*Article* **Risk Assessment for Musculoskeletal Disorders in Forestry: A Comparison between RULA and REBA in the Manual Feeding of a Wood-Chipper**

**Margherita Micheletti Cremasco 1, Ambra Giustetto 1, Federica Caffaro 2, Andrea Colantoni 3, Eugenio Cavallo 2,\* and Stefano Grigolato 4**

Received: 10 December 2018; Accepted: 27 February 2019; Published: 5 March 2019

**Abstract:** The analysis of the postural attitude of workers during the interaction with workstation's elements and working environment is essential in the evaluation and prevention of biomechanical overload risk in workplaces. RULA (Rapid Upper Limb Assessment) and REBA (Rapid Entire Body Assessment) are the two easiest methods for postural risk assessment in the workplace. Few studies investigated postural risk in forestry sector with regard to human–machine interaction, in particular manually fed wood-chippers. The aim of this study was to evaluate the postures assumed by an operator during the manual feeding of a wood-chipper, and to compare RULA and REBA, in order to identify the more effective and appropriate method for the assessment of the risk of biomechanical postural overload. The results pointed out several postural issues of the upper limbs, and showed that RULA is a more precautionary method to protect operator's health during the targeted tasks. Implications to improve the human–wood-chipper interaction are discussed.

**Keywords:** ergonomics; forestry; postural risk assessment; RULA; REBA; wood-chipper

### **1. Introduction**

The analysis of the postural attitude of the worker during the interaction with workstation's elements and working environment is essential in the evaluation and prevention of biomechanical overload risks in workplaces [1]. Awkward working postures may decrease the workers' concentration and increase accidents frequency and biomechanical overload [2–6], giving rise to musculoskeletal disorders in the different body regions involved, as at the main limb joints level and the vertebral column [7].

Some standards [8–10] dealing with the biomechanical overload caused by incongruous static and dynamic postures have been developed to define the risk assessment methods to evaluate postural load referable to activity and workstation characteristics and to human–machine interaction. They are referenced in international [11] and national [12] legislations aimed at protecting workers' health and safety.

RULA (Rapid Upper Limb Assessment) [13] and REBA (Rapid Entire Body Assessment) [14] are two easy methods for occupational postural risk assessment. Indeed, previous studies [15,16] showed that observational methods are considered effective in the assessment of biomechanical work-related overload, having the advantage of being more versatile and less expensive in terms of time and resources required compared to objective laboratory measures. Both RULA and REBA allow to obtain a numerical index that represents the quantitative value of the risk at which the worker is exposed during the targeted work activity and to derive the priority level of intervention and the actions needed. The RULA method is suggested for the identification of postural disorders of the upper limbs, of the neck and of the back in relation to the muscular action and external loads applied to the body. The REBA method is applied to identify postural disorders of the whole body, in relation to the muscular action, to the external loads applied to the body and to the type of grip. They are referenced in the international standard for occupational risk assessment [9] and cited among the selected tools for Work-related Musculoskeletal Disorders (WMSDs) prevention according to International Ergonomics Association (IEA) and World Health Organization (WHO) [17]. These methods are also widely applied in several working contexts, mainly industrial work activities (secondary sector) and those producing services and goods (tertiary sector), characterized by a precise standardization of tasks, geometries, gestures and relative execution frequencies that allow a systematic and controlled forecasting and quantification of the biomechanical overload risk. Furthermore, the two methods have been combined and compared to assess postural risk in industry [18–20], construction [21], supermarkets [22], hospital and dental sector [23], work with video terminals [24], waste collection activity [25], for firefighters and emergency medical technicians [26], and artisans [27–29] and sawmill activities [30].

RULA and REBA have been adopted a few times in the agricultural [31,32] and forestry sectors [33,34] because the evaluation of the biomechanical overload risk in the primary sector activities is more difficult; due to the large variability of the tasks the operators have to perform, depending on crops, operations (seeding, weeding, pruning, harvesting, etc.), the machinery and tools adopted, the, sometimes extreme, weather conditions they have to be carried out with, and the daily and seasonal exposure, as well as the lack of a strict standardization of the work in general [35,36]. Interaction with machines, tools and environments in agriculture, and particularly in forestry, requires therefore a particular attention in the application of risk assessment methods conceived for other contexts, or the formulation of specific methods that consider the distinctive characteristics of these activities [37].

Kundu and Gaur [38] compared RULA and REBA in a study addressed at investigating how much these techniques were appropriate for evaluating the postures assumed by agricultural workers. They highlighted some shortfalls in using these techniques to study risk factors associated with agricultural field operations and suggested to add some factors as posture duration, field condition, environmental factors and nutritional status to better assess the occupational risks and possible remedies, especially for agricultural work in the fields.

Besides RULA and REBA, other methods have been used to assess the postural risk in manual agricultural activities. For example, Kong et al. [39] compared RULA, REBA, OWAS (Ovako Working Posture Analyzing System [40]) and ALLA (Agricultural Lower Limb Assessment [41]) for various agricultural tasks. They found that ALLA better estimated the risk because it identifies critical issues that the other methods do not analyse and, therefore, it always returns a higher level of risk, compared to the other methods. Ojha and Kwatra [42] combined REBA and VAS (Visual Analog Scale) methods in rice cultivation manual operations. REBA indicated postural load and suggested interesting corrective measures. However, in both studies [39,42], the object of the analysis was a manual activity, while, in developed countries, most of the farming operations require the use of machinery and workers spend many hours in interacting with them, making the investigation of the human–machine interaction more relevant [43].

Among the studies specifically dealing with the human–machine interaction in agriculture, RULA is the most frequently adopted method. Vyavahare and Kallurkar [44] used RULA to assess postures assumed during the interaction with agricultural machines such as maize dehusker-sheller. The study analysed and evaluated the risk of various key postures, such as squat, forward/lateral bending, hands flexion/extension, wrist/spine twisting. Putri et al. [45] used RULA to study the use of thresher machines for threshing rice plants. In the first study, RULA helped to optimize a digital human manikin

posture, resulting in precise risk assessment and better designed and widely accepted products and workplaces, while, in the second study, RULA provided indications to redesign the machine to reduce injuries and musculoskeletal overload caused by the mismatch between the dimensions of the engine and farmers' postures and dimensions.

In the forestry sector, the studies report postural risk evaluation mainly referred to manual tools. Gallo and Mazzetto [34] applied different methods including REBA and OWAS to evaluate the postural risks from the cutting operations with chainsaw, showing their good applicability for the assessment of WMSD in forestry. The comparison between OWAS and REBA showed that REBA has a higher level of detail of assessment because it considers the angles between body segments and extremities as the wrist, the neck, the elbow and the shoulders (parameters that are not considered by OWAS method) for both sides of the body and, furthermore, it assesses the type of handle coupling and the characteristics of the performed activities. REBA results to be suitable for suggesting interventions to be performed to decrease musculoskeletal overload, even though it has not been developed specifically for forestry operations and it presents some weaknesses during the assessment, as the lack of coded postures as the kneeling posture.

Forestry is recognized as a highly hazardous industry [46] and working postures are one of the most investigated risk factors for workers' safety, even though the postural aspects in the human–machine interaction have been under investigated in this sector [47,48]. With regard to machinery, manually fed wood-chippers are one of the most widely used machines in forestry, agricultural, landscaping and urban tree maintenance to reduce the volume of woods for following disposal or re-use in bio-energy production [47]. The attention to this machine is rising because of the significant increasing interest in biomass production as biofuel [49] and because they are often involved in fatal and non-fatal injuries [50]. Data on the occurrence of accidents provide an objective index of the danger of machinery as well as a valid reason for identifying the most critical features of these machines.

In the United States, between 1992 and 2002, Struttman [50] reported 31 fatal and 2042 non-fatal accidents involving wood-chippers. The estimated social cost of these fatal accidents were estimated in 2003 to US\$28.5 million; furthermore, an in-depth analysis disclosed that 58% of these fatal accidents involved groundskeepers and machinery operators in the forestry and agricultural sectors [50]. Analyses of the non-fatal accidents showed that 60% of the accidents caused immediate injury or amputation of parts of the upper-body limbs. For 25% of these injuries, the victims were unable to return to work for periods of up to 30 work days [51]. Further studies indicated that more than one-third of these accident victims had less than 11 months of experience in that particular job [50].

In the further period between 2008 and 2018, statistics from the United States Occupational Safety and Health Administration of Department of Labor [52] reported 56 incidents during chipping operations: 64% of the accidents dealt with feeding operations and 36% of them were fatal for the operators. From the same statistics, the accidents not related with feeding operations of wood-chippers had a lower rate, 16%, of fatal accidents.

Accurate statistics about accidents with wood-chippers are not available from most of the European Union countries [53]. Only the French Ministry of Agriculture, Agrifood, and Forestry reported that at least one severe accident related to the use of wood-chippers occurs every year in France and that, in most of the cases, the operators involved were young apprentices [54], one of the category of users for which warnings to eliminate hazardous behaviours when intrinsically safe machine are used, are less effective [55,56].

Based on the literature review, the aim of this study was to identify the more effective and appropriate method between RULA and REBA for the assessment of the risk of biomechanical postural overload, evaluating the postures assumed by an operator during the manual load of a wood-chipper, in a controlled experimental setting. The study further discusses which of the two methods is more precautionary in evaluating postural risk and reports higher risk indices, for the specific activity considered.

For the present study, a small manually fed wood-chipper has been selected since: (1) small-size wood-chippers are widespread among forestry, agricultural and urban green maintenance operators; (2) similar types of wood-chippers are used for occasional and accessory operations to the main activity [47]; (3) often experienced, but not specialized, operators are involved in the wood-chipping operations and this category of operators are known to be the most exposed to safety risks [57]. As a consequence, the manual feeding seems to be critical both in terms of safety and health risks for the operators [58,59].

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

#### *2.1. The Wood-Chipper*

The manually fed wood-chippers generally consist of a feeding system, a chipping units based on rotating knives (drum or disc mounted) and on a discharge system. Alternative solutions for the comminution devices are available for production of quality wood chips for fuel [60]. In the case of manually-fed wood-chippers, the operators feed small trees, part of trees and/or branches into the infeed chute by hand with a following risk of biomechanical postural overload, which is typical of manually-loaded wood machines [61].

