Al line [116, 117, 122]. Two mechanisms can explain this unique behavior. One is that the hydrostatic stress increases with increasing the width of Cu lines based on the result of stress simulation [123]. Hence, a stronger driving force for void formation is produced in wide Cu lines than in narrow Cu lines. The other mechanism can be explained by the theory of “active diffusion volume” [124]. In this theory, the formation void is related to the number of vacancies, which are available within a diffusion length of the via. The wider Cu lines can provide a greater number of vacancies to form void under the bottom of the via. Thus, the wider Cu lines take less time to form a “killer void” and have a weak resistance against stress-induced void. To solve stress-induced void reliability issue on the narrow via and wide line, a design solution is provided by inserting redundant vias in the wide Cu line [125, 126]. By this approach, the stress gradient is reduced and the volume of the killer void is increased, thus enhancing stress-induced void.
5.2.2. Cu surface effect
Stress-induced void in Cu lines are mostly observed under vias [116]. A high tensile stress in the metal at the edge of the via and a weak adhesion between the barrier metal and the underlying Cu at the bottom of the via are responsible for this failure mode. A high tensile stress in the metal at the edge of the via was detected through stress simulation modeling [127, 128]. At this point, if the tensile stress exceeds the critical stress, a void will nucleate and then grow along the interface between the barrier metal and the underlying Cu at the bottom of the via. Once a void forms, the critical stress will be reduced, making the stress field surrounding the void becomes less tensile. The resulting stress gradient favors vacancy diffusion toward the void resulting in further growth.
To solve this failure mode of stress-induced void, providing a better Cu interface is the main strategy. Therefore, the approaches to optimize Cu interfaces applied for EM improvement also provide great help for stress-induced void [129, 130, 131].
5.2.3. Cu grain boundary effect
Stress-induced void can also be observed at grain boundaries in Cu lines [132]. Thus, grain boundary is another diffusion path. Decreasing the grain boundaries in Cu lines (i.e. maximizing Cu grain size) can minimize the fail rate of stress-induced void, similar to the improvement in EM reliability. The most effective method to maximize Cu grain size is by the use of an annealing process. The operation timing is after Cu plating and before Cu CMP step. It is noted that the maximum annealing temperature must be limited after dielectric capping layer deposition because high-temperature annealing after dielectric capping layer deposition can lead to high rates of stress-induced void formation due to either confined grain growth or due to increased stress in the Cu line [122, 132]. Additionally, the use of metal capping layers [133] and/or Cu alloying lines [106, 107], which are used to improve EM has also shown to reduce the failure rate of stress-induced void.
5.2.4. Via barrier effect
The early failure of stress-induced void occurs inside the via due to a defect in the via with a lower tensile stress [124]. Since Cu atoms will migrate to the regions of higher tensile stress, the vacancies will diffuse to the regions of lower tensile stress. If there is a defect in the via, then void nucleation will be further enhanced in the via. Poor coverage of seed layer and undesirable gap filling of electroplating are the precursors for void formation. As the dimensions of Cu interconnects shrink, these two processes are becoming more challenging. To ensure low resistance of the metal line in the advanced technology nodes, the thickness of Cu barrier layer is required to thin down as much as possible. However, the issues of Cu diffusion into the dielectric, metal barrier layer coverage on the bottom and sidewalls of trenches and vias and Cu plating gap filling are important.
This failure mode of stress-induced voids can be eliminated with good metal barrier layer coverage on the bottom and sidewalls of trenches and vias and void-free Cu-filling process. To achieve these goals, pore sealing on porous low-k dielectrics [134], good control of the via and trench profiles [135], use of ALD barrier technology [136], and optimization of the additives in the Cu plating process [37] have been demonstrated.
5.3. Time-dependent dielectric breakdown (TDDB)
During a prolonged stress at high electric fields, electric damage can occur in dielectric materials. This induces the loss of the insulating properties for a dielectric material for which the resistance state is converted from high to low. Finally, an electrical breakdown occurs as a conducting path is formed. This loss of reliability is called “time-dependent-dielectric breakdown” (TDDB) [137, 138, 139, 140, 141].
The time-dependent dielectric breakdown can occur in gate dielectrics and BEOL dielectrics [142, 143]. The former has been an important reliability issue because the thickness of gate dielectrics is continuously decreased with the advance of technology node although the latter is not a key issue in Al interconnects because the applied electric field across the BEOL dielectric is low due to the relatively large spacing between the metal lines. However, as the technology node of ICs is continuously advancing, the lateral electric field across the BEOL dielectric significantly increases due to the reduction of interconnect dimension. Simultaneously, the used BEOL dielectric is transforming to low-k dielectrics with a lower dielectric constant than 4.0. The breakdown strength of low-k dielectrics is lower than that of SiO2 film and typically decreases with the reduction of the dielectric constant. These combined effects result in a critical challenge in time-dependent dielectric breakdown for BEOL dielectrics in the advanced technology nodes [144, 145].
The test structure for the TDDB reliability evaluation has two typical configurations: comb-comb or comb-serpentine layout [146, 147, 148, 149], as shown in Figure 11. Typically, metal-1 is the most commonly used metal level because it has the smallest pitch. During a test, one electrode (e.g., serpentine) is grounded and a constant positive voltage is applied to the other electrode (e.g., comb). The leakage current is measured with the stress time. The typical leakage current versus the stress time is the initial decrease in leakage current due to trapping of charge, followed by stress-induced leakage current, and finally breakdown [150]. The stress time with a sharp increase in the monitored leakage current, is corresponding to the breakdown time.
Figure 11.
Top view schematic of time-dependent dielectric breakdown test structures. (A) Comb-comb structure. (B) Comb-serpentine structure.
Since the time-dependent dielectric breakdown is used to assess the dielectric reliability, its performance is strongly dependent on the property of a dielectric. Additional investigations have indicated that a high density of defect sites in the as-deposited dielectric (especially for low-k materials) [151], damage or contamination of the dielectric from processes such as plasma and CMP processes [152, 153, 154], and patterning problems such as line edge roughness or via misalignment [155, 156] resulted in the low breakdown strength of BEOL dielectrics. Consequently, the optimization of the BEOL interconnect process can effectively improve time-dependent dielectric breakdown reliability.
Additionally, Cu metallization also influences the TDDB performance. Cu diffusion into the dielectric leads to serious degradation in BEOL dielectrics reliability [157, 158, 159]. Cu diffusion into the dielectric can be through the dielectric and metal barrier layers, which are interfacial diffusion and bulk diffusion, respectively. The interfacial diffusion is considered to be the dominant Cu diffusion path. This can be demonstrated by the fact that dielectric breakdown between neighboring Cu wires generally occurs at the interface between the capping layer and the dielectric [150, 160]. The interface is expected to have a higher trap density than the bulk dielectrics due to the bond mismatch between the different materials or due to contaminants from the Cu CMP process [153, 161]. Hence, the interface between the capping layer and the dielectric is the preferred diffusion and leakage path for Cu atoms. Moreover, the fabricated Cu lines are generally tapered shape (wider at the top than at the bottom), so the space is smallest at the top of the Cu line, leading to the highest electric field at this location. Due to the combination of high electric field and high-defect density, the interface is the dominant path for Cu diffusion.
Moreover, “Cu-diffusion-catalyzed breakdown” theory has been proposed to explain lower dielectric breakdown strength for Cu diffusion into the dielectric [162]. In this theory, Cu could act as a precursor for an ultimate dielectric breakdown. As the concentration of Cu in the dielectric reaches a critical value, the dielectric breakdown event occurs. Two possible mechanisms can account for Cu-induced dielectric breakdown. First, the diffused Cu atoms can catalyze the bond breakage reaction by inducing permanent bond displacement in the dielectric. The other mechanism is that the accumulated Cu atoms in the dielectric form clusters of nanoparticles. As these clusters are connected, a metallic shorting bridge or a local dielectric thinning is established, triggering a dielectric breakdown.
In addition to the reduced dielectric breakdown strength and failure time, Cu diffusion into the dielectric alters the TDDB electric field acceleration model, which is used to determine the fail rate or lifetime at the use conditions (the high-field stress data must be extrapolated to the lower fields at the use conditions). The “E-model” [163, 164, 165], which is a field-driven model and chemical bond breakage mechanism, fails to describe the low-k TDDB behavior with Cu diffusion. Instead, “E1/2-model” [146, 166] is the most appropriate model. It is postulated that the accelerated electrons, injected from the cathode, transport inside low-k dielectric by means of Schottky-Emission or Poole-Frenkel conduction. Some electrons undergo thermalization under high field and high temperature and impact the Cu atoms at the anode. This produces the positive Cu ions, which in turn inject into the dielectric under the field along a fast diffusion path. Since the current in the Schottky-Emission or Poole-Frenkel conduction is proportional to E1/2, the “E1/2-model” is the possible model to describe low-k time-dependent dielectric breakdown with Cu diffusion. However, the TDBD model is not yet fully accepted and so it remains an open issue.
To minimize Cu diffusion into the dielectric to avoid reliability degradation in TDDB, several process strategies have been proposed including using adequate metal barrier layers [167, 168], minimizing residues after post-CMP cleaning [169], and minimizing air exposure prior to capping of the Cu [150, 153]. Additionally, alternating polarity operation method instead of direct current stress could increase dielectric breakdown lifetime, resulting from recovery effect due to the backward migration of Cu ions during the reverse-bias stress [170, 171].
6. Conclusions
To improve the performance of ICs by reducing RC delay, the conductor with a lower resistivity in interconnects should be rechosen. In the past two decades, better performance of ICs was achieved by using Cu conductor in place of Al conductor. Currently, although Cu metallization has been successfully integrated into ICs, a different and complex process to fabricate Cu interconnects has many remaining issues, resulting in integration and reliability challenges.
In future, the interconnect process returning to subtractive metal process from dual damascene process is one possible solution. Furthermore, looking for an alternative to replace Cu is an on-going important topic for research and development. Silver, carbon nanotube, graphene, or photonic interconnects are possible candidates.
