Support for this work was obtained by the VA Rehabilitation Research &amp; Development National Center of Excellence for the Medical Consequences of Spinal Cord Injury (VA RR&amp;D #B9212C). Two of the four powered exoskeleton devices were used on a loaner basis at no cost from ReWalk Robotics, Inc. (Marlborough, Massachusetts). Additionally a portion of participants obtained Orthopedic shoes which were donated by Aetrex Worldwide Inc. (Teaneck, New Jersey).
Assistance from Denis Doyle-Green was invaluable during the training program and we thank him for this. We would also like to thank the physical therapists from the Rehabilitation and Spinal Cord Injury Services at the James J. Peters VA Medical Center for their advisement and consultations.

The authors have nothing to disclose.

During the past five years, our group has developed a successful screening and training program for participants to use the type of powered exoskeleton that requires crutches. We have trained individuals with motor complete paralysis as well as those with incomplete paralysis. This training program has the potential to be modified and built upon with additional devices that require the use of crutches, or newer versions of the existing devices.
Standardization of a training program is important to ensure participant safety, successful use of the device, identify staff resources, and to acquire consistent results. Key points in a good training program include appropriate candidate selection, proper fitting of the device, appropriate skill progression, and providing assistance on the shoulders or on an area with intact sensation to enable the user to recognize the required force and movement, promoting adaption of their movements during the subsequent stepping actions. It is important to practice this strategic dance between the trainer and the user in order to minimize trainer support, thus helping the user gain expertise and independence in the device. Trainers should avoid assisting below the participant&#39;s level of sensation since this action results in difficulty in becoming independent in the exoskeleton. Another key point to improve walking skill is to challenge the participant with walking on various surfaces and in different environments. Participants perceive walking indoors and on flat/smooth surfaces in the medical center to be easier than ambulating on a carpeted floor. Walking on carpeted floors, in turn, is reported to be easier than walking outdoors on uneven surfaces such as concrete or asphalt. Walking up and down different slope gradients force the participant to adapt their walking strategy because the method of weight shifting becomes more challenging due to the altered center of balance presented by the slope. All of these challenging environments are commonly encountered within the community and therefore, are very important to practice in a controlled setting to properly prepare the participant.
There have been several reports in persons with SCI who have learned to use a powered exoskeleton to safely ambulate overground16-19,21,36. Many of the participants in these reports had little to no residual function or sensation in their lower extremities. No serious adverse events were reported from these studies and the devices were deemed safe to use with the proper training. The adverse events reported included skin abrasions, bruising or redness of the skin, and fatigue of the upper extremities, especially during the initial training sessions16,19,36. It was noted that with continued training, participants noticed a reduction of upper extremity fatigue and skin abrasions resolved quickly with better fitting of the device. Future bruising and redness were avoided with adjustment of the straps and strategic placement of additional padding surrounding the affected area.
Proficiency in the use of the device is determined by the ability to achieve faster ambulation speeds, reduced levels of assistance, and safe ambulation in varied environments. Prior reports of walking ability showed that those who were more independent would ambulate faster than those who needed assistance. A report by van Hedel et al. categorized walkers as &#34;assisted walkers&#34; if they could ambulate with a minimum speed of 0.44 &#177; 0.14 m/sec; a speed associated with those who chose to walk outdoors with assistance over using their wheelchair42. This walking speed is similar to the 0.40 m/sec speed of the limited community ambulators reported in persons with stroke.43 Although only a few studies have reported ambulation speed and level of assistance using robotic exoskeletons, these studies indicated that many participants were able to achieve the 0.40 m/sec walking speed mentioned in these previous reports. A report using a powered exoskeleton showed that 7 of 12 participants were able to ambulate faster than 0.40 m/sec18. Another investigation using a different powered exoskeleton was able to illustrate 6 of 16 participants successfully ambulating greater than 0.40 m/sec36. Although reports using a third powered exoskeleton have not demonstrated walking speeds of 0.40 m/sec22,44, future reports may show increased walking speeds with further training and/or adaptions in that device. Thus far, all studies using powered exoskeletons have reported those needing greater levels of assistance walked at slower speeds. One thought discussed in these reports was that although some of the participants did not ambulate above the 0.40 m/sec speed, they were able to ambulate at the level of &#34;supervision&#34; as defined in the FIM scale. These reports suggest that, with additional training or modifications to the devices, ambulation at these faster speeds may be achieved.
Energy expenditure measured by oxygen consumption has been demonstrated to be increased with exoskeletal-assisted walking, but not above the threshold that is unduly fatiguing. Eight participants who ambulated in the powered exoskeleton at an average pace of 0.22 &#177; 0.11 m/sec demonstrated walking oxygen consumption rates of 11.2 &#177; 1.7 ml/kg/min and heart rates of 118 &#177; 21 bmp (48% &#177; 16% heart rate reserve), both of which were a significant increase from sitting and standing17, but significantly below the maximum predicted values. Another report using a different powered exoskeleton, evaluated oxygen consumption in 5 participants during 2 bouts of walking and reported 9.5 &#177; 0.8 ml/kg/min when walking at 0.19 &#177; 0.01 m/sec and 11.5 &#177; 1.4 ml/kg/min when walking at 0.27 &#177; 0.05 m/sec21. Both of these studies demonstrated that participants ambulating at a moderate intensity were above the minimal training intensity threshold determined by the American College of Sports Medicine to be effective for cardiorespiratory benefits45. This suggests that these devices have the potential to be used for longer periods of time, providing a form of activity that if performed regularly may be expected to lead to improvements in the user&#39;s fitness, body composition and lipid profile.
The powered exoskeletons offer a form of modified independence (level six as defined by the FIM) for standing and overground ambulation for persons with upper extremity function. Future devices may be designed to ambulate at faster speeds or provide a greater ability to vary the desired ambulation speed. Future exoskeletons may also be designed for those with limited hand and arm function (such as those with tetraplegia) by maintaining the user&#39;s balance with additional trunk support and providing another mechanism than holding a crutch for maintaining balance. Advancements in brain control may one day be available to be incorporated to control the walking movement20. Within this emerging field, the basic training concepts presented may be applicable to the current and future powered exoskeletons, but should be tailored to the user and the exoskeleton being used.
Standardized training strategies are currently utilized for successful participant exoskeletal-assisted walking; future modifications of these devices may need adaptions to the training paradigm. Teaching qualified SCI health-care professionals to appropriately train persons with SCI to perform exoskeletal-assisted walking is needed for the continued use and prescription of these devices. The future is bright for these devices; the use of powered exoskeletons by persons with SCI would become more widespread with the establishment of training programs in medical and rehabilitation centers throughout the world. Additionally, future research may show that regular exoskeletal-assisted walking improves many of the secondary medical complications that are associated with immobility and paralysis from spinal cord injury.