The study was carried out with a manually fed wood-chipper model Tirex, made available by the manufacturer Peruzzo (Peruzzo Srl, Curtarolo, Italy). The machine was connected to the rear three-point hitch of a 55 kW tractor and was powered by the rear Power Take-Off (PTO) (Figure 1). The feeding system consists of a feeding hopper with an infeed chute above 600 mm from the ground level and with a width of 1200 mm at the external edge. The feeding system consists of two horizontal rollers electro-hydraulically controlled by a load-limiting mechanism and located at the end of the infeed chute at a distance of 1200 mm from the external infeed chute limit, in accordance with EN 13,525:2005 + A2:2009 [62]. The machine is equipped with an electro-hydraulic feed control bar fixed on the bottom and along the two sides of the infeed chute. The chipping unit consists of a flywheel (diameter 620 mm, mm thickness 30 mm) rotating at 1500 rpm and with a torque of about 33 Nm. The flywheel is equipped with four knives (thickness 25 mm and length 200 mm) while an anvil is fixed to the frame of the machine. The manufacturer claims the machine is able to chip wood stems with a diameter up to 180–200 mm.

**Figure 1.** The manually fed wood-chipper used during the test.


**4. Advancements in non-conventional methods — Development of a highspeed, computer-controlled hybrid scanner**

From the performance of the acousto-optical imaging system shown in Figure 9, it was evident that to meet practical requirements and to advance the potential benefits, the following characteristics would be very desirable.

**Figure 11.** Imaging side-drilled holes in a test block

282 Advancements and Breakthroughs in Ultrasound Imaging

**Figure 12.** Image of a micro crack in a T-weld

*3.2.5. Practical limitations of the first prototype*

the test block.

Figure 12 below shows an actual T-weld being tested with the prototype system and the image of a crack in the weld. This micro-crack was actually visible from the ground side surface of

The results from the first prototype have clearly demonstrated the potential of direct ultrasonic imaging reaching performance close to theoretical limits. These include: maximum possible speed of imaging, maximum lateral resolution for the size of the arrays used, forming focused

However, there were still practical limitations. This system also had a small field of view approximately that covered laterally by the array aperture; in this case ~3 cm wide. This is too

images of the whole object field covered by the transducer aperture.

The first property adds a much greater degree of freedom to test uneven surfaces from selected or prepared locations. The resulting images would be similar to a B-mode sector scan, but the imaging modality is such that it produces image zones for required sectors as opposed to individual line serial scanning, with just a few overlapping sectors enabling much higher frame rates to be achieved. Since there is no requirement for focusing in the image medium, as that is already taken care of by the sonoptical design, the scanning simply means that only the insonifying beam need to be steered to illuminate the object sector.

Since the image reconstruction is implemented with a sonoptical system designed with paraxial ray equations, when imaging a wide field like a sector, there is likely to be a significant degree of peripheral geometrical aberration in the image field. It should be possible to correct these dynamically, since for each image zone, implementation of an appropriately precalculated delay in the firing of the stroboscopic light source, channel gain/phase manipulation is possible.

Inclusion of the above properties is the basis of the development [17-19] described below. Figure 13 shows the basic block diagram of the hybrid prototype developed, incorporating scanning hardware (SCH) controlled by a microcomputer (PC). Dynamic control hardware (DCH) is intended to provide gain equalisation during off-axis imaging and time varying gain (TVG) to produce a uniform image field. Field Focus Control (FFC) ensures that the strobo‐ scopic illumination is synchronised such that the image field is optically frozen at the instant of best focus. It is also intended to provide a degree of off-axis aberration correction with sectordependent focusing delay control.

Figure 14 shows a more detailed block diagram of the improved design with a possible use in

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**Figure 14.** A hybrid imaging Topology with computer controlled scanning and acoustooptical image reconstruction

Since the principle of acousto-optical imaging is very different to that of the conventional methods, some of the requirements of electronics and ultrasonic hardware is very different to that of the conventional systems in many ways. Since the received signals are converted back into ultrasound signals and retransmitted into an acoustic modulator to produce optical effects,

(Expanded block diagram)

**Figure 15.** The first prototype of the hybrid scanner (GB2278443B)

testing of welds. Figure 15 shows the first prototype hybrid scanner developed.

**Figure 13.** Basic block diagram of the hybrid scanner

#### **4.1. Principle of operation**

As mentioned previously, in contrast to conventional imaging, this system also operates on the basis of forming an ultrasonic map of the object field from received echoes inside a visible medium. Once an ultrasonic pulse illuminated the object medium, the acoustic signals intercepted by the receiving array is retransmitted into the image medium through the acoustic focusing system after conditioning and boosting signal power using signal conditioning hardware (SC). The re-transmitting section consists of an identical set of transducer array (except for the shape) to that of the receiving array, coupled to a specially designed ultrasonic focusing arrangement according to the requirements described in 8.1.1 (Appendix). Two important characteristics of this focusing lens design are that all the target echoes are brought into focus at the same instant of time irrespective of the target depth while maintaining image spatial linearity, so that a complete image frame is produced by just one insonifying pulse in real-time.

When the acoustic image field is at the time of best focus, an ultra-short stroboscopic light pulse of the order of a few nanoseconds is emitted, which produces a visible image. This can be viewed directly or captured by a video camera. Since the maximum repetition rate is now only determined by the time-of-flight of the sound pulse, the system achieves the highest speed of operation theoretically possible in ultrasonic imaging, eliminating the problem of temporal artefacts inherent with the existing systems.

Figure 14 shows a more detailed block diagram of the improved design with a possible use in testing of welds. Figure 15 shows the first prototype hybrid scanner developed.

**Figure 14.** A hybrid imaging Topology with computer controlled scanning and acoustooptical image reconstruction (Expanded block diagram)

**Figure 15.** The first prototype of the hybrid scanner (GB2278443B)

**Figure 13.** Basic block diagram of the hybrid scanner

284 Advancements and Breakthroughs in Ultrasound Imaging

artefacts inherent with the existing systems.

As mentioned previously, in contrast to conventional imaging, this system also operates on the basis of forming an ultrasonic map of the object field from received echoes inside a visible medium. Once an ultrasonic pulse illuminated the object medium, the acoustic signals intercepted by the receiving array is retransmitted into the image medium through the acoustic focusing system after conditioning and boosting signal power using signal conditioning hardware (SC). The re-transmitting section consists of an identical set of transducer array (except for the shape) to that of the receiving array, coupled to a specially designed ultrasonic focusing arrangement according to the requirements described in 8.1.1 (Appendix). Two important characteristics of this focusing lens design are that all the target echoes are brought into focus at the same instant of time irrespective of the target depth while maintaining image spatial linearity, so that a complete image frame is produced by just one insonifying pulse in

When the acoustic image field is at the time of best focus, an ultra-short stroboscopic light pulse of the order of a few nanoseconds is emitted, which produces a visible image. This can be viewed directly or captured by a video camera. Since the maximum repetition rate is now only determined by the time-of-flight of the sound pulse, the system achieves the highest speed of operation theoretically possible in ultrasonic imaging, eliminating the problem of temporal

**4.1. Principle of operation**

real-time.

Since the principle of acousto-optical imaging is very different to that of the conventional methods, some of the requirements of electronics and ultrasonic hardware is very different to that of the conventional systems in many ways. Since the received signals are converted back into ultrasound signals and retransmitted into an acoustic modulator to produce optical effects, the technique relies on the preservation of amplitude and phase characteristics of the signals to a high degree. Also, adequate signal power is required to produce acousto-optical modu‐ lation in the image medium to make the acoustic map of the object field visible.

essentially behaving as an active, acousto-optical imaging device operating in real time. Therefore, even in the scanning mode, this allows very high temporal resolution to be achieved since one beam produces a complete image field or image zone. Furthermore, the whole of the effective aperture is utilized for each image frame and therefore diffraction-limited lateral

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If the scanning angles are small, the system could produce images without the need for any compensation. This is the simplest mode of scanning operation. For larger beam angles in the scanning mode, compensation to account for the reduction in sensitivity and field uniformity may be applied by the control of insonifying energy, receiver gain and scan derived electronic

As mentioned above, although the prototype was equipped with 30 channels, the number of channels used for the feasibility study was just 8 with an effective aperture of only 1.6 cm (as marked on the receiving array in Figure 17a). This was because of a problem of some of the channels breaking into oscillations due to excessive capacitive feedback at the time of experi‐ ments. Nonetheless, it can be seen from Figure 17 that the images produced are still very good

As mentioned above, in the coaxial mode, the system resembles in operation to that shown in Figure 10 to 12. The speed of imaging is very high, e.g. in a test block of steel 40 cm deep, a complete image is formed within 0.15ms and the frame rate can be as high as 1kHz or more. Estimation of resolution capabilities previously obtained using the coaxial system of Figure 9 showed that the images can be produced to within one wavelength resolution in the axial direction and about 1.5 wavelengths laterally at about 60mm below the surface of the test block;

In order to test the operation in the scanning mode; a test block with side drilled holes covering a wide sector was prepared as shown in Figure 17(a). Figure 17 (b), (c) & (d) shows the images when the insonifying beam was steered statically (i.e. in manually selected angles) to image the 3 holes in the left, two in the centre and the two on the right-hand side of the test block. Dynamic scanning was also verified. However, since the scan sector required for the above

statically scanned, aberration can be controlled for each angle manually by selecting strobo‐

Although the prototype system was designed for NDT applications, attempts to image biological moving tissues, such as heart valves, revealed excellent temporal resolution as expected. However, since the transducer arrays used did not have the required element spacing characteristics and matching properties, and the sonoptics of the prototype was designed for industrial material, the prototype could not obviously produce true B-mode images for biological tissues although the potential for advancement in this area was clearly

scopic firing delays to achieve best focus. For smaller sectors (e.g. < 600

) peripheral geometrical aberration was significant. When

) aberration was not a

resolution approaches the theoretical maximum.

phase delays, which is an area for further development.

**4.2. Results from the 1st hybrid scanner prototype**

for the aperture used.

thus approaching theoretical limits.

test object was very large (~1200

significant factor.

evident.

For the first hybrid prototype system, the excitation pulses generated were 2MHz single sinusoidal pulses of the order of 120Vpp as shown in Figure 16 below. These pulses were shaped to obtain the near-ideal response from the transducer elements.