\n',keywords:"Cu interconnects, BEOL, damascene, resistivity, reliability",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/58180.pdf",chapterXML:"https://mts.intechopen.com/source/xml/58180.xml",downloadPdfUrl:"/chapter/pdf-download/58180",previewPdfUrl:"/chapter/pdf-preview/58180",totalDownloads:2591,totalViews:961,totalCrossrefCites:4,totalDimensionsCites:11,totalAltmetricsMentions:0,impactScore:4,impactScorePercentile:90,impactScoreQuartile:4,hasAltmetrics:0,dateSubmitted:"June 28th 2017",dateReviewed:"November 13th 2017",datePrePublished:"January 26th 2018",datePublished:"July 4th 2018",dateFinished:"December 13th 2017",readingETA:"0",abstract:"Resistance-capacitance (RC) delay produced by the interconnects limits the speed of the integrated circuits from 0.25 mm technology node. Copper (Cu) had been used to replace aluminum (Al) as an interconnecting conductor in order to reduce the resistance. In this chapter, the deposition method of Cu films and the interconnect fabrication with Cu metallization are introduced. The resulting integration and reliability challenges are addressed as well.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/58180",risUrl:"/chapter/ris/58180",book:{id:"6282",slug:"noble-and-precious-metals-properties-nanoscale-effects-and-applications"},signatures:"Yi-Lung Cheng, Chih-Yen Lee and Yao-Liang Huang",authors:[{id:"59549",title:"Prof.",name:"Yi-Lung",middleName:null,surname:"Cheng",fullName:"Yi-Lung Cheng",slug:"yi-lung-cheng",email:"yjcheng@ncnu.edu.tw",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Copper damascene metallization",level:"1"},{id:"sec_3",title:"3. Copper deposition method",level:"1"},{id:"sec_4",title:"4. Integration issues of copper metal",level:"1"},{id:"sec_4_2",title:"4.1. Width effect on resistivity of Cu",level:"2"},{id:"sec_5_2",title:"4.2. Cu diffusion into the dielectric",level:"2"},{id:"sec_6_2",title:"4.3. Cu oxidation",level:"2"},{id:"sec_7_2",title:"4.4. Cu chemical mechanical polishing",level:"2"},{id:"sec_9",title:"5. Reliability of copper metal",level:"1"},{id:"sec_9_2",title:"5.1. Electromigration (EM)",level:"2"},{id:"sec_9_3",title:"5.1.1. Scaling effect",level:"3"},{id:"sec_10_3",title:"5.1.2. Cu interface effect",level:"3"},{id:"sec_11_3",title:"5.1.3. Microstructure effect",level:"3"},{id:"sec_12_3",title:"5.1.4. Dielectric effect",level:"3"},{id:"sec_13_3",title:"5.1.5. Cu seed layer doping effect",level:"3"},{id:"sec_15_2",title:"5.2. Stress-induced voiding (SIV)",level:"2"},{id:"sec_15_3",title:"5.2.1. Scaling effect",level:"3"},{id:"sec_16_3",title:"5.2.2. Cu surface effect",level:"3"},{id:"sec_17_3",title:"5.2.3. Cu grain boundary effect",level:"3"},{id:"sec_18_3",title:"5.2.4. 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Journal of the Electrochemical Society. 2008;155(7):H485-H490'},{id:"B170",body:'Jung S-Y, Kim B-J, Lee NY, Kim B-M, Yeom SJ, Kwak NJ, Joo Y-C. Bias polarity and frequency effects of Cu-induced dielectric breakdown in damascene Cu interconnects. Microelectronic Engineering. 2011;89:58-61'},{id:"B171",body:'Cheng YL, Lee CY, Huang YL, Sun CR, Lee WH, Chen GS, Fang JS, Phan BT. Cu-induced dielectric breakdown of porous low dielectric constant film. Journal of Electronic Materials. 2017;46(6):3627-3633'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Yi-Lung Cheng",address:"yjcheng@ncnu.edu.tw",affiliation:'
Department of Electrical Engineering, National Chi-Nan University, Nan-Tou, Taiwan, ROC
Department of Electrical Engineering, National Chi-Nan University, Nan-Tou, Taiwan, ROC
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1. Introduction
A consensus agreement on the definition of cellular senescence may be stated as a viable but non-proliferative condition distinct from the G0 quiescent phase of the cell cycle and postmitotic terminal differentiation [1, 2]. While aged living organisms accumulate senescent cells, aging and senescence are not synonymous terms—the cellular and molecular pathways that eventuate in the senescent state can be activated by diverse mechanisms, not necessarily chronologic aging nor the limit of replicative cell division. It was the latter phenomenon, in fact, that led early investigators to the original concept of cellular senescence as an in vitro observation; that replicating fetal “skin tissue cells” stop dividing at a certain passage number, the so-called “Hayflick Limit” [3]. This review focuses on our current understanding of how cellular senescence occurs in the skin, its irreversible (and possibly reversible) characteristics, description of known trigger points involving genetic and epigenetic factors and their clinical implications in health and disease.
Cellular senescence is characterized by cell cycle arrest [4], the expression of senescence associated secretory phenotype (SASP) [5, 6], damage to DNA [7, 8, 9], deregulated metabolic profile [2, 10], changes to the epigenome [11] and transcriptome [12], resistance to apoptosis [13, 14], and altered immune surveillance [15, 16]. It can be triggered by multiple factors [2], the mechanisms of which appear to categorize the ‘type’ of senescence into two main groups; so-called replicative senescence (RS) due to shortened telomeric DNA resulting from excessive cell division cycles [17, 18, 19, 20]; and a state generally termed ‘premature senescence’ (PS), in which both oncogene-induced senescence (OIS), triggered by activation of oncogenes such as ras [21], and several other ‘molecular stresses’ [4] also eventuates in the senescent phenotype.
There are a variety of biomarkers for cellular senescence but not all senescent cells express the same biomarkers due to these differential molecular induction pathways. Several senescent biomarkers have been identified in the skin [22]; however, it is currently unclear how the multitude of cell types that comprise this tissue respond to senescence-inducing triggers and how this correlates with skin aging, other than the fact that senescent cells accumulate in all skin compartments with age, just like other organ systems. What is becoming clear, however, is that cellular senescence plays critical roles in the pathobiology of skin aging and disease [23].
2. Aging of the skin
Both intrinsic (time, genetic and hormonal) and extrinsic (environmental) factors contribute to skin aging. Old skin not only appears clinically different from young skin but has altered physiology due to a combination of molecular, cellular, and biochemical processes, and tracing the pathogenic origin of the ‘skin aging phenotype’ remains a work in progress. From a clinical perspective however, the skin of most people older than 6–7 decades of life, particularly in photo-exposed areas, is thinner, looser, less tethered to underlying tissue, more wrinkled, more translucent with more visible capillary vessels, more discolored, drier, and less padded by the subcutaneous layer [24, 25]. Scalp skin also ages, commonly observed as pigment loss (graying), and most people experience hair loss as another inevitable esthetic problem.
Anatomically, the structure of human integument tissue we call ‘skin’ is composed of ectodermal-derived epithelial cells layered as stratified squamous epithelium on top of mesenchymal-derived dermis separated by a specialized basement membrane zone (BMZ) called the dermal-epidermal junction (DEJ). Directly below the dermis is the fatty hypodermis (or subcutaneous layer) separating fascia and muscle from the skin. Epithelial-mesenchymal interactions that occur during embryogenesis (and wound repair) contribute to the formation of glandular structures buried within these compartments (called adnexa) which are comprised of eccrine, apocrine, sebaceous and hair follicle structures. Peripheral nerves and blood vessels traverse the subcutaneous and dermal layers and together with all other structures of the integument, serve the functions of barrier protection, retention of heat and water, sensation, contractility, and lubrication [26].
This multi-compartmental system is the largest organ of the body and is composed of about 20 different cell types responsible for skin function and its stratification [27], all of which also change with age to contribute to the overall ‘skin aging phenotype’. At the microscopic level, skin tissues of older individuals exhibit common characteristics regardless of whether sun protected or chronically exposed to ultraviolet (UV) light [28]. The most obvious and well documented structural changes include epidermal thinning, loss of rete ridges and flattening of dermal papillae [29, 30], keratinocyte and melanocyte architectural changes [31, 32], BMZ/DEJ alterations [33], less dense and altered reticular dermal collagen structure [34], accumulation of altered elastin and elastic fiber structural abnormalities [35], altered shape and loss of papillary dermal capillary loops [36, 37, 38], and size and structural alterations in glandular structures of the eccrine, apocrine and sebaceous units [39]. Concomitant with morphologic changes observed in aged skin, senescent cell populations increase in all skin compartments.
2.1 Effects of aging on epidermal structure and function
The epidermis, consisting of 5 different layers of keratinocytes, continuously renews itself on an approximate 27-day cycle by a differentiation program involving basal cells which are maintained and replenished by stem cells residing in the bulge region of the hair follicle and the interfollicular epidermis [40]. Of particular importance to aging of the epidermal compartment is the general concept that the cellular microenvironment (or niche) of stem cell populations plays a critical role in homeostatic resupply of transient amplifying basal cells [41]. The epidermis maintains a dynamic equilibrium by proliferating in the basal layer that is attached to the DEJ, then cell division ceases and basal keratinocytes undergo terminal differentiation while spatially migrating towards the top of the epidermis. During this transition, keratinocytes acquire specialized cytoskeletal components and create an intercellular diffusion barrier, eventually forming the outermost epidermal layer called the stratum corneum (SC). The SC is a specialized acidic, hydrophobic, protein-lipid-carbohydrate flexible ‘shell’ resistant to wear and tear, water loss, and invasion of microbes [42]. The “barrier function” of the skin is derived from the SC.
The epidermal compartment appears to deal with the ravages of extrinsic aging in a fundamentally different way than the dermal compartment because terminally differentiated (cell cycle arrested) keratinocytes are continuously shed, thus removing accumulated DNA and other macromolecular damage that otherwise trigger the senescent phenotype. But since the epidermis is continuously replenished by stem cells arising from the interfollicular niche, its alteration can affect epidermal biology in profound ways. In fact, epidermal stem cell niche can be affected during aging by both basal keratinocytes [43] and dermal fibroblasts [44]. Niche microenvironments can be altered by intrinsic and extrinsic aging at cell-cell, cell-matrix and paracrine signaling levels, leading to stem cell depletion and the ‘atrophic epidermal phenotype’ observed in intrinsic aged skin [45].
Many other cell types localize to the epidermis, including pigment-producing melanocytes found in the basal layer that protect against UV radiation. Pigment is synthesized within the melanocyte but transferred to neighboring basal keratinocytes (and specialized hair follicle-associated keratinocytes) via a complex melanosomal exo/phagocytosis mechanism localizing at the dendritic tips of melanocytes which interdigitate with up to 20 keratinocytes [46]. Melanocyte dysfunction associated with extrinsic aging (mostly photoaging) manifests clinically as abnormally dispersed and/or diminished melanin pigment (i.e., dyschromia, lentigines, and in the scalp, canities). Senescence of the melanocyte has been observed both in vitro and in vivo and the molecular pathways involved identified [47]. In fact, based on biomarker (e.g., P16) expression, senescent melanocytes appear to represent most senescent cells in aged epidermis [48] and their contributions to development of the epidermal atrophic phenotype via autocrine and paracrine (i.e., SASP) mechanisms have been identified [49].
The epidermal immune system is a network of resident antigen-presenting dendritic Langerhans cells (LC) thought to function as immune sentinels [50] together with trafficking lymphoid immune cells including resident memory T cells as CD8+ and CD4+ cells [51]. Of interest, a specialized CD4 + T cell (Treg) residing near the hair follicle bulge areas (located in the dermis) has been shown to play a role in hair growth cycling [52]. Skin aging is associated with variable deterioration of both adaptive and innate immune function, generally referred to as cutaneous ‘immunosenescence’. This term has become controversial in the literature [16] because immune cell senescence is, in part, a physiologic adaptive response to survival and fitness of the organism. Its use to describe altered skin immune responses with age appears appropriate in the context of inflammaging [53], since the concept of immunosenescence encompasses both systemic chronic, low-grade inflammation [i.e., elevated serum levels of interleukins IL-6 and IL-8 and increased tumor necrosis factor alpha (TNF-α), etc.] and the presence of dysfunctional immune responses in various skin compartments apparently related to both tissue level RS and PS. Currently unknown, however, is whether cutaneous inflammaging is a cause or an effect of dysfunctional innate immune responses observed in the elderly and whether cellular senescence is responsible.
Examples of cutaneous aged-related immune dysfunction include reported reductions in the number and functionality of LC in aged skin and this correlates with both age related defective epidermal barrier function and inflammaging [54]. Likewise, defective physiologic immune clearance of senescent cells that contribute to aged skin pathologies have been demonstrated in dermal fibroblasts by the observation that these cells express a nonclassical major histocompatibility antigen (HLA-E). Its increased expression appears to block activation of natural killer (NK) cells and CD8+ cells responsible for clearing damaged cells, suggesting that evasion of dermal immunosurveillance leads to persistence of senescent dermal fibroblasts [55].