Many persons with spinal cord injury (SCI) are unable to stand and ambulate with or without the use of an assistive device or physical assistance. For centuries, the only mobility option for those with severe SCI has been the wheelchair1. During the past few decades, persons with SCI have had the option to supplement their mobility by using passive orthotic devices such as a variety of reciprocating gait orthosis (RGO)2-7. These devices, however, have not become more widely used due to the physical effort required by the user to ambulate using these devices. The RGOs also have limitations in the ability to climb stairs, stand up, and sit down3,7. Efforts have been made to enhance the efficiency of these devices by incorporating Functional Electrical Stimulation (FES) to power the movement and help facilitate the forward swinging of the limb; however, these efforts have not progressed beyond concepts or prototypes8-12. In the 1970s, motors were incorporated with an orthosis to power the movement of the hip and knee joints and was successful in allowing a person with SCI to take steps13. However, inadequate battery and computer technology of the time limited the range of the device, and further development was abandoned10,13.
With recent technological advancements, several powered exoskeletons have been developed to enable persons with various pathologies to ambulate overground. These powered exoskeleton devices have been studied in persons with stroke14,15, persons with complete and incomplete SCI16-24, and other persons with disabilities causing reduced control of their lower extremities25-27. Although the devices differ, each one requires training and practice by the user for safe performance. Three of the referenced devices require the use of crutches to ambulate and maintain balance. The fourth one maintains balance and stability because of its large footplate and mass which enlarges the base of support and lowers the center of gravity20. The three devices that require crutching utilize the same principles even though there are some variations with the mechanics and methods of controlling the desired actions due to differences in the design of the devices.
A training program was developed at the James J. Peters VA Medical Center (JJPVAMC), Bronx, NY by a group of researchers consisting of a biomedical engineer, physiologist, physiatrist, exercise physiologist, neurologist and physical therapists. The training program was developed with one specific powered exoskeleton previously described 17,18 but it incorporates sets of skills that are applicable to other powered exoskeletons which require a set of crutches to maintain balance. All potential participants were screened prior to participating in the progressive training program. The importance of screening in persons with SCI is to ensure absence of contraindicated medical complications that may inhibit the safe use of these devices. One area of concern is low bone mineral density (BMD). Persons with SCI suffer dramatic bone loss immediately after injury28,29 which may continue throughout their lives30. This loss of BMD results in a high risk of long bone fractures. Currently, there is no effective treatment to mitigate bone loss for those with complete motor SCI. In addition, an established fracture threshold for person with SCI does not exist, but efforts have been made to identify criteria which may be used as a guide31-33 along with clinical judgement and fracture history. Other common contraindications may be treated and resolved, such as limited range of motion (ROM)34 and pressure ulcers35. Each of the different powered exoskeleton may require different conditions for eligibility, such as ROM criteria, to be a candidate to use the device, most of which have been described17-19,21,22,36.
Once a person has successfully passed all of the screening criteria, fitting the device to the user and training may proceed. Proper fitting of the device is important to avoid inappropriate contact of the lower extremities with the exoskeleton because poor fitting may lead to bruising and/or skin abrasions16. Users may have limited or no lower extremity sensation and proprioception; this lack of sensory and tactile feedback from the feet can contribute to an overall lack of awareness of their center of balance, slowing the user&#39;s ability to master the device. This lack of awareness of the center of balance may also lead to challenges with appropriate weight shifting such as difficulty in gauging the extent of the forward and lateral shift necessary during the gait cycle and inappropriately timed weight shifting, resulting in excess use of weight bearing on the arms and crutches for balance maintenance. Once the basics mechanisms of standing balance and weight shifting are acquired, the user is taught to walk in the device. Multiple sessions are needed to improve walking and other mobility skills. Initially, surfaces that are flat and smooth within the medical center are used for training. However, with improved skill level, the user is challenged with incrementally more difficult tasks by introducing different walking surfaces such as carpet, asphalt, concrete, grass, and unleveled surfaces with different degrees of slopes.
The purpose of this manuscript is to report the screening criteria, proper fitting and training procedures for use a powered exoskeleton for overground walking. This program was developed for one device specifically, which is described by others16-18, but it addresses aspects and challenges that are common for staff trainers and persons with SCI who participate in exoskeletal-assisted walking programs which may use another powered exoskeleton. Certain aspects of this protocol are specific to the device used at the JJPVAMC. Additionally, some of the components of the training program were developed by the manufacture which includes orientation of the device components, basic guidelines for a proper fit and basic standing and sitting skill instructions. The researchers at the JJPVAMC developed all training activities performed once the user is standing up. These include enhancement of the standing and sitting training instructions, standing balance skills, indoor walking progression skills, outdoor walking progression skills, and other mobility tasks for reaching, stopping, turning, and various types of door/threshold navigation.