The front-end receiving section of the hardware consists of wideband amplifiers (15 MHz) with a variable gain of up to 60dB. The output impedance is of the order of 50 Ohms with a maximum output swing capability of 120V pp. Input surge protection up to 1kV was provided with a very low recovery time to achieve minimal dead-zone. Although the above prototype scanner was equipped with 30 channels, only 8 channels were used for the feasibility study. This represented just 1.6 cm acoustic aperture. No dynamic compensation was used. However, the performance as can be seen from statically scanned images was still very good.

#### *4.1.1. Operational modes*

The system may be operated in the axial mode for very high-speed imaging or be used in the scanning mode to cover a wide field. In the coaxial mode, the system resembles in operation to that shown in Figure 10 -12, producing narrow-field linear array B-mode images covered by the aperture of the array. In the scanning mode the system is under the control of the computer and can be used to produce wide-field, high resolution B-mode sector scan images, or be programmed to scan along any specific areas of the target. In order to achieve field uniformity for off-axis imaging, scan-angle derived field-focus compensation (FFC) and dynamic compensation (DCH) may be applied. In the scanning mode, wideband insonifying pulses are beamed to target areas by phased array beam steering. However, when receiving the echoes from the targets, there is no requirement for beam forming at all, as the system essentially behaving as an active, acousto-optical imaging device operating in real time. Therefore, even in the scanning mode, this allows very high temporal resolution to be achieved since one beam produces a complete image field or image zone. Furthermore, the whole of the effective aperture is utilized for each image frame and therefore diffraction-limited lateral resolution approaches the theoretical maximum.

If the scanning angles are small, the system could produce images without the need for any compensation. This is the simplest mode of scanning operation. For larger beam angles in the scanning mode, compensation to account for the reduction in sensitivity and field uniformity may be applied by the control of insonifying energy, receiver gain and scan derived electronic phase delays, which is an area for further development.

#### **4.2. Results from the 1st hybrid scanner prototype**

the technique relies on the preservation of amplitude and phase characteristics of the signals to a high degree. Also, adequate signal power is required to produce acousto-optical modu‐

For the first hybrid prototype system, the excitation pulses generated were 2MHz single sinusoidal pulses of the order of 120Vpp as shown in Figure 16 below. These pulses were

The front-end receiving section of the hardware consists of wideband amplifiers (15 MHz) with a variable gain of up to 60dB. The output impedance is of the order of 50 Ohms with a maximum output swing capability of 120V pp. Input surge protection up to 1kV was provided with a very low recovery time to achieve minimal dead-zone. Although the above prototype scanner was equipped with 30 channels, only 8 channels were used for the feasibility study. This represented just 1.6 cm acoustic aperture. No dynamic compensation was used. However,

The system may be operated in the axial mode for very high-speed imaging or be used in the scanning mode to cover a wide field. In the coaxial mode, the system resembles in operation to that shown in Figure 10 -12, producing narrow-field linear array B-mode images covered by the aperture of the array. In the scanning mode the system is under the control of the computer and can be used to produce wide-field, high resolution B-mode sector scan images, or be programmed to scan along any specific areas of the target. In order to achieve field uniformity for off-axis imaging, scan-angle derived field-focus compensation (FFC) and dynamic compensation (DCH) may be applied. In the scanning mode, wideband insonifying pulses are beamed to target areas by phased array beam steering. However, when receiving the echoes from the targets, there is no requirement for beam forming at all, as the system

the performance as can be seen from statically scanned images was still very good.

lation in the image medium to make the acoustic map of the object field visible.

shaped to obtain the near-ideal response from the transducer elements.

**Figure 16.** Single sinusoidal excitation pulse

286 Advancements and Breakthroughs in Ultrasound Imaging

*4.1.1. Operational modes*

As mentioned above, although the prototype was equipped with 30 channels, the number of channels used for the feasibility study was just 8 with an effective aperture of only 1.6 cm (as marked on the receiving array in Figure 17a). This was because of a problem of some of the channels breaking into oscillations due to excessive capacitive feedback at the time of experi‐ ments. Nonetheless, it can be seen from Figure 17 that the images produced are still very good for the aperture used.

As mentioned above, in the coaxial mode, the system resembles in operation to that shown in Figure 10 to 12. The speed of imaging is very high, e.g. in a test block of steel 40 cm deep, a complete image is formed within 0.15ms and the frame rate can be as high as 1kHz or more. Estimation of resolution capabilities previously obtained using the coaxial system of Figure 9 showed that the images can be produced to within one wavelength resolution in the axial direction and about 1.5 wavelengths laterally at about 60mm below the surface of the test block; thus approaching theoretical limits.

In order to test the operation in the scanning mode; a test block with side drilled holes covering a wide sector was prepared as shown in Figure 17(a). Figure 17 (b), (c) & (d) shows the images when the insonifying beam was steered statically (i.e. in manually selected angles) to image the 3 holes in the left, two in the centre and the two on the right-hand side of the test block.

Dynamic scanning was also verified. However, since the scan sector required for the above test object was very large (~1200 ) peripheral geometrical aberration was significant. When statically scanned, aberration can be controlled for each angle manually by selecting strobo‐ scopic firing delays to achieve best focus. For smaller sectors (e.g. < 600 ) aberration was not a significant factor.

Although the prototype system was designed for NDT applications, attempts to image biological moving tissues, such as heart valves, revealed excellent temporal resolution as expected. However, since the transducer arrays used did not have the required element spacing characteristics and matching properties, and the sonoptics of the prototype was designed for industrial material, the prototype could not obviously produce true B-mode images for biological tissues although the potential for advancement in this area was clearly evident.

allows true signals from targets to be coherently re-constructed in the image medium while the noise and out of phase signals are being largely rejected as evident from Figure 18 below. However, just like all ultrasound equipment multiple reflections between closely spaced targets could still cause artefacts. Therefore, inclusion of selective insonification of the object medium as in item No. 2 above should enable the system to achieve even higher SNR while

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Another important factor as mentioned in section 2 for any imaging technique is the dynamic range. In this respect, one of the typical bottlenecks is the limited dynamic range of display equipment. The present development can provide a greater display dynamic range when images are viewed with the naked eye as the acousto-optical modulator can provide a higher display dynamic range than images captured by a camera and presented on a typical conven‐

Conventional ultrasonic imaging systems have inherent limitations such as low speed leading to temporal artefacts and in some cases limited lateral resolution; these being the result of lineserial scanning, lengthy processing and other limitations arising from the particular techniques used. The extent to which the above deficiencies affect performance has been analytically

In this respect, it has been shown that an alternative hybrid approach to imaging using acoustooptical image reconstruction could give clear advantages; reaching theoretical limits of performance in speed and resolution unachievable with the existing methods. This is mainly due to the combination of electronic and sonoptical image reconstruction, avoiding line-serial scanning and lengthy processing required by the conventional systems. The hybrid system reaches almost ultimate speed and resolution in the coaxial imaging mode. In the B-scan mode,

suppressing artefacts when imaging near-field targets.

**Figure 18.** Noise suppression

tional VDU.

investigated.

**6. Conclusions**

**Figure 17.** a) - Test Block. Statically scanned images of the test block


*2.4.1 Spaces for speech intelligibility*

Speech clearness is vital in a specific type of functions. Without speech intelligibility, classes' success could fail. Teacher explanations, court sessions, and board meetings are functional, practical examples where speech is the key role of their success. Large institutions need clear discussions to make efficient decisions. In the court, when the speech is not clear, many judicial problems could take part from innocently. Such spaces need clear speech with no reflection nor echoes. In such functions, the number of absorber materials should increase in favor of the reflectors. The absorption material scheme when exceed, will produce a harmful background and thus will require more effort from the sources to enable the audience to hear. Therefore the introduction of some diffusers is necessary to balance the acoustics performance (**Figure 5**).

**Figure 5.** *Examples of classroom, boardroom, and court room where the speech intelligibility needs many absorbers.*


Cerebellar Transcranial Direct Current Stimulation (ctDCS) Effect in Perception and Modulation of Pain

*Tommaso Bocci, Roberta Ferrucci, Alberto Priori, Massimiliano Valeriani and Ferdinando Sartucci*


*2.3. Identifying Di*ff*erentially Expressed Protein Phosphorylation Sites Induced by Coelonin Treatment*

To identify differentially expressed signaling-associated phosphorylated proteins between coelonin-treated and untreated RAW264.7 cells induced by LPS, the expression levels of phospho-antibody specific proteins were compared. Of the 1318 antibodies analyzed in microarray experiments, a total of 32 different phosphorylation proteins showed downregulated expression using a fold ratio ≥2 as the cutoff criterion (Figure 4, Table 1). In addition, we performed a protein network analysis using REACTOME (http://reactome.ncpsb.org/) to identify major interactions, three major cellular processes were significantly enriched (*p* value < 0.05) as follows: (1) Immune system, (2) signal transduction, and (3) cell cycle. LPS is the component of the outer membrane of Gram-negative bacteria, and is one of the most well characterized pathogen-associated molecular patterns (PAMPs), which can be recognized by Toll-like receptor 4 (TLR4), and trigger innate responses [19], such as enhancing the secretion of cytokines and chemokines, and promoting macrophages migration and proliferation [20]. Therefore, it is evident that the protein network analysis results imply coelonin may block LPS induced RAW264.7 cell signal transduction, immune response, and proliferation.

**Figure 4.** Protein and phosphorylation altered after coelonin treatment. From a total of 1318 differentially phosphorylated proteins, 32 were selected as significantly downregulated by coelonin using a volcano plot analysis.

**Table 1.** The phosphorylation of 32 proteins were significantly downregulated by coelonin treatment (*p* < 0.05).

Moreover, we entered the ENTREZ Gene IDs of the 32 genes into the DAVID Bioinformatics Resources 6.8 database. As depicted in Figure 5, the numbers of changed genes in the PI3K/AKT signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, the neurotrophin signaling pathway, the Sphingolipid signaling pathway and Ras signaling pathway were ranked in the top 15. In addition, we found that within these genes, the maximum number of genes belonged to the PI3K/AKT signal pathway. It is noteworthy that the phosphorylation of both subunits p65 and p105/50 of transcription factor NF-κB in this signaling pathway was reduced, which was closely related to the regulation of inflammatory gene expression [21].