Aging is a clinical comorbidity in many skin diseases pathogenically linked to defective cutaneous innate and adaptive immune responses [53]. The incidence and prevalence of autoimmune blistering disorders such as bullous pemphigoid (BP), pemphigus vulgaris, and epidermolysis bullosa acquisita are all increased in older populations, BP being the most common example [56]. Likewise, aging is a comorbidity in the development of skin cancers, and the loss of immunosurveillance due to dysfunctional LCs is thought to contribute to progression of both non-melanoma skin cancer (NMSC) [57] and melanoma [58].
Another unique cell type scattered along the DEJ, considered part of the epidermal compartment, and possessing mixed neuronal, endocrine, and immunologic functions (as well as embryonic origin) is the Merkel cell (MC) [59]. Its involvement in the skin’s somatosensory system is key to the sense of fine touch discrimination, which is decreased in the elderly [60]. In glabrous skin MC form complexes with intraepidermal sensory neurites found at the DEJ termed ‘touch domes’ or Merkel’s discs. Digital skin of aged humans contains less of these complexes, lower density of MC and decreased expression of the stretch-activated ion-channel component Piezo2 [61]. Occurring mainly in aged humans, a rare but very aggressive skin cancer, Merkel cell carcinoma (MCC) has attracted recent attention due to its mysterious etiopathogenesis. 80% of MCC is associated with integration of a newly identified polyoma virus (MCPyV) [62], whereas 20% appear linked to accumulation of UV light-induced somatic mutations [63]. As detailed in the next section on epigenetics, it is of interest that the majority of MCC display expected chronologic age but DNA methylation patterns of epigenetically youthful cells [64].
2.2 Aging and the dermis
The dermal compartment is divided into superficial, reticular, and deep dermis with unique cellular, vascular, extracellular matrix (ECM), and adnexal components that define each space. Much of the ‘business-end’ of the dermis is localized to the superficial dermal compartment and the DEJ is central to its structure and functionality. It is considered part of both the epidermis and the superficial dermis because cellular components of each layer contribute to its synthesis, maintenance, and renewal. Serving as an adhesive scaffolding for basal keratinocytes, a shear-resistant Velcro-like surface securing the dermis to the epidermis, a complex paracrine factor-sequestering and mechano-transducer signaling layer, the DEJ modulates a remarkable number of cutaneous cellular processes involved in skin structure, function, regeneration, and resistance to trauma [33]. Comprised mainly of Type IV collagen and laminin, like other BMZs, DEJ complexity has been dissected at the molecular level to reveal a complex network of other collagens (VII, XVII and XVIII), 4 different isoforms of laminin (511, 521, 311 and 332), perlecan, nidogens, SPARC, fibrulins-1 and -2, dystroglycans, and integrins a3b1 and a6b4. All of these DEJ components are altered during aging and these changes correlate with age dependent increases in both DEJ thickness and stiffness [65].
Immediately beneath the DEJ, forming nipple-like structures projecting into the epidermal compartment and containing unique ECM, microvasculature, specialized fibroblasts, and dermal mesenchymal stem cells is the papillary dermis (PD). Here, undulating dermal protrusions interdigitate with epidermal rete ridges (pegs) to increase surface area for nutrient transfer, trafficking of immune cells, and increased tensile strength. The transition of superficial to reticular dermis is static and defined mostly by changes in ECM structure but dynamic during repair and disease. The majority of space in reticular dermis is occupied by thick bundles of interstitial collagens I and III, elastin and fibrillin fibers, and amorphous ‘ground substance’ comprised of hyaluronic acid, proteoglycans and glycoproteins. Like the DEJ, most if not all these dermal ECM components are altered and/or dysfunctional due to aging [35]. Both intrinsic and extrinsic aging correlate with the loss of rete ridges and flattening of dermal papillae [29, 30]. Compared to young, non-exposed and old photo-protected skin, the PD of chronically photodamaged skin displays marked structural changes, the most dramatic feature of which is the presence of “solar elastosis” in the superficial dermis. Solar elastosis consists of pathologically altered elastin fibrils [66] that present as dense accumulations of amorphous material best visualized with trichrome staining.
The cellular composition of these dermal sub-compartments is a complex mix of fibroblasts, endothelial cells, myofibroblasts, macrophages, mast cells, trafficking immune cells, adipocytes, various stem cells, sensory neurites, and the differentiated cellular components of dermal adnexa (including the hair follicle). An example of such cellular complexity, the significance of which continues to evolve, is the apparent postnatal plasticity of the dermal fibroblast. Single cell RNA sequencing has revealed at least four different subpopulations of human dermal fibroblasts [67, 68], and skin aging has been demonstrated to have a strong effect on both dermal ‘fibroblast’ phenotype and functionality. For example, young papillary dermal fibroblasts can direct reformation of youthful DEJ and epidermal structure and function, whereas old papillary and/or reticular dermal fibroblast populations cannot [69]. Furthermore, it is the senescent PD fibroblast and CD271+, laminin 332-expressing interfollicular stem cells that contribute to age-associated pathologic remodeling of the DEJ [33, 70]. Recent attention has focused on specific dermal fibroblast subpopulations and their involvement in wound healing, fibrosis, and loss of epidermal stem cell ‘stemness’ due to niche signaling dysfunction. The homeobox gene engrailed-1 (EN-1) expression appears to distinguish two types of fibroblasts; those cells expressing EN-1 are associated with fibrotic healing phenotype whereas EN-1 negative fibroblasts promote physiologic remodeling [71, 72]. Epigenetic modulation of the fibrotic phenotype is reviewed in the next section.
2.3 Subcutaneous layer involvement in skin aging dysfunction
The subcutaneous compartment (hypodermis) is composed mainly of cellular lipid storage units (adipocytes) separated by thin weblike networks of specialized ECM stroma containing microvasculature, adipose derived mesenchymal stem cells (ADSC) and immune cells. It functions as a thermoregulatory and shock-resistant barrier, as well as a reservoir of bioactive factors involved in systemic lipid metabolism, energy balance, and endocrine function [73]. Subcutaneous fat also undergoes age-related changes that are generally like the epidermal and dermal compartments where an ‘atrophic’ phenotype becomes clinically evident. With aging, subcutaneous fat deposits in various body locations disappear and/or are redistributed to visceral locations elsewhere in the body, causing esthetic concerns; this redistribution is associated with a variety of systemic age-related disease states, including insulin resistance, metabolic syndrome, cardiovascular disease, and obesity [74, 75]. Of note, senescent cells have been shown to accumulate in aged adipose tissue [76], contributing to systemic inflammaging. Experimental clearance of senescent cells can dramatically affect the redistribution of fat from the visceral to the subcutaneous compartment and decrease SASP expression [77]. The mechanism(s) of adipose cell senescence has not been clearly defined; however, ADSC exhaustion, oxidative stress by reactive oxygen species (ROS), and niche disruption appear to play important roles [78].
The influence of adipogenic hormones in skin aging and senescence has received recent attention with the discovery that UVB-light induced PS in human keratinocytes can be rescued by adiponectin via its suppression of inflammatory signaling pathways and human beta defensin-2 (hBD2) expression [79]. Human dermal fibroblasts express adipokine receptors and both leptin and adiponectin have been shown to stimulate expression of the ECM components hyaluronic acid and interstitial collagen [80]. These adipogenic hormones secreted by subcutaneous fat cells thus appear to represent paracrine cutaneous anti-aging factors for both the epidermal and dermal compartments.
‘Fat grafting’ has become a popular procedure in esthetic medicine and a variety of other clinical indications [81, 82] with special attention focused on ADSC. These cells can be isolated from subcutaneous fat removed during liposuction procedures after the stromal vascular fraction (SVF) is either mechanically sorted or enzymatically digested with bacterial collagenase, decanted (or centrifuged), washed, and grafted [83]. SVF is composed of cellular components (pre-adipocytes, adipocytes, histiocytes, endothelial cell progenitor cells and ADSCs) and is a rich source of growth factors (i.e., bFGF, IGF-1, VEGFs, PDGF-BB), matrikines, and other paracrine cellular factors. The ADSC secretome has been well characterized, consisting of soluble protein factors and lipid membrane particles (exosomes and ectosomes) that are used internationally in multiple therapeutic clinical trials for a vast array of indications, including dermatologic conditions (esthetics, wound healing, fibrotic diseases, dermatoporosis, etc). It is the loss of ADSC stemness, decreased proliferative potential, and dysfunctional secretome expression accompanying skin aging that continues to draw intense interest [82].
3. Genetic influences on cutaneous cellular senescence
The two major molecular pathways resulting in RS and PS have been observed in the skin [23]. These are reviewed in the following section by examining first the genetic aspects of cutaneous cellular senescence, followed by epigenetic influences. It should be noted here that acquisition of these cellular senescence phenotypes plays a critical role in both normal organismal and tissue level physiology by, for example, dampening fibrotic responses during the remodeling phase of wound repair or suppressing tumor formation [84]. However, it also appears to be a major pathologic driver in age-related disease states [85].
3.1 DNA damage related to telomere biology
The senescent phenotype can be activated by DNA damage at the ends of all eukaryotic chromosomes, called telomeres, which consist of DNA loops containing noncoding repeats of guanine-rich sequences complexed with protective oligomeric proteins (Shelterins). Discovery that chromosomal replicative machinery responsible for somatic cell division cannot synthesize exact duplicates of these structures led to the concept of the ‘end-replication problem’ during serial passaging [20]; thus, telomeric DNA is subjected to attrition because DNA polymerase fails to replicate the 3′ lagging strands.
Telomeric DNA are shortened by approximately 50–200 bp per cell division and thus a molecular clock is achieved, reflecting the replicative history of primary cells [86]. A specialized DNA polymerase (telomerase) is responsible for fixing the ‘end replication problem’, maintaining telomeric length, but its expression and function are restricted to immortal postnatal cells; in vivo, comprising stem, progenitor, and cancer cells. When cells reach their ‘Hayflick Limit’ telomeres lose enough DNA [87] to trigger a genomic instability signal and chromosomes become ‘uncapped’ by loss of Shelterin. This genomic instability signal is a specialized DNA damage response (DDR) and generates telomere dysfunction-induced foci (TIFs). Approximately half of all persistent DNA damage foci are localized to telomeres, and these can trigger RS. But senescent cells can harbor many other forms of persistent chromosomal DNA damage foci, called DNA-SCARS (DNA segments with chromatin alterations reinforcing senescence) [9]. These dynamic structures can also trigger cell cycle arrest and SASP induction.
Independent of telomere length or uncapping by loss of Shelterins, guanine-rich telomeric DNA repeats can become damaged by ROS, generating DDR telomere-associated foci (TAFs), which are associated with triggering the senescence phenotype [19]. This observation has particular relevance to the state of chronic inflammation, SASP expression, and tissue aging (Inflammaging) in skin and other tissues [15, 88, 89], as discussed in Section 3.2.
While epidermal, dermal, and subcutaneous cellular compartments all harbor evidence of RS in aged skin tissue, direct evidence that telomeric DNA associated RS is involved in skin aging is supported by experiments involving ectopic expression of human telomerase (hTERT). We reported that neonatal human dermal microvascular endothelial cells (HDMEC) undergo RS in vitro but can become immortalized with viral transfer of the catalytic subunit of hTERT [90]. Furthermore, these telomerized HDMEC formed fully functional microvessels in vivo (perfused with murine blood) that exhibited superior durability with time after xenografting in immunodeficient mice versus vessels created with in vitro-aged primary HDMEC [91]. As previously reviewed, cutaneous microvasculature of aged papillary dermis is markedly reduced and abnormally structured versus young dermis [38], presenting clinically as telangiectasia and senile purpura/dermatoporosis. The roles of RS, OIS, and other senescent pathways on skin vasculature remain to be determined.