<table border="0" cellpadding="0" cellspacing="0" width="464">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:464px;">Powered Exoskeleton such as ReWalk&#8482;, Ekso&#8482;, REX&#174;, and Indego&#174;, etc.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Loft strand Crutches</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Comfortable sneakers</td>
		</tr>
	</tbody>
</table>

training persons with spinal cord injury to ambulate using a powered exoskeleton

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper extremity function and are able to maintain upright balance using forearm crutches. To ambulate in an exoskeleton, the user must acquire the ability to maintain balance while standing, sitting and appropriate weight shifting with each step. This can be a challenging task for those with deficits in sensation and proprioception in their lower extremities. This manuscript describes screening criteria and a training program developed at the James J. Peters VA Medical Center, Bronx, NY to teach users the skills needed to utilize these devices in institutional, home or community environments. Before training can begin, potential users are screened for appropriate range of motion of the hip, knee and ankle joints. Persons with SCI are at an increased risk of sustaining lower extremity fractures, even with minimal strain or trauma, therefore a bone mineral density assessment is performed to reduce the risk of fracture. Also, as part of screening, a physical examination is performed in order to identify additional health-related contraindications. 
Once the person has successfully passed all screening requirements, they are cleared to begin the training program. The device is properly adjusted to fit the user. A series of static and dynamic balance tasks are taught and performed by the user before learning to walk. The person is taught to ambulate in various environments ranging from indoor level surfaces to outdoors over uneven or changing surfaces. Once skilled enough to be a candidate for home use with the exoskeleton, the user is then required to designate a companion-walker who will train alongside them. Together, the pair must demonstrate the ability to perform various advanced tasks in order to be permitted to use the exoskeleton in their home/community environment.