**Figure 5.** Number of genes in signaling pathways changed by coelonin pretreatment.

*2.4. Validation of Proteomic Findings–Coelonin Treatment Inhibits Inflammatory Cytokines Secretion by Blocking NF-*κ*B Activation*

NF-κB is the most important signaling molecule induced by LPS through TLR4. The phosphorylated NF-κB can translocate to the nucleus, interacts with the κB elements and cause numerous cytokines secretion such as IL-1β, MCP-1 and TNF-α et al. [21]. As observed in Table 1, both NF-κB p65 and p105/50 phosphorylation levels were significantly reduced by 2.5 μg/mL coelonin treatment. Therefore, we performed western blot and immunofluorescence assays by nucleus translocation of p65 for validation. As shown in Figure 6A, the western blot results indicated that LPS stimulation significantly increased p65 accumulation in the nucleus, but the coelonin dose dependently reduced the effect of LPS. Moreover, confocal microscopic analysis reconfirmed that LPS stimulation significantly induced p65 translocation from the cytoplasm to the nucleus, which was remarkably inhibited by 2 μM of ammonium pyrrolidine dithiocarbamate (APDC), an inhibitor of NF-κB, and 5.0 μg/mL of coelonin pre-treatment (Figure 6B). In addition to cytokines, numerous studies have shown that NF-κB also regulates the expression of iNOS and COX2 genes [22,23]. Furthermore, over expression of iNOS and COX2 can lead to inflammation, tissue damage and even tumorigenesis [24,25]. As observed in Figure 6A, 200 ng/mL LPS treatment for 24 h caused a remarkable increased expression of iNOS and COX2. Pre-treatment with coelonin dose dependently reversed LPS-induced iNOS and COX2 expression. These results were consistent with the microarray results and confirmed that coelonin exerts its anti-inflammatory effect by inhibiting NF-κB activity.

#### *2.5. Colonin May Partially Inhibit the Activation of NF-*κ*B through PTEN*/*AKT Pathway*

It is well know that LPS can activate NF-κB through the Toll-like receptor 4/myeloid differentiation factor 88/IL-1 receptor associated kinase/TNF receptor associated factor 6/TGF beta-Activated Kinase 1/inhibitor of nuclear factor-κB kinase (TLR4/MyD88/IRAK/TRAF6/TAK1/IKKs) pathway [26] and now, several lines of evidence suggest that LPS can also activate NF-κB through the TLR4/MyD88/PI3K/AKT/IKKs pathway [27,28]. However, the PI3K/AKT pathway is negatively regulated by the PTEN [29,30]. Previous research has shown that the down regulation of PTEN can activate NF-κB activity by increasing p65 nucleus translocation in mouse mesangial cells and bovine alveolar macrophages, contrary, activate PTEN would reverse the effect [29,31]. The microarray results indicated that coelonin treatment significantly down-regulated the phosphorylation of PTEN at Ser380/Thr382/Thr383 (see Table 1), which implies that coelonin may inactivate NF-κB by restoring the activity of PTEN, as phosphorylation of PTEN will make it inactivated [30]. Thus, western blotting was carried out to verify this presumption. As show in Figure 6A, LPS significantly increased the phosphorylation of PTEN, AKT and inhibitor of NF-κB (IκBa), which was dose-dependently reduced by coelonin pre-treatment (Figure 6A). In order to further confirm the possible inhibition activity of coelonin against LPS-induced NF-κB activation mediated by PTEN/AKT pathway, RAW264.7 cells

were pre-treated with PTEN inhibitor SF1670. As show in Figure 7A, pre-treatment with SF1670 significantly increased LPS-induced AKT phosphorylation, which could not be downregulated by additional coelonin. Meanwhile, SF1670 also dramatically promoted LPS-induced secretion of IL-1β, IL-6 and TNF-α, and significantly reduced but could not completely block the inhibitory activity of coelonin. This result further indicates that PTEN did participate in PI3K/AKT/NF-κB activation pathway. However, it is note worthy that most levels of IL-1β and IL-6 were still significantly inhibited by coelonin co-treated with SF1670, suggesting that more critical pathways need to be identified besides the PTEN/AKT pathway.

**Figure 6.** (**A**) Coelonin inhibits LPS induced NF-κB activation in macrophage. RAW264.7 cells were pretreated with coelonin for 1 h and then treated with 200 ng/mL LPS for 30 min. Then, the nuclear protein was extracted, the content of p65 in the nucleus was determined, the total cellular protein was extracted, and the levels of phosphorylated PTEN, AKT and IκBα were determined. For iNOS and COX2 detection, RAW264.7 cells were pretreated with coelonin for 1 h, then treated with 200 ng/mL LPS for 24 h, then total cellular protein was extracted and analyzed. (**B**) Confocal microscopy analysis of p65 nucleus translocation. RAW264.7 cells were incubated with solvent (a–c) or 200 ng/mL LPS for 1 h in the absence (d–f) or presence 2 μM of the NF-κB inhibitor APDC (g–i) or 5 μg/mL of coelonin (j–l).

**Figure 7.** (**A**) Western blotting result of pAKT. RAW264.7 cells were incubated with solvent or 200 ng/mL LPS for 30 min in the absence or presence 5 μg/mL of coelonin or 2 μM of the PTEN inhibitor SF1670 or 2 μM of SF1670 combined with 5 μg/mL of coelonin. Then total cellular protein was extracted and analyzed by a simple western immunoblotting technique on a Peggy Sue system. (**B**–**D**) Effect of PTEN inhibitor SF1670 on anti-inflammatory activity of coelonin. RAW264.7 cells were incubated with solvent or 200 ng/mL LPS for 12 h in the absence or presence 2 μM of the PTEN inhibitor SF1670 or 5 μg/mL of coelonin or 2 μM of SF1670 combined with 5 μg/mL of coelonin. Culture supernatants was collected for IL-1β, IL-6 and TNF-α detection by CBA method. Data are expressed as mean ± SD (*n* = 6). \*\* *p* < 0.01.

#### *2.6. Coelonin Treatment Leads to G1 Cell Cycle Arrest through PTEN*

The PI3K-Akt pathway has been shown be involved in a variety of cellular processes, including inflammation response [27], cell survival and proliferation [32]. Many reports indicate that the PI3K/AKT pathway plays a pivotal role in regulating cell cycle progression through phosphorylation and degradation of cell cycle regulator p27Kip1 [33,34]. P27Kip1 can interact with cyclin-dependent kinase 2 (CDK2) and cyclinE to prevent cell entry into the S phase, and over-expression p27Kip1 would promote cell G1 phase cell cycle arrest [35]. In many cancer cells, such as cervical cancer cells, oral squamous cell carcinoma cells, and prostatic carcinoma cells, the expression protein of p27Kip1 was significantly reduced, the invasion and migration ability was enhanced, and the expression of p27Kip1 was negatively correlated with survival [33,36]. We noticed that the microarray results showed that coelonin significantly down regulated the phosphorylation of p27kip1, and interestingly, a previous study indicated that the up-regulation of PTEN prevents p27Kip1 phosphorylation and proteolysis [37]. Hence, we speculate that coelonin may inhibit p27Kip1 phosphorylation and degradation mediated by PTEN. Western blot confirmed that 5 μg/mL coelonin treatment significantly recovered the p27Kip1 level, which was even higher than the un-treatment group, while LPS stimulation significantly reduced the p27Kip1 level, and PTEN inhibitor SF1670 almost completely abrogated the effect of coelonin (see Figure 8A). Theoretically, high levels of p27Kip1 can block cells in the G1 phase cell-cycle, so we used flow cytometry to verify whether coelonin could induce G1 phase cell cycle arrest in RAW264.7 cells. As observed in Figure 8B, coelonin pre-treatment remarkably induced G1 phase cell cycle arrest and SF1670 completely inhibited the effect of coelonin. These data suggest that coelonin inhibits p27Kip1

degradation in a PTEN-dependent manner, thereby exerting its G1 phase cell cycle arrest effect in RAW264.7 cells.

**Figure 8.** (**A**) Western blotting result of p27kip1. RAW264.7 cells were incubated with solvent or 200 ng/mL LPS for 12 h in the absence or presence 5 μg/mL of coelonin or 2 μM of the PTEN inhibitor SF1670 or 2 μM of SF1670 combined with 5 μg/mL of coelonin. Then total cellular protein was extracted and analysed by a simple western immunoblotting technique on a Peggy Sue system. (**B**) Coelonin induce G1 cell cycle arrest through PTEN analysed by flow cytometry. RAW264.7 cells were incubated with solvent or 200 ng/mL LPS for 12 h in the absence or presence 5 μg/mL of coelonin or 2 μM of the PTEN inhibitor SF1670 or 2 μM of SF1670 combined with 5 μg/mL of coelonin. Cells were harvested, fixed and stained with PI, and cell cycle distributions were analyzed on a BD Accuri™ C6 flow cytometer in triplicate.

#### **3. Discussion**

To date, over 200 phenanthrene compounds have been isolated and identified, most of which come from the Orchidaceae family [38]. In *Bletilla striata*, a member of Orchidaceae, more than 30 phenanthrenes have been isolated from its pseudobulbs and fibrous roots, most of which showed antitumor and antimicrobial activities [39,40]. In addition, numerous studies have shown that phenanthrene and dihydrophenanthrene derivatives with remarkable anti-inflammation activity [38,41,42]. In this study, coelonin, a compound with a strong anti-inflammatory activity, was isolated and identified from the pseudobulbs of *Bletilla striat* under the guidance of biological activity screening. Few studies have reported its anti-inflammatory activity, and our studies show that coelonin can dose-dependently inhibit LPS-induced expression and secretion of IL-1β, IL-6 and TNF-α in RAW264.7 cells. Furthermore, we found that *Bletilla striata* has a high content of coelonin (0.020%–0.301% in different samples) [43]. Therefore, coelonin is probably one of the main anti-inflammatory active components of *Bletilla striata*.