3.2 Genotoxic and exposome insults
As noted in the introduction, cellular senescence can be induced in the absence of any telomeric damage or loss and this premature senescence (PS) has similar deleterious effects on aged tissues, including the skin. The triggers for the PS program generally fall into (a) accumulation of subcytotoxic, unrepairable, non-telomeric DNA damage, including mitochondrial DNA (mtDNA), (b) macromolecular insults to cytosolic and secreted proteins and lipids, and (c) metabolic dysfunction involving an altered mitochondrial-lysosomal axis [92]. All of these PS triggers have been demonstrated in skin cells in vitro and in vivo [93].
The molecular and cellular effects of chronic UV light exposure (photoaging) have also been well-documented and, in many ways, more extensively than intrinsically aged human skin. Both UVA (320–400 nm, less energy) and UVB (280–320 nm, more energy) light cause photoaging but UVB is mostly absorbed by the epidermis, where it causes sunburns. UVA penetrates the superficial and reticular dermal compartments and is considered a major factor in photoaging. While both UVA and UVB wavelengths generate reactive oxygen species (ROS), indirectly damaging DNA, UVB is also directly mutagenic, causing DNA defects called cyclobutene pyrimidine dimers and 6–4 photoproducts [94]. Remodeling of dermal ECM favoring an atrophic phenotype is triggered in unwounded skin by UV exposure via the activation of mitogen-activated protein kinase (MAPK) and activator protein 1 (AP-1) signaling pathways which causes downstream expression of matrix metalloproteinases (MMPs) in both the epidermal and dermal compartments [95]. These same pathways block transforming growth factor beta (TGF-β)/SMAD signaling via TGF-b type II receptor down regulation causing decreased collagen synthesis [96, 97]. Dissection of the molecular effects of chronic UV exposure on the DEJ and PD have been recently reviewed [33].
Our understanding of the role senescent cells play in cutaneous aging pathologies continues to evolve. In the past, senescent cells observed in the skin with biomarkers in vivo were believed to be passive, unresponsive bystanders recognized morphologically by their enlarged, seemingly flattened, abnormal shapes and senescence-associated (SA) β-galactosidase staining. But characterization of SASP expression in senescent cells (and their paracrine effects) provided compelling evidence that senescent cells are anything but passive.
It is now widely accepted that senescent cells remarkably influence surrounding non-senescent neighbors and ECM networks via secretion of inflammatory cytokines, chemokines, matrikines, MMPs, tissue inhibitors of metalloproteinases (TIMPs), and other proteinase-inhibitor systems that comprise the tissue ‘proteinase web’ [98]. One such example is the role played by plasminogen activator inhibitor-1 (PAI-1) in modulating senescence. PAI-1 is a soluble and matrix bound serine protease inhibitor with multiple matricellular functions and can be found at increased levels in both dermal fibroblasts from aged donors and premature aging syndrome patients [99, 100, 101]. Ectopic expression of PAI-1 in fibroblasts induces the senescent phenotype and is both necessary and sufficient for RS downstream of p53 [102]. Many other examples of SA ECM alterations have been reviewed [33].
The quintessential example of extrinsic aging involves the postmitotic dermal fibroblast population which responds to ‘expososomal’ damage [103] by activating DDR pathways, triggering PS and subsequent expression of macromolecular damage profiles involving mtDNA damage. One mechanism of mtDNA damage appears to involve UV light-induced deletion of a significant length of mtDNA, termed the ‘common deletion’ (CD) [104]. This 49 kb mtDNA fragment contains codons for electron transport chain (ETC) protein complexes I, IV and V which together express 72 ETC subunits, the loss of which cripples physiologic functions of mitochondrial energy metabolism, ROS protective mechanisms, and calcium homeostasis. Tracking the mtDNA CD in human skin revealed that both intrinsic (photo-protected) and extrinsic (chronic UV-damaged) skin contain this marker [105], and that dermal fibroblasts appear to be the culprit for subsequent age-related tissue damage of ECM [106, 107].
3.3 Genetic skin diseases associated with DNA repair pathway defects
Analysis of progeroid syndromes have provided insights into molecular mechanisms of intrinsic and extrinsic skin aging. Common skin phenotypic signs and symptoms shared by both these premature aging disorders and skin aging in the general population include skin atrophy, alopecia, fibrosis, telangiectasia, poikiloderma, canities and both NMSC and melanoma. Rare autosomal recessive patterns of different mutations in DNA repair genes group these heritable disorders into those involving; (1) multiple defects in nucleotide excision repair (NER) genes [e.g., DNA polymerase eta (POLH) among six others] coding for repair proteins in xeroderma pigmentosum (XP) [108], (2) transcription and transcription-coupled NER genes in Cockayne syndrome (CS) and (3) mutations in the gene family of RecQ helicases involved in DNA double strand break repair in Werner syndrome, Bloom syndrome, and Rothman-Thomson syndrome [109]. In the latter three disorders, mitochondrial defects have been well documented and correlate with cellular senescence phenotypes [110, 111]. In XP-V null mouse models, loss of POLH leads to obesity and marked adipose tissue senescent phenotype expression [112].
3.4 SNPs and transcriptomics
Several genome-wide association studies (GWAS) and meta-analyses performed on young and old populations have identified single nucleotide polymorphisms (SNPs) in genes thought to be correlated with skin aging [113, 114, 115, 116, 117, 118] or ‘perceived’ facial age’ [119]. These large cohort-based studies suggest specific allelic variants of pigmentation gene (MC1R), aryl hydrocarbon receptor gene (AHR), basonuclin 2 gene (BCN2), type-1 collagen alpha-2 gene (Col1A2) or SNPs within or near the DIAPH2, KCND2 and EDEM1 loci all appear to correlate with both intrinsic and extrinsic skin aging phenotypes and/or youthful skin appearance.
Of all these identified genes and their allelic variants, the biology of MC1R gene has received perhaps the most recent attention due to its central role in modulating human (and murine) skin pigmentation systems, the clinical influence of which led to the categorization of Fitzpatrick Skin Phototypes. MC1R signaling is associated with both skin cancer and skin aging via its mixed role in UV induced PS in melanocytes and promotion of efficient DNA damage repair [120]. Genetic variants of MC1R (coding for G protein-coupled transmembrane melanocortin receptor-1 on melanocytes) are strongly linked to increased risk of both NMSC and melanoma in both red and brown Caucasian phototype cohorts [121]. Meta-analysis of several GWAS studies demonstrated SNPs in or near MC1R (and SLC45A2 and IRF4) correlated with different skin aging phenotypes using a skin surface topographic scoring system of solar elastosis [116] and the MC1R gene may also affect inflammaging via generation of ROS independent of its function in melanin production [122].
Gene expression studies of the skin aging phenotype have revealed several important observations about the complexities of distinguishing intrinsic from extrinsic mechanisms, as they appear to overlap in many important ways. In human skin, gene profiling and transcriptomic analyses [115, 123, 124, 125, 126, 127, 128] have identified thousands of upregulated and downregulated genes in old vs. young and intrinsically aged vs. extrinsically aged (photoaged) skin. One transcriptomic study showed genes associated with mitigating oxidative stress, control of lipid synthesis, and epidermal differentiation were all downregulated in both exposed and photo-protected skin, whereas, elastin expression was increased in exposed skin (consistent with formation of solar elastosis), and interstitial collagen expression decreased in sun protected skin (consistent with intrinsic aging) [127]. Similarly, confirming histologic studies, expression profiling of human aging that spanned subjects between the ages of 24–70 years demonstrated younger-appearing skin upregulated expression of the LAMA5 gene (DEJ component) and epidermal cell-cell adhesion complex (desmosomal) genes DSC3 and CDH1 [126].
Race, sex, and skin tone of subjects also all play a role in the genetic correlates of skin aging. The expression of some aging related genes was found to be sex-dependent in a Caucasian sample [129], and studies in a Han Chinese sample showed distinct genetic variants and phenotypes from that in a Caucasian population [118]. These discrete expression patterns further highlight the complexity of cataloging aging mechanisms in the skin and suggest that much more information would be required from a wider diversity of samples to understand any potential global age-related changes.
Altogether, genetic factors, including telomere DNA loss, genotoxic accumulation of mutations in both genomic and mtDNA, DDR signaling, DNA repair dysfunction, and allelic variations in key cutaneous protective genes controlling pigmentation, inflammation, dermal, epidermal, and subcutaneous physiology all converge on our emerging understanding of the central role cellular senescence plays in skin aging. What follows is a review of how epigenetic factors also influence cellular senescence in cutaneous biology and the aging phenotype.
4. Epigenetic influences on cutaneous cellular senescence
Though the evolution to senescence is usually characterized as a genomically driven phenotype, its manifestation can be largely characterized as an epigenetically entrenched state. The baseline definition of a senescent cell is an otherwise mitotic cell that has entered permanent cell cycle arrest, but this also begets a broader shift in cell behavior and protein production. For these changes to be permanent they must be encoded in long-term gene expression tendencies, i.e., in the cellular epigenome. The epigenome is the composite architecture consisting of chemical and physical modifications to the DNA that do not alter the underlying coding and noncoding sequences but instead modify its oligomeric structure and transcription. These modifications span multiple layers from the local control of specific gene promoters to large scale regulation of entire domains of genes.
Canonically, the epigenome is most strongly associated with cell identity, as it makes accessible the portions of genomic DNA needed for the cell’s functional role while segregating and silencing irrelevant regions. Thus, the most dramatic epigenetic shifts are observed when cells differentiate from stem or progenitor states. In this full and dramatic state transition, the function of the cell is redefined, affecting everything from its morphology to its protein production and factor secretion [130]. For skin this can mean, for instance, a transiently amplifying cell in the basal epidermis fully differentiating to a corneocyte through natural turnover or endothelial progenitor cells differentiating into new endothelium in response to an angiogenic signal. Conversely, cells undergo constant but more minor epigenetic events as they are exposed to regular stimuli from the environment, which can lead to upregulation or downregulation of certain behaviors [131]. For instance, methylation sequencing of the same cell type across patients (e.g., epidermal keratinocytes) will show a distribution with perturbation on the mean population value based on internal and local external stimuli [132]. This heterogeneity evolves with different stressors, and with aging itself becomes more prominent. Eventually the stressors can lead to enough diversity to characterize pseudostates and pseudostate transitions that may by-and-large retain the cell’s identity but with a different grade of functionality across multiple genes. We will discuss a few additional examples of this in the following sections, like fibrotic versions of connective tissue cells and pro-/anti-inflammatory versions of macrophages; however, the focus of this section that could also be considered an epigenetic pseudostate is senescence. It meets the criteria in that it still retains core cell identity but also dramatically affects a multitude of genes to alter protein production and secretion profile, and thus requires a core epigenetic component. In this section we will review the multitude of epigenetic changes that accompanies this pseudostate evolution in skin cells, what role they play in establishing the senescent phenotype and how they may potentially be engaged therapeutically.