The following measurements are obtained throughout the training. Two handed and one handed crutch balance skills are each assessed for 1 min as &#34;able&#34; or &#34;not able&#34; to maintain balance (Figure 2). Walking assessments for time and distance are obtained throughout the training sessions using the 6MWT, 10MWT and the TUG. Exoskeletal-assisted walking on commonly encountered surfaces are tested indoors (Figures 3 and 4) and outdoors (Figures 5-6). Other mobility skills such as navigating doors (Figures 7 and 8), reaching over head into a cabinet (Figure 9) and sitting outside on a park bench (Figure 10) are assessed as &#34;able&#34; to perform or &#34;not able&#34; to perform.
Average walking speeds during the 10MWT in 10 session intervals for the first 60 sessions are depicted (Figure 11). This graph shows participants have varying initial ability to use the powered exoskeleton and varying rates of improvement among users. The average &#177; standard deviation of the best fit line&#39;s slope is 0.0048 &#177; 0.004 m/sec and values ranged from 0.00026 to 0.015 m/sec. This indicates that although each participant improved at variable rates they walked an average of 0.0048 m/sec faster each session. The average &#177; standard deviation of the best fit intercept is 0.16 &#177; 1.8 m/sec and the values ranged from -0.026 to 0.50 m/sec. This indicates that on average participants have an average initial speed of 0.16 m/sec; with some participants having almost no ability to ambulate and others have a very good ability in the early stages of training.
Trainer assistance affects performance; those who need a greater level of assistance walk slower than those who are more proficient and independent in using the system18. The three walking test measurements, although similar, provide different proficiency information. The 10MWT provides an indication of the best effort for speed (m/sec) that the user is able to ambulate in the device. The 6MWT distance, when converted to speed in m/sec, provides an average walking speed and is an indication of the consistency of walking in the exoskeleton. Since the timer continues when the user accidently stops walking, the speed from a 6MWT that is closer to the best effort 10MWT indicates that the person had consistent walking and fewer stops. The TUG requires many skills to be performed in consecutive combination. The TUG is a measure of the person&#39;s overall ability to incorporate standing up, walking, turning, stopping, and sitting down in the powered exoskeleton. An overview of the 6MWT, 10MWT and the TUG measurements have been described previously by Yang18 and are presented in Table 1 along with the patient demographic information of the participants.
<img alt="Figure 1" src="/files/ftp_upload/54071/54071fig1.jpg" /> 
Figure 1. Two handed crutch balance. This figure demonstrates a person standing still and balancing with both crutches. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54071/54071fig2.jpg" /> 
Figure 2. One handed crutch balance. This figure demonstrates a person standing still and balancing with only 1 crutch. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54071/54071fig3.jpg" /> 
Figure 3. Walking indoors on a smooth surface. This figure demonstrates a person walking indoors on a flat surface. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54071/54071fig4.jpg" /> 