In order to better elucidate the potential anti-inflammatory molecular mechanism of coelonin, PEX100 protein microarrays containing 1318 antibodies were used. The results indicated that 32 different phosphorylated proteins were significantly downregulated by pre-treatment with coelonin, which were closely related to the response of LPS-induced signal transduction, immune response and cell proliferation. Additionally, most of these genes were focused on the PI3K/AKT signal pathway, and we

performed verification on the phosphorylation levels of PTEN, NF-κB and p27Kip1. NF-κB is considered as a central regulator of LPS-induced pro-inflammatory response in macrophage activation [44], which is usually formed as p65:p50 heterodimer. In addition, PEX100 microarray results indicated that the phosphorylation levels of both p65 and p50 were significantly downregulated by coelonin pre-treatment (Table 1), which was further confirmed by western blot and confocal microscopic analysis on p65 nucleus translocation (Figure 6). Studies have shown that numerous dihydrophenanthrene derivatives exert anti-inflammatory effects by inhibiting NF-κB pathway [38,41,42], which was significantly correlated with the presence of phenolic hydroxyl groups [45]. Interestingly, coelonin has two phenolic hydroxyl groups, which is in accordance with its anti-inflammatory effect. However, few reports have studied the upstream targets that regulate the activity of NF-κB by dihydrophenanthrene derivatives. According to the results of the PEX100 microarray, we propose that coelonin may play an anti-inflammatory role by inhibiting the activity of NF-κB through the PTEN/AKT pathway. However, some studies have indicated that the PI3K/AKT pathway negatively regulates LPS induced NF-κB activation [46]. In contrast, other studies have shown that the PI3K/AKT pathway does positively regulate LPS-induced gene expression [27]. We found that LPS stimulation could dramatically induce AKT phosphorylation, IκBa degradation and NF-κB nucleus translocation (Figure 6), and the PI3K inhibitor LY294002 significantly inhibited LPS inducted IL-1β, IL-6 and TNF-α expression in RAW264.7 cells (see Supplementary Figure S5), which is consistent with the findings that the PI3K/AKT pathway is required for LPS induction of gene expression in RAW264.7 cells. Meanwhile, PTEN inhibitor SF1670 significantly increased LPS-induced AKT phosphorylation and cytokines secretion (Figure 7), which is consistent with the report of Zhao et al. [47]. These results indirectly supported that both PI3K/AKT and PTEN/AKT pathways are involved in LPS inducted NF-κB activation. However, the phosphorylation level of Akt did not decrease after co-treatment with PTEN inhibitor SF1670 and coelonin, but coelonin still could significantly down-regulate the expression of inflammatory cytokines induced by LPS. This contradictory result not only indicated that coelonin has other pathways to exert its anti-inflammation activity, but also indicated that AKT pathway has complex mechanisms in the regulation of inflammation. In fact, the exact mechanism of activation of NF-κB by AKT pathway remains controversial. Some reports indicated that activation AKT lead to IKK-dependent IκBα degradation and nucleus translocation of NF-κB, while others shown that AKT-dependent activation of NF-κB by stimulating the transactivation potential of the p65 subunit, rather than inducing IκBα degradation [48]. Obviously, although coelonin could inhibit IκBα phosphorylation and degradation in a dose-dependent manner, it is not clear whether coelonin plays a role through the AKT pathway or TLR4/MyD88/TRAF6/TAK1 pathway, the canonical pathway of NF-κB activation (Figure 9). Therefore, it is difficult to draw a definite conclusion only through the intervention of inhibitors, and more scientific and reasonable experiments need to be designed to elucidate the alternative mechanism of coelonin inactivating NF-κB.

In addition, our study also detected the degradation of p27Kip1 after the LPS challenge, which is a downstream target of PTEN/AKT. This effect was dramatically inhibited by coelonin pre-treatment. However, the PTEN inhibitor SF1670 completely abrogated the effect of coelonin (Figure 8). We noticed that LPS treatment also induced RAW264.7 cells G1 phase arrest (Figure 8B), which contradicts a previous report [20], but is consistent with report [49]. The exact molecular mechanism underlying these contradictory results need to be further studied, but it is clear that LPS-induced G1 phase arrest in RAW264.7 cells did not occur through the up-regulation of p27Kip1. However, LPS treatment did promote p27Kip1 degradation (Figure 8A), and which may be dependent on PTEN inactivation (Figure 9). While coelonin could block the LPS-induced degradation of p27Kip1 by restoring PTEN activity (Figure 9). Studies have shown that alveolar macrophage proliferation may play an important role in the formation of multinucleated giant cells and granuloma induced by silica or asbestos [50]. These results suggest that, in addition to inhibiting the secretion of inflammatory factors by macrophages, inhibiting the proliferation of macrophages may also play a role in alleviating silica or asbestos-induced lung pathological changes to an extent.

**Figure 9.** Proposed mechanism of coelonin inhibiting LPS-stimulated activation of NF-κB and inducing G1 phase cell-cycle arrest in RAW264.7 cells. Coelonin inhibits the expression of inflammatory cytokines IL-1β, IL-6 and TNF-α in RAW264.7 cells treated by LPS may partially through the PTEN/AKT pathway. However, it induces G1 cell cycle arrest of RAW264.7 cells by inhibiting the degradation of p27Kip1 in a PTEN-dependent manner. The black arrow section has been verified, while the red arrow section needs further validation. The question mark indicated that coelonin may inactivate NF-κB by inhibiting TLR4/MyD88/TRAF6/TAK1 signal pathway, the canonical pathway of NF-κB activation.


**<IP_ADDRESS> Type II – BIO (Bending and Independent Opening)**

This type II device modified version is intended for use in ascending aorta substitution in absence of dissection. The two ends of the device can vary their reciprocal axis up to 90° to

New Approaches for Treatment and Prevention of Aortic Aneurysms 279

For this purpose, on one side, the wireframe consistency was decreased by variably reducing the gauge of nitinol wires and their respective position within wireframe and, on the other side, the external ligature was carried out with the thinnest possible prolene (5-0) encircling suture, transfixing full thickness the device and aortic wall at three equidistant points. The limit of the former was the ability to sustain the external ligature without wireframe deformation/collapse at pressure providing "airtight" seal; the limit of the later was the ability

The present versions of the devices with minimal consistence and great compliance minimize the risk of mechanical conflict and erosion with the confining tissue/organs; moreover wireframe consistency was settled in such a way that is minimal at the device end

Concerning the external ligature and its many possible ways of application, it may be useful to recall that vascular anastomosis must guarantee haemostatic sealing and stability of the coupling which, contrariwise to standard suture, rely on different mechanisms with this

Thus even though external devices surface is provided with short needles perpendicularly positioned around its circumference at 4-6 equidistant points, reduction of the wireframe

To optimize coupling stability in this lighter expandable wireframe we successfully tested 5- 0 prolene encircling suture, transfixing full thickness the device and aortic wall at three equidistant points (fig. 18). In this way in fact the minimal expandable wireframe consistence to sustain the external ligature can be further significantly reduced and stability increased, by splitting the external ligature circumference in three separate and equivalent segments. In this embodiment the expandable ring will be then really only a bit more

to provide stability of the aortic stump-expandable device coupling at stretch test.

consistency may probably decrease their reliability in keeping coupling stability.

To provide reliable coupling stability, instead of simple external ligature, 5-0 prolene encircling suture, transfixing full thickness the device and aortic wall at three equidistant points, was passed and tied as shown, in a sort of revival of the Carrel triangulation historical technique. Video at http://www.fondazionecarrel.org/tsb2/tsb2.html

dislocation on the expandable wireframe throughout the entire circumference.

This coupling stability was proved by a stretch test consisting in a sequence of manoeuvres carried out on the anastomosis that includes: **1)** complete compression of the anastomosis in orthogonal directions and then **2)** vigorous manual stretching of the anastomosis in the coaxial plane separately at four points of its circumference. The sequence was repeated three times and the anastomosis checked for any significant vascular stump backwards

where the possible conflict with aortic wall could be higher.

type of coupling (Fig. 8).

consistent of a common graft tube.

Fig. 18. Carrel's triangulation - back to the future.

better fit with the curvature of the ascending aorta; their aperture can be independently controlled in order to comply with possible differences in diameter of the proximal and distal stumps. In this way the ascending aorta substitution can be carried out very quickly by one single device.

Fig. 17. Device Type II – BIO.

This type II device version allows the bending up to a 90° degree angle of the axis of their ends, whose aperture can be independently controlled. This allows to fit curved anatomy of ascending aorta and possible variations in diameter of the vascular stumps. Video at: http://www.fondazionecarrel.org/tsb2/tsb2.html

In case of dissection however type III device previously connected with graft tube should be better used for distal anastomosis. Proximal anastomosis can be carried out with standard suture, time being not here a critical factor, or by a second type III device if dissection involves also the proximal anastomosis line.

This device version can also be used in isthmus rupture repair, having care of entering the aorta through or close to the laceration in order to preserve integrity of the vascular wall at site of device ends deployment.

#### **<IP_ADDRESS> Recent refinements**

The extensive past experience provided full mechanical reliability of devices in all their versions and proved their easy applicability to aortic stump in all conditions. While the expandable device allows a much easier, quicker and more efficient ("airtight" seal) graftaortic stump coupling than standard suture (Nazari 2010), it implies however the permanence of endovascular tubular wireframe and external ligature that could, at least theoretically, mechanically conflict with aortic wall, particularly at device ends, and with confining tissues/organs.

Aorto-digestive fistula is an infrequent but well documented occurrence after aortic open (Geraci et al 2008; Luo et al, 2010) as well as after endovascular (Ruby & Cogbill, 2007; Marone et al, 2007; Chiesa et al, 2010) repair. While graft or/and suture line contamination/infection may occasionally be suspected as the primary etiological factor, pure mechanical erosion from systolic movements of graft and even from the suture line only (Tanaka et al, 2009) may probably represent the first triggering factor in a portion of cases difficult to quantify.

The pure mechanical effect of these movements on the confining tissue/organ is predictably higher the harder/less compliant is the prosthetic material as well as the more conflicting is its orientation in relation to the confining tissue/organ.