4.1 Sequence specific modulation
One modality of epigenetic is sequence specific meaning it targets specific regions of the genetic code—either DNA or RNA. DNA base pair methylation is a well-known example of this modality. Cytosine is the most commonly methylated base in eukaryotic cells and when methylated, often serves to block the activity of RNA polymerase, as in the context of CpG islands. Found in the promoter region of many genes, CpG islands are clusters of methylated cytosine followed by guanine, wherein the methylation inhibits (silences) the transcription of that gene [132]. Widespread hypomethylation has been documented in aging and senescent fibroblasts, and in some cases, impairs cell cycling pathways through the suppression of cyclin pathways. Specifically, a lack of methylated sites leads to the upregulation of p16INK4a which inhibits cyclin D/CDK4 to suppress G1 phase progression, while upregulation of p14ARF leads to activation of p53/p21 and inhibits cyclin E/CDK4 to prevent S phase progression [7]. The global methylation status of fibroblasts is directly and strongly correlated with donor chronological age through regression algorithms known as ‘epigenetic clocks.’ These algorithms calculate a weighted linear combination of the beta coefficients (the percent signal from the methylated out of the total unmethylated and methylated alleles) [133]. When dermal fibroblasts were passaged towards replicative senescence (RS) these epigenetic clocks show aggregated methylomic evolution. The cell cycle was reengaged by overexpressing the telomerase gene hTERT, causing cells to progress to further doubling. However, the epigenetic clock did not reverse and the cells continued to age, bypassing RS, further hinting that a broader epigenetic change was occurring through the progression to senescence rather than just the suppression of a few mitotic arrest genes [134].
Conversely, sequence specific epigenetic regulation on the level of transcribed RNA is accomplished through feedback mechanisms by families of non-coding RNA-including microRNAs, siRNAs, long and short non-coding RNAs, and others. These non-coding RNAs will interact with other DNA, RNA, and proteins to regulate their expression, further enhancing the complexity of the transcriptome over the more rigid landscape of the methylome [135]. Thus, non-coding species are often used to not just reinforce but also propagate the senescence response. The particular influence of miRNAs, short sequences that complement and bind to specific regions of mRNAs to limit their stability and thus their translation likelihood, has been explored in the context of cutaneous cell senescence [136]. For instance, UV-induced senescent fibroblasts are known to produce miR-34 which targets a number of transcripts within these cells for cell cycle regulatory genes like MYC and BCL2 as well as genes for other epigenetic factors such as E2H and SIRT1 [137]. Meanwhile, in wounding-induced senescence the extracellular secretion of miR-21 as part of the SASP phenotype triggers the activation of resident macrophages to drive the local inflammatory response [138], but these represent only a few of a handful of drivers. Senescent keratinocytes, for instance, have displayed upregulation of over a hundred different microRNAs correlated with expression of the senescence biomarkers p16, p53, and senescence-associated β-galactosidase (SA-β-Gal) [136]. Together, these mechanisms represent the precise regulation of specific genomic targets and interfering with transcription machinery as one mode of enforcing the senescent epigenetic state.
4.2 Compaction
For regulation across gene domains (~150 base pairs or greater), the epigenome uses methods of physical compaction to close off regions of the genome from transcription. The negative charge of the DNA attracts it to wrap around the positively charged protein octamer spools called histones, which segregate the sequences away from transcription machinery. Chemical modifications like methylation, acetylation, and ubiquitination of the amino acid residues on the tails of these histone proteins alter the charge interaction with DNA and with other histones influencing oligomeric structure [139]. Senescence engages in this mechanism by modulating the enzymatic activity that regulates these histone tails. For example, the activity of methyltransferases like EZH2, which adds trimethylation to the lysine residue 27 of histone 3 (H2K27me3), is reduced in senescent cells. This reduction in the resulting H3K27me3, especially at the INK4a/ARF locus mentioned previously, reinforces the discontinuation of the cell cycle [8]. Other forms of histone tail modification include acetylation, which tends to promote more transcription. One of the most well-studied classes of deacetylation enzymes is the sirtuin family of proteins. In both fibroblasts and keratinocytes, Sirt1 and Sirt6 directly respond to DNA damage and inflammation, but their expression is diminished in senescent cells [140]. Interestingly, both Sirt1 and Sirt6 also play an active role in regulating collagen balance, thus their downregulation could be conceptually likened to senescence of the dermal ECM and its turnover, just like that of cellular turnover.
Histones can also be modified through changes within the core octamer proteins themselves and a hallmark example of this phenomenon is the variant species of the H2A protein known as H2A.J. This modified protein is prevalent in a lot of senescent skin cell types where it weakens the binding of another histone in the complex, H1, triggering a signaling cascade that preempts the interferon response and contributes to initiation of SASP expression [141]. In senescent epidermal keratinocytes in particular, the increase in H2A.J variants is correlated with arrested cell cycle and maturation of the basal cells into mature corneocytes, thus it may play a direct role in the morphologic phenomena of epidermal thinning seen with age [142]. The broader contribution of these histone changes, along with local DNA methylation shifts, is the transition to wide-reaching genome compaction in senescent cells, for example the condensation of senescence associated heterochromatin foci, as in H3K9me3 rich regions of nuclease resistant compact facultative heterochromatin [11]. These foci are seen across skin cell types like fibroblasts and keratinocytes and are thought to entrench the senescent state by long term segregation and silencing of mitotic genes [143]. However, the evolution of these foci seems to be specific to the type of senescence induction, most prominent in OIS, suggesting that senescence itself may even be a family of pseudo-states rather than a distinct, singular manifestation [144]. Nevertheless, in general, these forms of epigenetic modification which bias entire regions of genes from active to passive and vice versa truly embody a cell state/pseudostate.
4.3 Alternative epigenetic pseudostates
The natural and prevalent engagement of senescence, even in young tissues, reflects its role as a form of stress response. In fact, a major function of senescence is to prevent the evolution of alternate, more detrimental states of the cells and tissue under these conditions. One such competing epigenetic pseudostate is fibrosis. The fibrotic transition is a common feature in the pathological evolution of many tissues, i.e., hypertrophic scarring and keloids in the skin, idiopathic pulmonary fibrosis in the lung, cirrhosis in the liver [145]. A key component of fibrosis is the differentiation of various cell types including fibroblasts, adipocytes, epithelial cells, and endothelial cells into a population known as myofibroblasts [146]. As mentioned, differentiation is canonically an epigenetic event as cells convert and specify their functional gene regions while silencing other unused regions. It involves the same modalities of control—methylation, histone tags, chromatin structure, etc.—often with more dramatic and permanent modifications. These activated myofibroblasts are critical for the repair response in that they secrete superfluous extracellular matrix (ECM) components (Collagen 1, alpha-smooth muscle actin (α-SMA), fibronectin, etc) that accumulate in the connective tissue [147]. At the same time, these cells diminish the process of anabolic degradation of ECM through reduction of MMPs [148]. Unbridled overgrowth of these myofibroblasts, as evidenced by the overactivation of growth factors like connective tissue growth factor (CTGF), leads to the buildup and disorganization of the connective tissue [149]. Senescence in this context is thought to be a responsive, secondary epigenetic evolution that is engaged to shut down this population and stop the overgrowth [150]. These processes—from the epigenetic cell identity shift (e.g., epithelial-to-mesenchymal or fibroblast-to-myofibroblast transitions, depending on the starting cell types) to the epigenetic proliferation-suppressed state (induction of senescence)—represent relatively fast epigenetic turnovers. As such, a key mediator of this rapid transition is thought to be the slew of non-coding RNAs, like let-7 g to engage TGFbeta driven myoblast conversion and miR-127-3p to induce p53/p21 drivers of senescence [151, 152].
Another alternative pseudostate that competes with senescence is of course cancer and more particularly for skin, melanoma. Like senescence, cancer is a state transition that involves bypassing apoptotic pathways, yet these aberrant cells also bypass the suppression of their cell cycle gene networks [14]. It is thought that melanoma cells are able to undo the senescence epigenetics and re-engage the cell cycle due to the deleterious recruitment of epigenetic enzymes, like histone demethylases and Jumonji proteins [153]. This means a host of pathways whose methylation would otherwise lead to cell cycle suppression, like the p15INK4B or the p27Kip1 pathways, are methylated without cell cycle arrest in melanoma [154, 155, 156]. The use of inhibitors to target these epigenetic enzymes seems to be a promising methodology to restore the cell cycle arrest and control the cancerous growth [157].
An interesting intersection of epigenetic and oncogenic pseudostates is highlighted in Merkel cell carcinoma (MCC). This aggressive, non-melanoma skin cancer is rare but occurs primarily in the elderly and immunosuppressed. Interestingly, methylation clock analysis of MCC cells shows their epigenetic age as significantly younger than the chronologic age of the patients from which they were derived—a stark contrast from the continually progressing epigenetic age of senescent cells. Further analysis of these MCC cells did not indicate any signs of pluripotency [64]. The mechanism by which MCCs reverse their epigenetic age is still unknown, however, it may be related to other epigenetic alterations recently discovered in this cell type, including decreased H3K27me3 expression [158, 159] and overactivity of the lysine-specific histone demethylase 1A [160, 161]. These are just some examples of this fundamental need to tightly control and disengage mitotic networks and why senescence requires a complex regulatory architecture like the epigenome.
4.4 Enablers of senescence
The phenomenon of senescence is promoted by the epigenetics of not just the arrested cells in question, but also that of the other resident cells that enable this transition. Though senescence is thought to be a permanent state, the persistence of senescence in the tissue is only meant to be transient. This is because the key function of this state is to respond to stressors by retaining cells, despite their damage, to maintain the tissue temporarily while preventing them going down the more detrimental alternate routes mentioned, all the while signaling the immune system and other repair mechanisms. When the immune system is young and efficient its cells are recruited to the skin and other tissues to clear out the senescent cells [162]. With aging, however, the number and lifetime of these senescent populations increases due to the altered epigenetic pseudostates of the senescent clearing cells as well, contributing to innate immunosurveillance dysfunction of the skin. One example of this is in the dominance of the pro-inflammatory M1 macrophage pseudostate over the anti-inflammatory M2 macrophage pseudostate [163]. There are a number of histone methylation and acetylation modifiers that play a role in pseudostate fate decision, for instance histone deacetylase 3 promoting M1 macrophages or the SYMD family of methyltransferases promoting M2 macrophages [164]. With the accumulation of stressors over a lifetime, the more pro-inflammatory epigenetic pseudo-states are favored in skin and other tissues, especially in response to factors like SASP or inflammaging [165]. In addition, in some disease states like type 2 diabetes, the wound healing response and inflammation tends to exaggerate the M1 state response with focal DNA methylation components at sites like peroxisome proliferator activated receptor gamma (PPARγ) or and elevation of miR-125b [164]. This epigenetic shift in the balance of macrophage cells then ties back to senescence as the M1 macrophage predominantly engages in more phagocytic clearance of foreign pathogens, while the M2 macrophages carry out more phagocytic clearance of damaged host cells (efferocytosis) [166]. This, coupled with the fact that senescent cells develop ways to better evade apoptosis, means that they are more likely to accumulate [167] in aged tissue. There are additional immune cell types that are similarly driven by the pro-inflammatory transition, yet become impaired at senescent cell clearance, including NK cells and neutrophils [167]. Altogether, this epigenetic evolution of the regulator cells, part of inflammaging, proves just as critical to the manifestation of a sustained senescence pressure in cutaneous tissue as epigenetic changes engaged in the non-dividing cells themselves.