Figure 4. Walking on carpet. This figure demonstrates a person walking indoors on a carpeted surface. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54071/54071fig5.jpg" /> 
Figure 5. Walking outdoors on grass. This figure demonstrates a person walking outdoors on grass. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54071/54071fig6.jpg" /> 
Figure 6. Walking on slopes. This figure demonstrates a person walking outdoors down a curb cutout. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54071/54071fig7.jpg" /> 
Figure 7. Navigating an elevator. This figure demonstrates a person walking out of a timed door setting such as an elevator door. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54071/54071fig8.jpg" /> 
Figure 8. Walking out of a revolving door. This figure demonstrates a person walking out of a revolving door. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/54071/54071fig9.jpg" /> 
Figure 9. Overhead cabinet and countertop reaching. This figure demonstrates a person taking items out of an overhead cabinet. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/54071/54071fig10.jpg" /> 
Figure 10. Sitting outside on a park bench. This figure demonstrates a person sitting outside on a park bench. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" src="/files/ftp_upload/54071/54071fig11.jpg" /> 
Figure 11. Averaged Ten-session 10MWT Speeds. The data demonstrates the 10MWT speeds for the first 60 sessions of training averaged by ten session intervals. The x-axis describes the sessions and the y-axis describes the average speed (m/sec) calculated from the 10MWT result obtained during the participants training session. A linear best fit line was overlaid on each participant&#39;s results. <a href="https://www.jove.com/files/ftp_upload/54071/54071fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="8">Demographic Characteristics</td>
			<td colspan="7">Walk Tests (WT) and Levels of Assistance (LOA)</td>
		</tr>
		<tr>
			<td rowspan="2">SID</td>
			<td rowspan="2">Age 
			(y)</td>
			<td rowspan="2">Ht 
			(cm)</td>
			<td rowspan="2">Wt 
			(kg)</td>
			<td rowspan="2">Gender</td>
			<td rowspan="2">DOI 
			(y)</td>
			<td rowspan="2">LOI</td>
			<td rowspan="2">AIS</td>
			<td colspan="2">10 m WT</td>
			<td colspan="2">6-min WT</td>
			<td>TUG&#160;</td>
			<td rowspan="2">(LOA)</td>
			<td rowspan="2">Assess- 
			ment Session&#160;</td>
		</tr>
		<tr>
			<td>(sec)</td>
			<td>(m/sec)</td>
			<td>(m)</td>
			<td>(m/sec)</td>
			<td>(sec)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>34</td>
			<td>173</td>
			<td>66.7</td>
			<td>Male</td>
			<td>9</td>
			<td>T4</td>
			<td>B</td>
			<td>39</td>
			<td>0.26</td>
			<td>90</td>
			<td>0.25</td>
			<td>83</td>
			<td>Min</td>
			<td>89</td>
		</tr>
		<tr>
			<td>2</td>
			<td>48</td>
			<td>168</td>
			<td>68</td>
			<td>Male</td>
			<td>4</td>
			<td>T10</td>
			<td>A</td>
			<td>62</td>
			<td>0.16</td>
			<td>51</td>
			<td>0.14</td>
			<td>NP</td>
			<td>Min</td>
			<td>18</td>
		</tr>
		<tr>
			<td>3</td>
			<td>44</td>
			<td>183</td>
			<td>77.1</td>
			<td>Male</td>
			<td>4.5</td>
			<td>T4</td>
			<td>A</td>
			<td>20</td>
			<td>0.50</td>
			<td>209</td>
			<td>0.58</td>
			<td>56</td>
			<td>MI</td>
			<td>63</td>
		</tr>
		<tr>
			<td>4</td>
			<td>58</td>
			<td>160</td>