We then recently focused on optimal consistency of the expandable wireframe and on means to provide external fixation with the final aim of achieving mechanical forces of coupling as much as possible similar to those taking place in standard hand suture anastomosis.

better fit with the curvature of the ascending aorta; their aperture can be independently controlled in order to comply with possible differences in diameter of the proximal and distal stumps. In this way the ascending aorta substitution can be carried out very quickly

This type II device version allows the bending up to a 90° degree angle of the axis of their ends, whose aperture can be independently controlled. This allows to fit curved anatomy of

In case of dissection however type III device previously connected with graft tube should be better used for distal anastomosis. Proximal anastomosis can be carried out with standard suture, time being not here a critical factor, or by a second type III device if dissection

This device version can also be used in isthmus rupture repair, having care of entering the aorta through or close to the laceration in order to preserve integrity of the vascular wall at

The extensive past experience provided full mechanical reliability of devices in all their versions and proved their easy applicability to aortic stump in all conditions. While the expandable device allows a much easier, quicker and more efficient ("airtight" seal) graftaortic stump coupling than standard suture (Nazari 2010), it implies however the permanence of endovascular tubular wireframe and external ligature that could, at least theoretically, mechanically conflict with aortic wall, particularly at device ends, and with

Aorto-digestive fistula is an infrequent but well documented occurrence after aortic open (Geraci et al 2008; Luo et al, 2010) as well as after endovascular (Ruby & Cogbill, 2007; Marone et al, 2007; Chiesa et al, 2010) repair. While graft or/and suture line contamination/infection may occasionally be suspected as the primary etiological factor, pure mechanical erosion from systolic movements of graft and even from the suture line only (Tanaka et al, 2009) may

The pure mechanical effect of these movements on the confining tissue/organ is predictably higher the harder/less compliant is the prosthetic material as well as the more conflicting is

We then recently focused on optimal consistency of the expandable wireframe and on means to provide external fixation with the final aim of achieving mechanical forces of coupling as

probably represent the first triggering factor in a portion of cases difficult to quantify.

much as possible similar to those taking place in standard hand suture anastomosis.

its orientation in relation to the confining tissue/organ.

ascending aorta and possible variations in diameter of the vascular stumps.

Video at: http://www.fondazionecarrel.org/tsb2/tsb2.html

involves also the proximal anastomosis line.

by one single device.

Fig. 17. Device Type II – BIO.

site of device ends deployment. **<IP_ADDRESS> Recent refinements**

confining tissues/organs.

For this purpose, on one side, the wireframe consistency was decreased by variably reducing the gauge of nitinol wires and their respective position within wireframe and, on the other side, the external ligature was carried out with the thinnest possible prolene (5-0) encircling suture, transfixing full thickness the device and aortic wall at three equidistant points. The limit of the former was the ability to sustain the external ligature without wireframe deformation/collapse at pressure providing "airtight" seal; the limit of the later was the ability to provide stability of the aortic stump-expandable device coupling at stretch test.

The present versions of the devices with minimal consistence and great compliance minimize the risk of mechanical conflict and erosion with the confining tissue/organs; moreover wireframe consistency was settled in such a way that is minimal at the device end where the possible conflict with aortic wall could be higher.

Concerning the external ligature and its many possible ways of application, it may be useful to recall that vascular anastomosis must guarantee haemostatic sealing and stability of the coupling which, contrariwise to standard suture, rely on different mechanisms with this type of coupling (Fig. 8).

Thus even though external devices surface is provided with short needles perpendicularly positioned around its circumference at 4-6 equidistant points, reduction of the wireframe consistency may probably decrease their reliability in keeping coupling stability.

To optimize coupling stability in this lighter expandable wireframe we successfully tested 5- 0 prolene encircling suture, transfixing full thickness the device and aortic wall at three equidistant points (fig. 18). In this way in fact the minimal expandable wireframe consistence to sustain the external ligature can be further significantly reduced and stability increased, by splitting the external ligature circumference in three separate and equivalent segments. In this embodiment the expandable ring will be then really only a bit more consistent of a common graft tube.

Fig. 18. Carrel's triangulation - back to the future.

To provide reliable coupling stability, instead of simple external ligature, 5-0 prolene encircling suture, transfixing full thickness the device and aortic wall at three equidistant points, was passed and tied as shown, in a sort of revival of the Carrel triangulation historical technique. Video at http://www.fondazionecarrel.org/tsb2/tsb2.html

This coupling stability was proved by a stretch test consisting in a sequence of manoeuvres carried out on the anastomosis that includes: **1)** complete compression of the anastomosis in orthogonal directions and then **2)** vigorous manual stretching of the anastomosis in the coaxial plane separately at four points of its circumference. The sequence was repeated three times and the anastomosis checked for any significant vascular stump backwards dislocation on the expandable wireframe throughout the entire circumference.

New Approaches for Treatment and Prevention of Aortic Aneurysms 281

In synthesis this method ideally allows to change any vascular-graft anastomosis ≥ 6 mm in diameter from the current facing ends Carrel suture into a simpler, quicker and more efficient ("airtight" seal) telescoping anastomosis, sealed and fixed by single thread external ligature passed full thickness at three points (or more when appropriate), in a sort of ideal

The hypothesizable potential impact (table 1) may exceed that expected on complication rate of open prosthetic substitution of all aortic tracts and in particular in those higher risk conditions as acute dissection. In fact the technical simplification with increased reliability of anastomosis haemostasis and dissection layer approximation with false lumen permanent seal has the logical direct consequence, for example, of enabling also lesser expert cardiovascular surgeons to deal with these clinical cases very often requiring immediate surgical attention, thus increasing surgical team efficiency and hospital unit productivity.

Table 1. Potential impact of expandable device aortic anastomosis compared with current

hand suture technique

re-elaboration of the historical Carrel triangulation technique.

Fig. 19. Stretch test.

Stability coupling was validate by stretching test consisting in vigorous manual stretching of the anastomosis in the coaxial plane separately at four points of its circumference; the anastomosis was than checked for any significant vascular stump backwards dislocation on the expandable wireframe throughout the entire circumference. Video at: http://www.fondazionecarrel.org/tsb2/tsb2.html

Device type III stability was also increased by further buttressing the inner and outer Dacron wrapping by 4-0 prolene suture transfixing the circumference in 3-6 points (fig. 20), a manoeuvre that however can be predictably carried out after perfusion is resumed. While it's certainly easier and also a bit quicker to use expandable device wireframe strong enough to block the aortic stump between simple external ligature and outer surface of the device fixing needles, I think that it's much more technically appropriate and probably safer to realize a vascular/graft coupling where the forces applied are as much as possible similar to those involved in standard vascular suture, even though this can mean a minimal increase of ischemic time, required for placing the 3 full thickness stitches around the positioned device.

Fig. 20. Type III security fixation.

A simpler in and out 4-0 prolene buttressing in 4-6 points of the inner and outer device wireframe will greatly increase coupling stability.

Video at http://www.fondazionecarrel.org/tsb2/tsb2.html

Stability coupling was validate by stretching test consisting in vigorous manual stretching of the anastomosis in the coaxial plane separately at four points of its circumference; the anastomosis was than checked for any significant vascular stump backwards dislocation on

Device type III stability was also increased by further buttressing the inner and outer Dacron wrapping by 4-0 prolene suture transfixing the circumference in 3-6 points (fig. 20),

While it's certainly easier and also a bit quicker to use expandable device wireframe strong enough to block the aortic stump between simple external ligature and outer surface of the device fixing needles, I think that it's much more technically appropriate and probably safer to realize a vascular/graft coupling where the forces applied are as much as possible similar to those involved in standard vascular suture, even though this can mean a minimal increase of ischemic time, required for placing the 3 full thickness stitches around the positioned device.

a manoeuvre that however can be predictably carried out after perfusion is resumed.

A simpler in and out 4-0 prolene buttressing in 4-6 points of the inner and outer device

the expandable wireframe throughout the entire circumference. Video at: http://www.fondazionecarrel.org/tsb2/tsb2.html

Fig. 19. Stretch test.

Fig. 20. Type III security fixation.

wireframe will greatly increase coupling stability.

Video at http://www.fondazionecarrel.org/tsb2/tsb2.html

In synthesis this method ideally allows to change any vascular-graft anastomosis ≥ 6 mm in diameter from the current facing ends Carrel suture into a simpler, quicker and more efficient ("airtight" seal) telescoping anastomosis, sealed and fixed by single thread external ligature passed full thickness at three points (or more when appropriate), in a sort of ideal re-elaboration of the historical Carrel triangulation technique.

The hypothesizable potential impact (table 1) may exceed that expected on complication rate of open prosthetic substitution of all aortic tracts and in particular in those higher risk conditions as acute dissection. In fact the technical simplification with increased reliability of anastomosis haemostasis and dissection layer approximation with false lumen permanent seal has the logical direct consequence, for example, of enabling also lesser expert cardiovascular surgeons to deal with these clinical cases very often requiring immediate surgical attention, thus increasing surgical team efficiency and hospital unit productivity.

Table 1. Potential impact of expandable device aortic anastomosis compared with current hand suture technique

New Approaches for Treatment and Prevention of Aortic Aneurysms 283

Fig. 21. The Rational. The experimental hypothesis is based on the fact that the net prosthesis positioned and maintained in stable contact with the aortic walls (A) is spontaneously, gradually covered by the neo-intima (B), invaded by fibroblasts and thus stably associated to the aortic wall. If the net mesh is appropriately dimensioned, it may be expected that the blood

flow through the collateral branches is not affected (arrows) (from Nazari et al, 1996c)

*Left square.* After 6 weeks the net prosthesis, a braided polipropilene fitted with a co-axial thin stainless steel coil surgically positioned in swine upper descending aorta, was found to be covered by the new intima and well attached to the aortic wall. In the lower right side of the prosthesis segment the net is no longer visible and presumably, with little more time, this smooth, normal intimal surface would extend to completely cover the whole net prosthesis (metallic coil was removed). *Right square.* Histology shows the net prosthesis completely included into the intima layer and lying in contact with the media. The square area shows the magnified polypropylene mesh net pattern. (mod from Nazari et al 1996 c)

The fabric framework linked to the aortic wall would then condition its significant, regular and uniform mechanical strengthening that fractionates and absorbs the centrifuge systolic stress of the bloodstream. The significant mechanical strengthening achieved in this way was measurable by comparison of compliance (∆V/∆P) of the treated aortic tract with that

Fig. 22. Swine experimental model.

Video http://www.fondazionecarrel.org/tsb2/tsb2.html

of the immediately confining segment (fig 23)

#### **3. New endovascular "net" prosthesis for aortic wall strengthening without blocking aortic branches perfusion to early stop aneurysm progression or to prevent its formation in high risk patients**

#### **3.1 Background**

While endovascular procedures already proved lower mortality and complications rate than open surgery and as good long term results in infrarenal and upper descending aorta, in all other aortic tracts endovascular techniques are much more complex and long term results less predictable mainly due to the presence of significant collateral branches, that require additional maneuvers to avoid their obstruction by the endoprosthesis (i.e. hybrid procedure with open surgery for supraortic trunks de-branching).

Moreover endovascular procedures failed to protect from spinal chord ischemia and consequent paraplegia in extended descending aorta prosthetic substitution; it has been hypothesized that this could be due, at least in some case, to the fact that while endoprosthesis prevents intercostal branches to be physiologically perfused, cannot prevent, at least for a certain time in the initial phase, backwards blood flow into the space between endoprosthesis and aortic wall, thus generating conditions for a blood flow "steal" from perfusion of the spinal chord (Kawaharada et al, 2010).

On the other hand current data (Gopaldas et al, 2010) show that only less than 1/4 of ideal candidates to endovascular treatment (uncomplicated, non genetic, isolated, elective descending aortic aneurysms) underwent endovascular procedure, while the remaining 3/4 still underwent open surgical repair in US.

#### **3.2 The rational**

The relative slowness of aneurysm formation and progression to rupture indicates that the decrease in the strength of the arterial wall under the aneurysm formation threshold may be very gradual and limited. Consequently one can imagine that measures to increase, even moderately, the mechanical strength of the arterial wall, for example by means of a dacron fabric network, should be successful in preventing aneurysm formations and thus its complications (dissection and rupture), without requiring complete prosthetic substitution. Moreover the conclusive understanding that a destruction of the elastin fibrils network is at the basis of aneurysm formation [Gott et al, 1996], might acknowledge the re-establishment of a uniform and regular network strengthening of the vascular wall with a net prosthesis as a logic, direct correction of the primary defect. On the other hand this mechanical approach since long time is already in use in many other technology fields for prevention of deformation of elastic/floppy tubes or structures subject to inner high pressure, as for example gardening tubes, tires, rubber boat etc.

In previous animal experiments we showed that the endovascular positioning of polypropilen "net" prosthesis, if kept in contact with the inner vascular surface, is spontaneously covered by new intima and included in the aortic wall in few weeks (fig 21 and fig 22), still keeping perfusion of some intercostals vessels, even though the net meshes used in those experiments were certainly too tight to assure their long term patency.

The experimental hypothesis is based on the fact that the net prosthesis positioned and maintained in stable contact with the aortic walls (A) is spontaneously, gradually covered by the neo-intima (B), invaded by fibroblasts and thus stably associated to the aortic wall. If the net mesh is appropriately dimensioned, it may be expected that the blood flow through the collateral branches is not affected (arrows) (from Nazari et al, 1996c)

While endovascular procedures already proved lower mortality and complications rate than open surgery and as good long term results in infrarenal and upper descending aorta, in all other aortic tracts endovascular techniques are much more complex and long term results less predictable mainly due to the presence of significant collateral branches, that require additional maneuvers to avoid their obstruction by the endoprosthesis (i.e. hybrid

Moreover endovascular procedures failed to protect from spinal chord ischemia and consequent paraplegia in extended descending aorta prosthetic substitution; it has been hypothesized that this could be due, at least in some case, to the fact that while endoprosthesis prevents intercostal branches to be physiologically perfused, cannot prevent, at least for a certain time in the initial phase, backwards blood flow into the space between endoprosthesis and aortic wall, thus generating conditions for a blood flow "steal" from

On the other hand current data (Gopaldas et al, 2010) show that only less than 1/4 of ideal candidates to endovascular treatment (uncomplicated, non genetic, isolated, elective descending aortic aneurysms) underwent endovascular procedure, while the remaining 3/4

The relative slowness of aneurysm formation and progression to rupture indicates that the decrease in the strength of the arterial wall under the aneurysm formation threshold may be very gradual and limited. Consequently one can imagine that measures to increase, even moderately, the mechanical strength of the arterial wall, for example by means of a dacron fabric network, should be successful in preventing aneurysm formations and thus its complications (dissection and rupture), without requiring complete prosthetic substitution. Moreover the conclusive understanding that a destruction of the elastin fibrils network is at the basis of aneurysm formation [Gott et al, 1996], might acknowledge the re-establishment of a uniform and regular network strengthening of the vascular wall with a net prosthesis as a logic, direct correction of the primary defect. On the other hand this mechanical approach since long time is already in use in many other technology fields for prevention of deformation of elastic/floppy tubes or structures subject to inner high pressure, as for

In previous animal experiments we showed that the endovascular positioning of polypropilen "net" prosthesis, if kept in contact with the inner vascular surface, is spontaneously covered by new intima and included in the aortic wall in few weeks (fig 21 and fig 22), still keeping perfusion of some intercostals vessels, even though the net meshes used in those experiments

The experimental hypothesis is based on the fact that the net prosthesis positioned and maintained in stable contact with the aortic walls (A) is spontaneously, gradually covered by the neo-intima (B), invaded by fibroblasts and thus stably associated to the aortic wall. If the net mesh is appropriately dimensioned, it may be expected that the blood flow through

**3. New endovascular "net" prosthesis for aortic wall strengthening without blocking aortic branches perfusion to early stop aneurysm progression or to**

**prevent its formation in high risk patients**

procedure with open surgery for supraortic trunks de-branching).

perfusion of the spinal chord (Kawaharada et al, 2010).

still underwent open surgical repair in US.

example gardening tubes, tires, rubber boat etc.

were certainly too tight to assure their long term patency.

the collateral branches is not affected (arrows) (from Nazari et al, 1996c)

**3.1 Background**

**3.2 The rational**

Fig. 21. The Rational. The experimental hypothesis is based on the fact that the net prosthesis positioned and maintained in stable contact with the aortic walls (A) is spontaneously, gradually covered by the neo-intima (B), invaded by fibroblasts and thus stably associated to the aortic wall. If the net mesh is appropriately dimensioned, it may be expected that the blood flow through the collateral branches is not affected (arrows) (from Nazari et al, 1996c)

Fig. 22. Swine experimental model.

*Left square.* After 6 weeks the net prosthesis, a braided polipropilene fitted with a co-axial thin stainless steel coil surgically positioned in swine upper descending aorta, was found to be covered by the new intima and well attached to the aortic wall. In the lower right side of the prosthesis segment the net is no longer visible and presumably, with little more time, this smooth, normal intimal surface would extend to completely cover the whole net prosthesis (metallic coil was removed). *Right square.* Histology shows the net prosthesis completely included into the intima layer and lying in contact with the media. The square area shows the magnified polypropylene mesh net pattern. (mod from Nazari et al 1996 c) Video http://www.fondazionecarrel.org/tsb2/tsb2.html

The fabric framework linked to the aortic wall would then condition its significant, regular and uniform mechanical strengthening that fractionates and absorbs the centrifuge systolic stress of the bloodstream. The significant mechanical strengthening achieved in this way was measurable by comparison of compliance (∆V/∆P) of the treated aortic tract with that of the immediately confining segment (fig 23)

New Approaches for Treatment and Prevention of Aortic Aneurysms 285

The structural properties of the aortic wall associated to the intraluminal net prosthesis rely on three factors (fig 24): 1) the structural properties of the net prosthesis, 2) the structural properties of the aortic wall, 3) the strength of the bonds between the aortic wall and the net prosthesis, based substantially on fibroblastic invasion of the net fabric and its permanent integration with aortic wall. Given the structural adequacy of the net prosthesis (polypropylene net prosthesis squared mesh 5x5 mm with a thread diameter of 0.5 mm sustaining a pressure of 300 mmHg (0.04 Nmm2) is charged at 50% of its failure tension (Nazari at al, 1996b, 1996c) and the mechanical effect of fractioning the aortic wall in the small area of the net meshes (fig 24, lower part), the important point of this model is the strength of the links biologically established between the threads of the mesh and the aortic

It may be hypothesized that its strength relies mainly in the degree of integration of the net thread with the intimal layer mainly due to fibroblastic invasion and is then predictably

stronger with porous material (PTFE) or multifilament braided polyester (Dacron).

Fig. 25. Variation of the circumferential stress through the aortic wall thickness.

from Robicsek and Thubrikar 1994)

For a thick cylinder (thickness > 8% of the radius (R)) such as the aorta, the stress is not uniform throughout the wall; it is maximal on the inner wall, it decreases through the thickness of the wall and is minimal on the outer wall. This explains the pathogenic mechanism of dissection, where only stress to the inner part of the wall is strong enough to cause tear; moreover it adds arguments to the rationale of intraluminal positioning of the net prosthesis in order to achieve the structural strengthening just where this is most needed, i.e. where the circumferential stress is maximal. This allows for the required

strengthening with optimization of the amount of prosthetic material (thinner threads) (Mod

The latter is the crucial point of this model. In fact if this link is sufficiently strong and stable in the time, it can be hypothesized to be able to stabilize aortic wall and prevent dilatation even independently from the "net" prosthesis own diameter, which could not be further distended after that their meshes have been firmly integrated into the aortic wall (Redaelli & Fiore, 2011). The practical consequence is that the precise equivalence between diameter of the net prosthesis and aortic diameter is not necessary; the prosthesis in fact can be

wall tissues (point 3 in fig. 24) during the 4-6 weeks integration process.

Fig. 23. Aortic wall compliance (ΔV/ΔP).