4.4.1 Distinction from temporary cell cycle arrest
Though sometimes associated with senescence, somatic stem cells (as opposed to differentiated cells) are typically associated with another form of cell cycle arrest, known as quiescence. Because their role is to remain as a niched tissue reserve, they often enter periods of temporary cell cycle arrest with a prolonged G0, instead of a permanent one, until they are called to activate, to proliferate and differentiate, by a stressor [168]. One major epigenetic distinction that enables this temporary quiescence vs. permanent senescence is the utilization of bivalent domains. These are regions of genes that are regulated by both a repressive histone tag as well as an activating one, that allows the region to rapidly switch from one state to another depending on stimulus [169]. A prime example of the use of this is in the coinciding utilization of repressive H3K27me3 and activating H3K4me3, which maintains a tenuous baseline suppression of the gene region. This pair forming a bivalent domain is widely used throughout the embryonic stem cell genome, establishing its broad potency as a cell type with the potential to express a lot of different proteins [170]. But when the same domains were searched for in dermal hair follicle stem cells (HFSC), they were found to be substantially restricted to lineage-specific factors like Sox9 and Nfatc1 and growth factor FGF18 [171]. Then, when these HFSC were stimulated to activate, many of the genes with H3K4me3 activating markers, which are located primarily near the gene promoters, were further reinforced by additional H3K79 dimethylation in the gene body, to tip the scale from suppression to activation [171]. These genes included many cell cycle regulators which, when combined with the cell lineage factors, properly executed differentiation. Thus, this mechanism of readily switchable suppression to expression establishes a major distinction in epigenetic regulation from cell cycle in quiescence from that of senescence where the cell cycle genes are more permanently, epigenetically suppressed.
4.4.2 Manipulability of senescent epigenetics
Earlier, we mentioned how drugs targeting epigenetic enzymes represent one methodology for modulating some of the epigenetic changes that drive senescence, such as senolytic therapies. However, a broader and more dramatic approach of epigenetic evolution is through the process of cellular reprogramming. This technology was inspired by the core epigenetic reset that occurs during the process of reproduction in which sperm and egg, two cells with very precise roles and epigenetic identities, are reprogrammed to make embryonic cells—epigenetically plastic cells that can differentiate into any cell in the body. The isolation and recapitulation of this process in any desired cell type was achieved through the discovery of core transcription factors [172]. When overexpressed in cells, this set of core transcription factors would drive a full epigenetic remodeling to produce embryonic-like cells with all their differentiation potential. This process is called induced pluripotent stem cell reprogramming (iPSC) and has been utilized in a variety of different cell types with dermal fibroblasts being the gold standard for many studies [173]. Even fully senescent fibroblast populations established from 51 population doublings and maintained for two months in culture, successfully showed iPSC reprogramming, as evidenced by revived proliferation, reduced p16 and p21, and re-differentiation after reaching the pluripotent state [174]. Crucially, the re-differentiated progeny were once again able to be passaged into senescence, thus suggesting that malignant transformation was not induced during the entire process. Furthermore, one of the key reprogramming factors Oct4, has been shown to independently re-engage senescent hair follicle mesenchymal stem cells back into cycling by engaging a host of DNA methyltransferase to inhibit the p21 pathway [175]. More recently researchers have shown that the prevalence of senescence in a population can be reduced with even a transient application of the reprogramming factors [176, 177, 178]. Though whether this means a re-engagement of senescent cells in the cell cycle or simply competitive growth advantage of healthy cells remains to be seen. This represents an enticing new possibility in that epigenetic manipulation may possibly counter the accumulation of senescent cells in many aged and diseased tissues, including the skin.
5. Conclusion
The skin represents an excellent organ system in which the effects of cellular senescence manifest as observed clinical changes in organismal health and disease. A myriad of processes drives the genomic erosion that instigates the transition to senescence. Some of these processes are more stereotyped, engineered into the cell by design, and are observed in chronologically aged skin, while others are stochastic and driven by environmental conditions, exemplified by exposomal damage. Either way, the result is an evolution of the entire state of the cell. This means more than just the direct arrestation of the cell cycle, but also entails changes through the cellular transcriptome, proteome, and secretome as encoded by alterations to the core cellular epigenome. This also involves a myriad of changes to the many layers of architecture that encode a cell’s function and identity. The global process is critical for the skin’s ability to retain functional integrity upon stress and insult as the first line of defense for the body, and in many ways, senescence represents the least of multiple evils.
This review gives a glimpse of how and why intrinsic and extrinsic factors trigger cutaneous cellular senescent phenotypes, leaving several important questions unanswered. For example, which genetic and epigenetic factors determine the dominant decision pathways favoring senescence vs. apoptosis or any other disease states for different skin compartments? How are the various types of senescence manifestations comparable in terms of evolution and manipulability? What are the molecular and cellular consequences of therapeutic re-engagement of senescent cells into the cell cycle? As the focus on aging grows as an ever more prominent factor in clinical and investigative dermatology, insights on these questions into the nature of senescence become a critical step towards both dermatologic therapeutic advancement specifically and translational medicine in general.
Acknowledgments
Intellectual and financial support by Turn Biotechnologies, Inc. is gratefully appreciated.
Conflict of interest
The authors declare no conflict of interest.
\n',keywords:"skin, DNA damage, telomeres, epigenetics, immunosenescence, inflammaging",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/79295.pdf",chapterXML:"https://mts.intechopen.com/source/xml/79295.xml",downloadPdfUrl:"/chapter/pdf-download/79295",previewPdfUrl:"/chapter/pdf-preview/79295",totalDownloads:105,totalViews:0,totalCrossrefCites:0,dateSubmitted:"July 1st 2021",dateReviewed:"October 10th 2021",datePrePublished:"November 11th 2021",datePublished:null,dateFinished:"November 11th 2021",readingETA:"0",abstract:"Skin is the largest human organ system, and its protective function is critical to survival. The epithelial, dermal, and subcutaneous compartments are heterogeneous mixtures of cell types, yet they all display age-related skin dysfunction through the accumulation of an altered phenotypic cellular state called senescence. Cellular senescence is triggered by complex and dynamic genetic and epigenetic processes. A senescence steady state is achieved in different cell types under various and overlapping conditions of chronological age, toxic injury, oxidative stress, replicative exhaustion, DNA damage, metabolic dysfunction, and chromosomal structural changes. These inputs lead to outputs of cell-cycle withdrawal and the appearance of a senescence-associated secretory phenotype, both of which accumulate as tissue pathology observed clinically in aged skin. This review details the influence of genetic and epigenetic factors that converge on normal cutaneous cellular processes to create the senescent state, thereby dictating the response of the skin to the forces of both intrinsic and extrinsic aging. From this work, it is clear that no single biomarker or process leads to senescence, but that it is a convergence of factors resulting in an overt aging phenotype.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/79295",risUrl:"/chapter/ris/79295",signatures:"Tapash Jay Sarkar, Maiko Hermsmeier, Jessica L. Ross and G. Scott Herron",book:{id:"10935",type:"book",title:"Senescence",subtitle:null,fullTitle:"Senescence",slug:null,publishedDate:null,bookSignature:"Dr. Hassan M. Heshmati",coverURL:"https://cdn.intechopen.com/books/images_new/10935.jpg",licenceType:"CC BY 3.0",editedByType:null,isbn:"978-1-83969-051-8",printIsbn:"978-1-83969-050-1",pdfIsbn:"978-1-83969-052-5",isAvailableForWebshopOrdering:!0,editors:[{id:"313921",title:"Dr.",name:"Hassan M.",middleName:null,surname:"Heshmati",slug:"hassan-m.-heshmati",fullName:"Hassan M. Heshmati"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Aging of the skin",level:"1"},{id:"sec_2_2",title:"2.1 Effects of aging on epidermal structure and function",level:"2"},{id:"sec_3_2",title:"2.2 Aging and the dermis",level:"2"},{id:"sec_4_2",title:"2.3 Subcutaneous layer involvement in skin aging dysfunction",level:"2"},{id:"sec_6",title:"3. Genetic influences on cutaneous cellular senescence",level:"1"},{id:"sec_6_2",title:"3.1 DNA damage related to telomere biology",level:"2"},{id:"sec_7_2",title:"3.2 Genotoxic and exposome insults",level:"2"},{id:"sec_8_2",title:"3.3 Genetic skin diseases associated with DNA repair pathway defects",level:"2"},{id:"sec_9_2",title:"3.4 SNPs and transcriptomics",level:"2"},{id:"sec_11",title:"4. Epigenetic influences on cutaneous cellular senescence",level:"1"},{id:"sec_11_2",title:"4.1 Sequence specific modulation",level:"2"},{id:"sec_12_2",title:"4.2 Compaction",level:"2"},{id:"sec_13_2",title:"4.3 Alternative epigenetic pseudostates",level:"2"},{id:"sec_14_2",title:"4.4 Enablers of senescence",level:"2"},{id:"sec_14_3",title:"4.4.1 Distinction from temporary cell cycle arrest",level:"3"},{id:"sec_15_3",title:"4.4.2 Manipulability of senescent epigenetics",level:"3"},{id:"sec_18",title:"5. Conclusion",level:"1"},{id:"sec_19",title:"Acknowledgments",level:"1"},{id:"sec_22",title:"Conflict of interest",level:"1"}],chapterReferences:[{id:"B1",body:'Rodier F, Campisi J. Four faces of cellular senescence. The Journal of Cell Biology. 2011;192(4):547-556'},{id:"B2",body:'Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C, et al. Cellular senescence: Defining a path forward. Cell. 2019;179(4):813-827'},{id:"B3",body:'Hayflick L, Moorhead PS. 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Molecular Cell Biology. 2014;15(7):482-496'},{id:"B155",body:'Worm J, Bartkova J, Kirkin AF, Straten P, Zeuthen J, Bartek J, et al. Aberrant p27Kip1 promoter methylation in malignant melanoma. Oncogene. 2000;19(44):5111-5115'},{id:"B156",body:'Sarkar D, Leung EY, Baguley BC, Finlay GJ, Askarian-Amiri ME. Epigenetic regulation in human melanoma: Past and future. Epigenetics. 2015;10(2):103-121'},{id:"B157",body:'Rowdo FPM, Barón A, Gallagher SJ, Hersey P, Emran AA, Von Euw EM, et al. Epigenetic inhibitors eliminate senescent melanoma BRAFV600E cells that survive long-term BRAF inhibition. International Journal of Oncology. 2020;56(6):1429-1441'},{id:"B158",body:'Busam KJ, Pulitzer MP, Coit DC, Arcila M, Leng D, Jungbluth AA, et al. Reduced H3K27me3 expression in Merkel cell polyoma virus-positive tumors. Modern Pathology. 2017;30(6):877-883'},{id:"B159",body:'Gujar H, Mehta A, Li H-T, Tsai YC, Qiu X, Weisenberger DJ, et al. Characterizing DNA methylation signatures and their potential functional roles in Merkel cell carcinoma. Genome Medicine. 2021;13(1):130'},{id:"B160",body:'Park DE, Cheng J, McGrath JP, Lim MY, Cushman C, Swanson SK, et al. Merkel cell polyomavirus activates LSD1-mediated blockade of non-canonical BAF to regulate transformation and tumorigenesis. Nature Cell Biology. 