			<td>64.4</td>
			<td>Female</td>
			<td>1.5</td>
			<td>C8/T8</td>
			<td>A (NT)</td>
			<td>24</td>
			<td>0.42</td>
			<td>139</td>
			<td>0.39</td>
			<td>59</td>
			<td>MI&#160;</td>
			<td>43</td>
		</tr>
		<tr>
			<td>5</td>
			<td>61</td>
			<td>175</td>
			<td>72.6</td>
			<td>Male</td>
			<td>14</td>
			<td>T11</td>
			<td>A</td>
			<td>23</td>
			<td>0.44</td>
			<td>137</td>
			<td>0.38</td>
			<td>66</td>
			<td>MI&#160;</td>
			<td>37</td>
		</tr>
		<tr>
			<td>6</td>
			<td>24</td>
			<td>185</td>
			<td>74.8</td>
			<td>Male</td>
			<td>5</td>
			<td>T5</td>
			<td>A</td>
			<td>56</td>
			<td>0.18</td>
			<td>60</td>
			<td>0.17</td>
			<td>NP</td>
			<td>Min</td>
			<td>12</td>
		</tr>
		<tr>
			<td>7</td>
			<td>40</td>
			<td>183</td>
			<td>88.5</td>
			<td>Male</td>
			<td>1.5</td>
			<td>T1</td>
			<td>B</td>
			<td>61</td>
			<td>0.16</td>
			<td>51</td>
			<td>0.14</td>
			<td>70</td>
			<td>S</td>
			<td>102</td>
		</tr>
		<tr>
			<td>8</td>
			<td>56</td>
			<td>175</td>
			<td>83.9</td>
			<td>Male</td>
			<td>3</td>
			<td>T9</td>
			<td>A</td>
			<td>22</td>
			<td>0.46</td>
			<td>151</td>
			<td>0.42</td>
			<td>116</td>
			<td>S</td>
			<td>51</td>
		</tr>
		<tr>
			<td>9</td>
			<td>50</td>
			<td>183</td>
			<td>99.8</td>
			<td>Male</td>
			<td>11</td>
			<td>T7</td>
			<td>A</td>
			<td>17</td>
			<td>0.59</td>
			<td>208</td>
			<td>0.58</td>
			<td>56</td>
			<td>MI</td>
			<td>56</td>
		</tr>
		<tr>
			<td>10</td>
			<td>37</td>
			<td>170</td>
			<td>65.8</td>
			<td>Male</td>
			<td>6</td>
			<td>T2</td>
			<td>A</td>
			<td>22</td>
			<td>0.46</td>
			<td>150</td>
			<td>0.42</td>
			<td>63</td>
			<td>Min</td>
			<td>59</td>
		</tr>
		<tr>
			<td>11</td>
			<td>64</td>
			<td>173</td>
			<td>72.8</td>
			<td>Male</td>
			<td>3</td>
			<td>T2</td>
			<td>A</td>
			<td>78</td>
			<td>0.13</td>
			<td>46</td>
			<td>0.13</td>
			<td>NP</td>
			<td>Mod</td>
			<td>28</td>
		</tr>
		<tr>
			<td>12</td>
			<td>37</td>
			<td>152</td>
			<td>65.8</td>
			<td>Female</td>
			<td>19</td>
			<td>C8</td>
			<td>C (NT)</td>
			<td>14</td>
			<td>0.71</td>
			<td>256</td>
			<td>0.71</td>
			<td>42</td>
			<td>MI&#160;</td>
			<td>39</td>
		</tr>
	</tbody>
</table>
Table 1. Characteristics of the Participants and Walk Test Results. SID=subject identification number; y=years; cm=centimeters; kg=kilograms; DOI=duration of injury; LOI=level of injury; AIS=American Spinal Injury Association Impairment Scale; LOA=level of assistance; s=seconds; m=meters; NP=Not-Performed and NT=non-traumatic SCI. LOA was adapted from the FIM as one of the following: moderate assistance (Mod) - participant performs 50% to 74% of the task; minimal assistance (Min) - the user performs 75% or more of the task; supervision (S) - the trainer is not touching the participant but is close enough to reach in to provide support for balance or guidance as needed; and modified independence (MI) - the trainer does not provide any assistance, and the participant is fully independent while walking in device. Re-print with permission, from Yang A, Asselin P, Knezevic S, Kornfeld S, Spungen A. Assessment of in-hospital walking velocity and level of assistance in a powered exoskeleton in persons with spinal cord injury. Top Spinal Cord Inj Rehabil. 2015; 21(2):100-109. Copyright (C) 2015 Thomas Land Publishers, Inc.