To quantify aortic wall strengthening induced by the net prosthesis its compliance (ΔV/ΔP). was compared with that of the confining segments that was found significantly higher. (from Nazari et al 1996b) Video at http://www.fondazionecarrel.org/tsb2/tsb2.html

The structural properties of the aortic wall associated to the intraluminal net prosthesis rely on three factors: **1)** the structural properties of the net prosthesis, **2)** the structural properties of the aortic wall, **3)** the strength of the bonds between the aortic wall and the net prosthesis, based substantially on fibroblastic invasion of the net fabric and its permanent integration with aortic wall. The latter is the crucial point and can be viewed as the true "functional unit" of this model. In fact if this link is sufficiently strong and stable in the time it can be hypothesized to be able to stabilize aortic wall and prevent dilatation even independently from the "net" prosthesis own diameter, which could not be further distended after that their fabric threads have been integrated into the aortic wall. (mod. from Nazari et al 1996b)

To quantify aortic wall strengthening induced by the net prosthesis its compliance (ΔV/ΔP). was compared with that of the confining segments that was found significantly higher. (from Nazari et al 1996b) Video at http://www.fondazionecarrel.org/tsb2/tsb2.html

The structural properties of the aortic wall associated to the intraluminal net prosthesis rely on three factors: **1)** the structural properties of the net prosthesis, **2)** the structural properties of the aortic wall, **3)** the strength of the bonds between the aortic wall and the net prosthesis, based substantially on fibroblastic invasion of the net fabric and its permanent integration with aortic wall. The latter is the crucial point and can be viewed as the true "functional unit" of this model. In fact if this link is sufficiently strong and stable in the time it can be hypothesized to be able to stabilize aortic wall and prevent dilatation even independently from the "net" prosthesis own diameter, which could not be further distended after that their fabric threads have been integrated into the aortic

Fig. 23. Aortic wall compliance (ΔV/ΔP).

Fig. 24. Aortic wall strengthening factors.

wall. (mod. from Nazari et al 1996b)

The structural properties of the aortic wall associated to the intraluminal net prosthesis rely on three factors (fig 24): 1) the structural properties of the net prosthesis, 2) the structural properties of the aortic wall, 3) the strength of the bonds between the aortic wall and the net prosthesis, based substantially on fibroblastic invasion of the net fabric and its permanent integration with aortic wall. Given the structural adequacy of the net prosthesis (polypropylene net prosthesis squared mesh 5x5 mm with a thread diameter of 0.5 mm sustaining a pressure of 300 mmHg (0.04 Nmm2) is charged at 50% of its failure tension (Nazari at al, 1996b, 1996c) and the mechanical effect of fractioning the aortic wall in the small area of the net meshes (fig 24, lower part), the important point of this model is the strength of the links biologically established between the threads of the mesh and the aortic wall tissues (point 3 in fig. 24) during the 4-6 weeks integration process.

It may be hypothesized that its strength relies mainly in the degree of integration of the net thread with the intimal layer mainly due to fibroblastic invasion and is then predictably stronger with porous material (PTFE) or multifilament braided polyester (Dacron).

Fig. 25. Variation of the circumferential stress through the aortic wall thickness. For a thick cylinder (thickness > 8% of the radius (R)) such as the aorta, the stress is not uniform throughout the wall; it is maximal on the inner wall, it decreases through the thickness of the wall and is minimal on the outer wall. This explains the pathogenic mechanism of dissection, where only stress to the inner part of the wall is strong enough to cause tear; moreover it adds arguments to the rationale of intraluminal positioning of the net prosthesis in order to achieve the structural strengthening just where this is most needed, i.e. where the circumferential stress is maximal. This allows for the required strengthening with optimization of the amount of prosthetic material (thinner threads) (Mod from Robicsek and Thubrikar 1994)

The latter is the crucial point of this model. In fact if this link is sufficiently strong and stable in the time, it can be hypothesized to be able to stabilize aortic wall and prevent dilatation even independently from the "net" prosthesis own diameter, which could not be further distended after that their meshes have been firmly integrated into the aortic wall (Redaelli & Fiore, 2011). The practical consequence is that the precise equivalence between diameter of the net prosthesis and aortic diameter is not necessary; the prosthesis in fact can be

New Approaches for Treatment and Prevention of Aortic Aneurysms 287

Possible prototypes versions were realized by 5x5mm meshes of braided fabric embodied with very thin nitinol wider helicoidal wireframe (*left*:blu lines, red arrows, 12-15 mm). Ascending aorta and arch would require curved prosthesis (*right*), maybe bespoke to the

A more straight, ready, maybe oversimplified method, but still mechanically efficient, could consist in the simple substitution of the Dacron tube with a fabric net prosthesis in any of

The prototypes resulting from the above project phase will undergo "in vivo" animal experiments. Ideally these experiments should include long term (≥12 months) evaluation in an appropriate animal (sheep) models. These may consist in prototypes positioning in the thoraco-abdominal aorta (model 1) and in the aortic arch (model 2) surgically or, possibly, as endovascular procedure if the endovascular delivery system is realized and suitable with the animal model peripheral vascular diameter. Half surviving animals could be sacrificed

regular integration of the net prosthesis into the intimal layer, looking for site and

the real structural strengthening achieved by measuring the aortic wall compliance

The remaining animals could be followed indefinitely and examined in case late

Ideally during the experimental phase will be identified and monitored Marfan patients with still normal or initial dilatation of any tract of the aorta in order to select those to be offered/accepting the prophylactic endovascular aortic strengthening. Obviously associated risk factors, in particular familiar history of aneurism and rupture, as well as other inclusion and exclusion criteria indicated by the Marfan Associations and clinical Institution Marfan

Marfan diseases is a condition where pure prophylaxis of aortic aneurism would be in most cases "per se" appropriate, due to its very high incidence and rather early in the patient life; on the other hand prophylactic surgical aortic substitution is an already considered option

extension of possible areas on lacking integration with aortic wall

(∆V/∆P) compared to that of the confining aortic segment (Fig. 5) absence of complications such as migration, ulceration, perforation etc

Centers, will form the basis for planning the possible future clinical trial.

preserved perfusion of all collateral branches for the extent of the net prosthesis

Fig. 26. "Net" prosthesis prototypes.

patient aortic anatomy on CT scans.

the current endovascular devices.

at 12 months and examined to verify:

complication or death.

**3.3.3 Preparation of clinical trial**

**3.3.2 Experimental protocol**

significantly redundant in respect to the effective aortic diameter, thus allowing for its easier adaptability to geometrical irregularity of aneurysm wall without consequences on its efficiency in keeping stable aortic diameter. The final "functional unit" of this model for aortic wall strengthening is then the single mesh of the net integrated into the aortic wall, whose efficiency relies then on the bonds between net threads and aortic tissue.

Interestingly enough in this model the endovascular net prosthesis provides aortic wall mechanical support just where the mechanical stress is higher (Robicsek & Thubrikar, 1994) (fig. 25), and thus just where it's most needed both to prevent further dilatation, theoretically preventable also by external aortic wall wrapping (Pepper et al 2010), and to avoid partial (dissection) or total rupture, with optimization of the amount of prosthetic material. The reduced volume of the net prosthesis in comparison with current endoprosthesis (≤1/6th approximately) further greatly enhances its introduction through peripheral vessel often tortuous and restricted by atherosclerosis.

#### **3.3 Methodology**

The previous experimental work as well as structural mechanical theoretical considerations allow to predict applicability and reliability of the method in the clinical setting of preventing aneurysm formation or arresting its progression at an early stage.

The organizative plan for achieving this final goal however includes several further distinct steps.

#### **3.3.1 Ideal "net" prosthesis prototypes setup**

This part of the project includes

The nitinol wireframe adds to the net further structural strengthening then greatly exceeding that required; an accurate structural mechanics computation would theoretically allow the achievement of the necessary aortic wall structural strengthening to permanently fix aortic diameter with really a very thin and wide meshes net prosthesis.

Fig. 26. "Net" prosthesis prototypes.

286 Front Lines of Thoracic Surgery

significantly redundant in respect to the effective aortic diameter, thus allowing for its easier adaptability to geometrical irregularity of aneurysm wall without consequences on its efficiency in keeping stable aortic diameter. The final "functional unit" of this model for aortic wall strengthening is then the single mesh of the net integrated into the aortic wall,

Interestingly enough in this model the endovascular net prosthesis provides aortic wall mechanical support just where the mechanical stress is higher (Robicsek & Thubrikar, 1994) (fig. 25), and thus just where it's most needed both to prevent further dilatation, theoretically preventable also by external aortic wall wrapping (Pepper et al 2010), and to avoid partial (dissection) or total rupture, with optimization of the amount of prosthetic material. The reduced volume of the net prosthesis in comparison with current endoprosthesis (≤1/6th approximately) further greatly enhances its introduction through

The previous experimental work as well as structural mechanical theoretical considerations allow to predict applicability and reliability of the method in the clinical setting of

The organizative plan for achieving this final goal however includes several further distinct

the selection of the best fabric material (PTFE, multifilament polypropylene, etc) with

 identification of its appropriated dimensioning and mechanical features, including elasticity, to provide the best support to the stream pressure stress up to its extreme

 identification of the most appropriate net prosthesis embodiment able to maintain it in permanent contact with intimal surface, which is essential to its quick and spontaneous incorporation into the aortic wall. This may include a thin nitinol wireframe associated to the main net prosthesis; a prototype for descending aorta currently in study is illustrated at fig. 26. However fluid-dynamic shapes of the net threads will be also evaluated in mechanical models with the aim to verify if it is possible to achieve

 Ascending aorta and Arch require variably curved net prosthesis (fig 26, right); for these tracts however bespoke prosthesis on the individual TC scan, maybe with wider meshes for the supraortic trunks area, is an option to be considered for clinical use; bespoke prosthesis however have being already realized in a external ascending aorta

The nitinol wireframe adds to the net further structural strengthening then greatly exceeding that required; an accurate structural mechanics computation would theoretically allow the achievement of the necessary aortic wall structural strengthening to permanently

whose efficiency relies then on the bonds between net threads and aortic tissue.

peripheral vessel often tortuous and restricted by atherosclerosis.

the best chances of the strongest links with aortic wall

conduit inner wall contact by means of pure fluid stream

fix aortic diameter with really a very thin and wide meshes net prosthesis.

**3.3.1 Ideal "net" prosthesis prototypes setup**

wrapping protocol (Pepper et al, 2010)). Endovascular delivery system is also to be realized

This part of the project includes

pathological limit (300 mm hg)

preventing aneurysm formation or arresting its progression at an early stage.

**3.3 Methodology**

steps.

Possible prototypes versions were realized by 5x5mm meshes of braided fabric embodied with very thin nitinol wider helicoidal wireframe (*left*:blu lines, red arrows, 12-15 mm). Ascending aorta and arch would require curved prosthesis (*right*), maybe bespoke to the patient aortic anatomy on CT scans.

A more straight, ready, maybe oversimplified method, but still mechanically efficient, could consist in the simple substitution of the Dacron tube with a fabric net prosthesis in any of the current endovascular devices.