2020;22(5):603-615'},{id:"B161",body:'Leiendecker L, Jung PS, Krecioch I, Neumann T, Schleiffer A, Mechtler K, et al. LSD1 inhibition induces differentiation and cell death in Merkel cell carcinoma. EMBO Molecular Medicine. 2020;12(11):e12525'},{id:"B162",body:'Elder SS, Emmerson E. Senescent cells and macrophages: Key players for regeneration? Open Biology. 2020;10(12):200309'},{id:"B163",body:'Krzyszczyk P, Schloss R, Palmer A, Berthiaume F. The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes. Frontiers in Physiology. 2018;9:419'},{id:"B164",body:'Davis FM, Gallagher KA. Epigenetic mechanisms in monocytes/macrophages regulate inflammation in cardiometabolic and vascular disease. Arteriosclerosis, Thrombosis, and Vascular Biology. 2019;39(4):623-634'},{id:"B165",body:'Zhuang Y, Lyga J. Inflammaging in skin and other tissues—The roles of complement system and macrophage. Inflammation & Allergy Drug Targets. 2014;13(3):153-161'},{id:"B166",body:'Korns D, Frasch SC, Fernandez-Boyanapalli R, Henson PM, Bratton DL. Modulation of macrophage efferocytosis in inflammation. Frontiers in Immunology. 2011;2:57'},{id:"B167",body:'Kale A, Sharma A, Stolzing A, Desprez P-Y, Campisi J. Role of immune cells in the removal of deleterious senescent cells. Immunity & Ageing. 2020;17(1):16'},{id:"B168",body:'Watt FM, Jensen KB. Epidermal stem cell diversity and quiescence. EMBO Molecular Medicine. 2009;1(5):260-267'},{id:"B169",body:'Voigt P, Tee W-W, Reinberg D. A double take on bivalent promoters. Genes & Development. 2013;27(12):1318-1338'},{id:"B170",body:'Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006;125(2):315-326'},{id:"B171",body:'Lien W-H, Guo X, Polak L, Lawton LN, Young RA, Zheng D, et al. Genome-wide maps of histone modifications unwind in vivo chromatin states of the hair follicle lineage. Cell Stem Cell. 2011;9(3):219-232'},{id:"B172",body:'Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872'},{id:"B173",body:'Malik N, Rao MS. A review of the methods for human iPSC derivation. Methods in Molecular Biology (Clifton, N.J.). 2013;997:23-33'},{id:"B174",body:'Lapasset L, Milhavet O, Prieur A, Besnard E, Babled A, Aït-Hamou N, et al. Rejuvenating senescent and centenarian human cells by reprogramming through the pluripotent state. Genes & Development. 2011;25(21):2248-2253'},{id:"B175",body:'Lu Y, Qu H, Qi D, Xu W, Liu S, Jin X, et al. OCT4 maintains self-renewal and reverses senescence in human hair follicle mesenchymal stem cells through the downregulation of p21 by DNA methyltransferases. Stem Cell Research & Therapy. 2019;10(1):28'},{id:"B176",body:'Ocampo A, Reddy P, Martinez-Redondo P, Platero-Luengo A, Hatanaka F, Hishida T, et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell. 2016;167(7):1719-1733.e12'},{id:"B177",body:'Kurita M, Araoka T, Hishida T, O’Keefe DD, Takahashi Y, Sakamoto A, et al. In vivo reprogramming of wound-resident cells generates skin epithelial tissue. Nature. 2018;561(7722):243-247'},{id:"B178",body:'Sarkar TJ, Quarta M, Mukherjee S, Colville A, Paine P, Doan L, et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nature Communications. 2020;11(1):1-12'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Tapash Jay Sarkar",address:null,affiliation:'
Turn Biotechnologies, Inc., Mountain View, California, USA
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Turn Biotechnologies, Inc., Mountain View, California, USA
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Turn Biotechnologies, Inc., Mountain View, California, USA
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Physical Sciences, Technology and Engineering Board
\\n\\n
Chemistry
\\n\\n
\\n\\t
Ayben Kilislioglu - Department of Chemical Engineering Istanbul University, İstanbul, Turkey
\\n\\t
Goran Nikolic - Faculty of Technology, University of Nis, Leskovac, Serbia
\\n\\t
Mark T. Stauffer - Associate Professor of Chemistry, The University of Pittsburgh, USA
\\n\\t
Margarita Stoytcheva - Autonomous University of Baja California Engineering Institute Mexicali, Baja California, Mexico
Joao Luis Garcia Rosa - Associate Professor Bio-inspired Computing Laboratory (BioCom) Department of Computer Science University of Sao Paulo (USP) at Sao Carlos, Brazil
\\n\\t
Jan Valdman - Institute of Mathematics and Biomathematics, University of South Bohemia, České Budějovice, Czech Republic Institute of Information Theory and Automation of the ASCR, Prague, Czech Republic
\\n
\\n\\n
Earth and Planetary Science
\\n\\n
\\n\\t
Jill S. M. Coleman - Department of Geography, Ball State University, Muncie, IN, USA
\\n\\t
İbrahim Küçük Erciyes - Üniversitesi Department of Astronomy and Space Sciences Melikgazi, Kayseri, Turkey
\\n\\t
Pasquale Imperatore - Electromagnetic Environmental Sensing (IREA), Italian National Council of Research (CNR), Naples, Italy
\\n\\t
Mohammad Mokhtari - Director of National Center for Earthquake Prediction International Institute of Earthquake Engineering and Seismology (IIEES), Tehran, Iran
\\n
\\n\\n
Engineering
\\n\\n
\\n\\t
Narottam Das - University of Southern Queensland, Australia
\\n\\t
Jose Ignacio Huertas - Energy and Climate Change Research Group; Instituto Tecnológico y Estudios Superiores de Monterrey, Mexico
Likun Pan - Engineering Research Center for Nanophotonics and Advanced Instrument, Ministry of Education, Department of Physics, East China Normal University, China
\\n\\t
Mukul Chandra Paul - Central Glass & Ceramic Research Institute Jadavpur, Kolkata, India
\\n\\t
Stephen E. Saddow - Electrical Engineering Department, University of South Florida, USA
\\n\\t
Ali Demir Sezer - Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, İstanbul, Turkey
\\n\\t
Krzysztof Zboinski - Warsaw University of Technology, Faculty of Transport, Warsaw, Poland
\\n
\\n\\n
Materials Science
\\n\\n
\\n\\t
Vadim Glebovsky - Senior Researcher, Institute of Solid State Physics, Chernogolovka, Russia Expert of the Russian Fund for Basic Research, Moscow, Russia
\\n\\t
Jianjun Liu - State Key Laboratory of High Performance Ceramics and Superfine Microstructure of Shanghai Institute of Ceramics, Chinese Academy of Sciences, China
\\n\\t
Pietro Mandracci - Department of Applied Science and Technology, Politecnico di Torino, Italy
\\n\\t
Waldemar Alfredo Monteiro - Instituto de Pesquisas Energéticas e Nucleares Materials Science and Technology Center (MSTC) São Paulo, SP, Brazil
Toshio Ogawa - Department of Electrical and Electronic Engineering, Shizuoka Institute of Science and Technology, Toyosawa, Fukuroi, Shizuoka, Japan
\\n
\\n\\n
Mathematics
\\n\\n
\\n\\t
Paul Bracken - Department of Mathematics University of Texas, Edinburg, TX, USA
\\n
\\n\\n
Nanotechnology and Nanomaterials
\\n\\n
\\n\\t
Muhammad Akhyar - Farrukh Nano-Chemistry Lab. Registrar, GC University Lahore, Pakistan
\\n\\t
Khan Maaz - Chinese Academy of Sciences, China & The Pakistan Institute of Nuclear Science and Technology, Pakistan
\\n
\\n\\n
Physics
\\n\\n
\\n\\t
Izabela Naydenova - Lecturer, School of Physics Principal Investigator, IEO Centre College of Sciences and Health Dublin Institute of Technology Dublin, Ireland
\\n\\t
Mitsuru Nenoi - National Institute of Radiological Sciences, Japan
\\n\\t
Christos Volos - Physics Department, Aristotle University of Thessaloniki, Greece
\\n
\\n\\n
Robotics
\\n\\n
\\n\\t
Alejandra Barrera - Instituto Tecnológico Autónomo de México, México
\\n\\t
Dusan M. Stipanovic - Department of Industrial and Enterprise Systems Engineering, University of Illinois at Urbana-Champaign
\\n\\t
Andrzej Zak - Polish Naval Academy Faculty of Navigation and Naval Weapons Institute of Naval Weapons and Computer Science, Gdynia, Poland
Petr Konvalina - Faculty of Agriculture, University of South Bohemia in České Budějovice, Czech Republic
\\n
\\n\\n
Biochemistry, Genetics and Molecular Biology
\\n\\n
\\n\\t
Chunfa Huang - Saint Louis University, Saint Louis, USA
\\n\\t
Michael Kormann - University Children's Clinic Department of Pediatrics I, Pediatric Infectiology & Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tübingen, Tübingen, Germany
\\n\\t
Bin WU - Ph.D. HCLD Scientific Laboratory Director, Assisted Reproductive Technology Arizona Center for Reproductive Endocrinology and Infertility Tucson, Arizona , USA
\\n
\\n\\n
Environmental Sciences
\\n\\n
\\n\\t
Juan A. Blanco - Senior Researcher & Marie Curie Research Fellow Dep. Ciencias del Medio Natural, Universidad Publica de Navarra Campus de Arrosadia, Pamplona, Navarra, Spain
\\n\\t
Mikkola Heimo - University of Eastern Finland, Kuopio, Finland
\\n\\t
Bernardo Llamas Moya - Politechnical University of Madrid, Spain
\\n\\t
Toonika Rinken - Department of Environmental Chemistry, University of Tartu, Estonia
\\n
\\n\\n
Immunology and Microbiology
\\n\\n
\\n\\t
Dharumadurai Dhanasekaran - Department of Microbiology, School of Life Sciences, Bharathidasan University, India
Isabel Gigli - Facultad de Agronomia-UNLPam, Argentina
\\n\\t
Milad Manafi - Department of Animal Science, Faculty of Agricultural Sciences, Malayer University, Malayer, Iran
\\n\\t
Rita Payan-Carreira - Universidade de Trás-os-Montes e Alto Douro, Departamento de Zootecnia, Portugal
\\n
\\n\\n
Medicine
\\n\\n
\\n\\t
Mazen Almasri - King Abdulaziz University, Faculty of Dentistry Jeddah, Saudi Arabia Dentistry
\\n\\t
Craig Atwood - University of Wisconsin-Madison, USA Stem Cell Research, Tissue Engineering and Regenerative Medicine
\\n\\t
Oreste Capelli - Clinical Governance, Local Health Authority, Modena, Italy Public Health
\\n\\t
Michael Firstenberg - Assistant Professor of Surgery and Integrative Medicine NorthEast Ohio Medical University, USA & Akron City Hospital - Summa Health System, USA Surgery
\\n\\t
Parul Ichhpujani - MD Government Medical College & Hospital, Department of Ophthalmology, India
Amidou Samie - University of Venda, SA Infectious Diseases
\\n\\t
Shailendra K. Saxena - CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India Infectious Diseases
\\n\\t
Dan T. Simionescu - Department of Bioengineering, Clemson University, Clemson SC, USA Stem Cell Research, Tissue Engineering and Regenerative Medicine
\\n\\t
Ke Xu - Tianjin Lung Cancer Institute Tianjin Medical University General Hospital Tianjin, China Oncology
\\n
\\n\\n
Ophthalmology
\\n\\n
\\n\\t
Hojjat Ahmadzadehfar - University Hospital Bonn Department of Nuclear Medicine Bonn, Germany Medical Diagnostics, Engineering Technology and Telemedicine
\\n\\t
Miroslav Blumenberg - Department of Ronald O. Perelman Department of Dermatology; Department of Biochemistry and Molecular Pharmacology, Dermatology, NYU School of Medicine, NY, USA Dermatology
\\n\\t
Wilfred Bonney - University of Dundee, Scotland, UK Medical Diagnostics, Engineering Technology and Telemedicine
\\n\\t
Christakis Constantinides - Department of Cardiovascular Medicine University of Oxford, Oxford, UK Medical Diagnostics, Engineering Technology and Telemedicine
\\n\\t
Atef Mohamed Mostafa Darwish - Department of Obstetrics and Gynecology , Faculty of Medicine, Assiut University, Egypt Gynecology
\\n\\t
Ana Polona Mivšek - University of Ljubljana, Ljubljana, Slovenia Midwifery
\\n\\t
Gyula Mozsik - First Department of Medicine, Medical and Health Centre, University of Pécs, Hungary
\\n\\t
Shimon Rumelt - Western Galilee-Nahariya Medical Center, Nahariya, Israel Ophthalmology
\\n\\t
Marcelo Saad - S. Paulo Medical College of Acupuncture, SP, Brazil Complementary and Alternative Medicine
\\n\\t
Minoru Tomizawa - National Hospital Organization Shimoshizu Hospital, Japan Gastroenterology
\\n\\t
Pierre Vereecken - Centre Hospitalier Valida and Cliniques Universitaires Saint-Luc, Belgium Dermatology
\\n
\\n\\n
Gastroenterology
\\n\\n
\\n\\t
Hany Aly - Director, Division of Newborn Services The George Washington University Hospital Washington, USA Pediatrics
\\n\\t
Yannis Dionyssiotis - National and Kapodistrian University of Athens, Greece Orthopedics, Rehabilitation and Physical Medicine
\\n\\t
Alina Gonzales- Quevedo Instituto de Neurología y Neurocirugía Havana, Cuba Mental and Behavioural Disorders and Diseases of the Nervous System
\\n\\t
Margarita Guenova - National Specialized Hospital for Active Treatment of Haematological Diseases, Bulgaria
\\n\\t
Eliska Potlukova - Clinic of Medicine, University Hospital Basel, Switzerland Edocrinology
\\n\\t
Raymond L. Rosales -The Royal and Pontifical University of Santo Tomas, Manila, Philippines & Metropolitan Medical Center, Manila, Philippines & St. Luke's Medical Center International Institute in Neuroscience, Quezon City, Philippines Mental and Behavioural Disorders and Diseases of the Nervous System
\\n\\t
Alessandro Rozim - Zorzi University of Campinas, Departamento de Ortopedia e Traumatologia, Campinas, SP, Brazil Orthopedics, Rehabilitation and Physical Medicine
\\n\\t
Dieter Schoepf - University of Bonn, Germany Mental and Behavioural Disorders and Diseases of the Nervous System
\\n
\\n\\n
Hematology
\\n\\n
\\n\\t
Hesham Abd El-Dayem - National Liver Institute, Menoufeyia University, Egypt Hepatology
\\n\\t
Fayez Bahmad - Health Science Faculty of the University of Brasilia Instructor of Otology at Brasilia University Hospital Brasilia, Brazil Otorhinolaryngology
\\n\\t
Peter A. Clark - Saint Joseph's University Philadelphia, Pennsylvania, USA Bioethics
\\n\\t
Celso Pereira - Coimbra University, Coimbra, Portugal Immunology, Allergology and Rheumatology
\\n\\t
Luis Rodrigo - Asturias Central University Hospital (HUCA) School of Medicine, University of Oviedo, Oviedo, Spain Hepatology & Gastroenterology
\\n\\t
Dennis Wat - Liverpool Heart and Chest Hospital NHS Foundation Trust, UK Pulmonology
\\n
\\n\\n
Social Sciences and Humanities Board
\\n\\n
Business, Management and Economics
\\n\\n
\\n\\t
Vito Bobek - University of Applied Sciences, FH Joanneum, Graz, Austria
Joao Luis Garcia Rosa - Associate Professor Bio-inspired Computing Laboratory (BioCom) Department of Computer Science University of Sao Paulo (USP) at Sao Carlos, Brazil
\n\t
Jan Valdman - Institute of Mathematics and Biomathematics, University of South Bohemia, České Budějovice, Czech Republic Institute of Information Theory and Automation of the ASCR, Prague, Czech Republic
\n
\n\n
Earth and Planetary Science
\n\n
\n\t
Jill S. M. Coleman - Department of Geography, Ball State University, Muncie, IN, USA
\n\t
İbrahim Küçük Erciyes - Üniversitesi Department of Astronomy and Space Sciences Melikgazi, Kayseri, Turkey
\n\t
Pasquale Imperatore - Electromagnetic Environmental Sensing (IREA), Italian National Council of Research (CNR), Naples, Italy
\n\t
Mohammad Mokhtari - Director of National Center for Earthquake Prediction International Institute of Earthquake Engineering and Seismology (IIEES), Tehran, Iran
\n
\n\n
Engineering
\n\n
\n\t
Narottam Das - University of Southern Queensland, Australia
\n\t
Jose Ignacio Huertas - Energy and Climate Change Research Group; Instituto Tecnológico y Estudios Superiores de Monterrey, Mexico
Likun Pan - Engineering Research Center for Nanophotonics and Advanced Instrument, Ministry of Education, Department of Physics, East China Normal University, China
\n\t
Mukul Chandra Paul - Central Glass & Ceramic Research Institute Jadavpur, Kolkata, India
\n\t
Stephen E. Saddow - Electrical Engineering Department, University of South Florida, USA
\n\t
Ali Demir Sezer - Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, İstanbul, Turkey
\n\t
Krzysztof Zboinski - Warsaw University of Technology, Faculty of Transport, Warsaw, Poland
\n
\n\n
Materials Science
\n\n
\n\t
Vadim Glebovsky - Senior Researcher, Institute of Solid State Physics, Chernogolovka, Russia Expert of the Russian Fund for Basic Research, Moscow, Russia
\n\t
Jianjun Liu - State Key Laboratory of High Performance Ceramics and Superfine Microstructure of Shanghai Institute of Ceramics, Chinese Academy of Sciences, China
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Pietro Mandracci - Department of Applied Science and Technology, Politecnico di Torino, Italy
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Waldemar Alfredo Monteiro - Instituto de Pesquisas Energéticas e Nucleares Materials Science and Technology Center (MSTC) São Paulo, SP, Brazil
Toshio Ogawa - Department of Electrical and Electronic Engineering, Shizuoka Institute of Science and Technology, Toyosawa, Fukuroi, Shizuoka, Japan
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Mathematics
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Paul Bracken - Department of Mathematics University of Texas, Edinburg, TX, USA
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Nanotechnology and Nanomaterials
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Muhammad Akhyar - Farrukh Nano-Chemistry Lab. Registrar, GC University Lahore, Pakistan
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Khan Maaz - Chinese Academy of Sciences, China & The Pakistan Institute of Nuclear Science and Technology, Pakistan
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Physics
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Izabela Naydenova - Lecturer, School of Physics Principal Investigator, IEO Centre College of Sciences and Health Dublin Institute of Technology Dublin, Ireland
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Mitsuru Nenoi - National Institute of Radiological Sciences, Japan
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Christos Volos - Physics Department, Aristotle University of Thessaloniki, Greece
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Robotics
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Alejandra Barrera - Instituto Tecnológico Autónomo de México, México
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Dusan M. Stipanovic - Department of Industrial and Enterprise Systems Engineering, University of Illinois at Urbana-Champaign
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Andrzej Zak - Polish Naval Academy Faculty of Navigation and Naval Weapons Institute of Naval Weapons and Computer Science, Gdynia, Poland
Petr Konvalina - Faculty of Agriculture, University of South Bohemia in České Budějovice, Czech Republic
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Biochemistry, Genetics and Molecular Biology
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Chunfa Huang - Saint Louis University, Saint Louis, USA
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Michael Kormann - University Children's Clinic Department of Pediatrics I, Pediatric Infectiology & Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tübingen, Tübingen, Germany
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Bin WU - Ph.D. HCLD Scientific Laboratory Director, Assisted Reproductive Technology Arizona Center for Reproductive Endocrinology and Infertility Tucson, Arizona , USA
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Environmental Sciences
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Juan A. Blanco - Senior Researcher & Marie Curie Research Fellow Dep. Ciencias del Medio Natural, Universidad Publica de Navarra Campus de Arrosadia, Pamplona, Navarra, Spain
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Mikkola Heimo - University of Eastern Finland, Kuopio, Finland
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Bernardo Llamas Moya - Politechnical University of Madrid, Spain
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Toonika Rinken - Department of Environmental Chemistry, University of Tartu, Estonia
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Immunology and Microbiology
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Dharumadurai Dhanasekaran - Department of Microbiology, School of Life Sciences, Bharathidasan University, India
Isabel Gigli - Facultad de Agronomia-UNLPam, Argentina
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Milad Manafi - Department of Animal Science, Faculty of Agricultural Sciences, Malayer University, Malayer, Iran
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Rita Payan-Carreira - Universidade de Trás-os-Montes e Alto Douro, Departamento de Zootecnia, Portugal
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Medicine
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Mazen Almasri - King Abdulaziz University, Faculty of Dentistry Jeddah, Saudi Arabia Dentistry
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Craig Atwood - University of Wisconsin-Madison, USA Stem Cell Research, Tissue Engineering and Regenerative Medicine
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Oreste Capelli - Clinical Governance, Local Health Authority, Modena, Italy Public Health
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Michael Firstenberg - Assistant Professor of Surgery and Integrative Medicine NorthEast Ohio Medical University, USA & Akron City Hospital - Summa Health System, USA Surgery
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Parul Ichhpujani - MD Government Medical College & Hospital, Department of Ophthalmology, India
Amidou Samie - University of Venda, SA Infectious Diseases
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Shailendra K. Saxena - CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India Infectious Diseases
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Dan T. Simionescu - Department of Bioengineering, Clemson University, Clemson SC, USA Stem Cell Research, Tissue Engineering and Regenerative Medicine
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Ke Xu - Tianjin Lung Cancer Institute Tianjin Medical University General Hospital Tianjin, China Oncology
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Ophthalmology
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Hojjat Ahmadzadehfar - University Hospital Bonn Department of Nuclear Medicine Bonn, Germany Medical Diagnostics, Engineering Technology and Telemedicine
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Miroslav Blumenberg - Department of Ronald O. Perelman Department of Dermatology; Department of Biochemistry and Molecular Pharmacology, Dermatology, NYU School of Medicine, NY, USA Dermatology
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Wilfred Bonney - University of Dundee, Scotland, UK Medical Diagnostics, Engineering Technology and Telemedicine
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Christakis Constantinides - Department of Cardiovascular Medicine University of Oxford, Oxford, UK Medical Diagnostics, Engineering Technology and Telemedicine
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Atef Mohamed Mostafa Darwish - Department of Obstetrics and Gynecology , Faculty of Medicine, Assiut University, Egypt Gynecology
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Ana Polona Mivšek - University of Ljubljana, Ljubljana, Slovenia Midwifery
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Gyula Mozsik - First Department of Medicine, Medical and Health Centre, University of Pécs, Hungary
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Shimon Rumelt - Western Galilee-Nahariya Medical Center, Nahariya, Israel Ophthalmology
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Marcelo Saad - S. Paulo Medical College of Acupuncture, SP, Brazil Complementary and Alternative Medicine
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Minoru Tomizawa - National Hospital Organization Shimoshizu Hospital, Japan Gastroenterology
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Pierre Vereecken - Centre Hospitalier Valida and Cliniques Universitaires Saint-Luc, Belgium Dermatology
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Gastroenterology
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Hany Aly - Director, Division of Newborn Services The George Washington University Hospital Washington, USA Pediatrics
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Yannis Dionyssiotis - National and Kapodistrian University of Athens, Greece Orthopedics, Rehabilitation and Physical Medicine
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Alina Gonzales- Quevedo Instituto de Neurología y Neurocirugía Havana, Cuba Mental and Behavioural Disorders and Diseases of the Nervous System