Watch this Scientific Journal Video about Training Persons with Spinal Cord Injury to Ambulate Using a Powered Exoskeleton at JoVE.com

Training Persons with Spinal Cord Injury to Ambulate Using a Powered Exoskeleton

Training a person with paralysis to ambulate using a powered exoskeleton may present challenges. The goals are to present the candidate selection criteria and the training procedures for exoskeletal-assisted walking and other mobility skills that can be progressed as the participant's skill level improves.

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

20.5K Views.  James J. Peters VA Medical Center. Training a person with paralysis to ambulate using a powered exoskeleton may present challenges. The goals are to present the candidate selection criteria and the training procedures for exoskeletal-assisted walking and other mobility skills that can be progressed as the participant's skill level improves. 

Video: Training Persons with Spinal Cord Injury to Ambulate Using a Powered Exoskeleton

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is desired. A novel neural-machine interface (NMI) based on neuromuscular-mechanical fusion developed in our previous study has demonstrated a great potential to accurately identify the intended movement of transfemoral amputees. However, this interface has not yet been integrated with a powered prosthetic leg for true neural control. This study aimed to report (1) a flexible platform to implement and optimize neural control of powered lower limb prosthesis and (2) an experimental setup and protocol to evaluate neural prosthesis control on patients with lower limb amputations. First a platform based on a PC and a visual programming environment were developed to implement the prosthesis control algorithms, including NMI training algorithm, NMI online testing algorithm, and intrinsic control algorithm. To demonstrate the function of this platform, in this study the NMI based on neuromuscular-mechanical fusion was hierarchically integrated with intrinsic control of a prototypical transfemoral prosthesis. One patient with a unilateral transfemoral amputation was recruited to evaluate our implemented neural controller when performing activities, such as standing, level-ground walking, ramp ascent, and ramp descent continuously in the laboratory. A novel experimental setup and protocol were developed in order to test the new prosthesis control safely and efficiently. The presented proof-of-concept platform and experimental setup and protocol could aid the future development and application of neurally-controlled powered artificial legs.

Neural-machine interfaces (NMI) have been developed to identify the user's locomotion mode. These NMIs are potentially useful for neural control of powered artificial legs, but have not been fully demonstrated. This paper presented (1) our designed engineering platform for easy implementation and development of neural control for powered lower limb prostheses and (2) an experimental setup and protocol in a laboratory environment to evaluate neurally-controlled artificial legs on patients with lower limb amputations safely and efficiently.

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during an impact are important factors in protecting the helmet wearer from impact-induced injury. This manuscript aims to investigate impact-induced injury mechanisms in different helmet fit scenarios through analysis of simulated helmeted impacts with an anthropometric test device (ATD), an array of headform acceleration transducers and neck force/moment transducers, a dual high speed camera system, and helmet-fit force sensors developed in our research group based on Bragg gratings in optical fiber. To simulate impacts, an instrumented headform and flexible neck fall along a linear guide rail onto an anvil. The test bed allows simulation of head impact at speeds up to 8.3 m/s, onto impact surfaces that are both flat and angled. The headform is fit with a crash helmet and several fit scenarios can be simulated by making context specific adjustments to the helmet position index and/or helmet size. To quantify helmet retention, the movement of the helmet on the head is quantified using post-hoc image analysis. To quantify head and neck injury potential, biomechanical measures based on headform acceleration and neck force/moment are measured. These biomechanical measures, through comparison with established human tolerance curves, can estimate the risk of severe life threatening and/or mild diffuse brain injury and osteoligamentous neck injury. To our knowledge, the presented test-bed is the first developed specifically to assess biomechanical effects on head and neck injury relative to helmet fit and retention.

Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact.

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Mouse Model of Pressure Ulcers After Spinal Cord Injury

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.

Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients.