**4. In-flight countermeasures for lumbopelvic deconditioning in space**

On the International Space Station (ISS), astronauts take part in up to 2.5 h of exercise each day including running, cycling and strength training. These exercise countermeasure programmes are known to be relatively successful at preventing bone loss and loss of muscle mass in some regions of the body. However, as mentioned earlier, they are not specifically targeted at preventing lumbopelvic muscle loss.

A number of bed rest studies have investigated potential inflight countermeasures, reporting on their effects on lumbopelvic musculoskeletal parameters. However, no studies have tested the operational countermeasures currently in use on the ISS [50]. Tested countermeasures included lower body negative pressure treadmill running, resistance exercises with external vibration, resistance exercise alone, rowing like exercises using a flywheel device and selfperformed exercises designed to mobilise the spine. In a recent systematic review of these bed rest studies, Winnard et al. [50] identified that no single potential countermeasure can successfully prevent all lumbopelvic musculoskeletal adaptations to simulated microgravity. For example, resistive vibration exercise was the only countermeasure, which is able to protect against lumbopelvic muscle adaptations, but it did not prevent spinal morphology changes such as loss of lumbar lordosis. No other countermeasure tested was able to do more than partially prevent (at best) the lumbopelvic muscle adaptations. This demonstrates a need for further research into new interventions to better protect the spine during microgravity exposure. It might be possible to translate ground-based interventions into new countermeasures or develop new ones based on the current lumbopelvic deconditioning rehabilitation theory. Any new interventions will need testing in ground-based microgravity simulations before incurring costs associated with actual spaceflight testing. Lower body negative pressure treadmill was the most effective currently researched countermeasure against lumbopelvic deconditioning. As this countermeasure is not yet used in operational spaceflight and no countermeasure is fully effective, there remains a need for rehabilitation.

### **5. Rehabilitation following actual and simulated spaceflight**

maximal voluntary contraction. Contractions are taught in postures such as supine, prone and four-point-kneeling while patients are asked to perform abdominal drawing in using TrA while maintaining a neutral lumbar lordosis. In addition to this, patients are taught: differentiation of lumbar, pelvic and hip movements and diaphragmatic breathing and maintenance

Live biofeedback with use of palpation, ultrasound imaging or possibly electromyography can be included to help isolate TrA and LM activation [49]. Treatment is then progressed to the second stage where the deep muscle recruitment learned in stage one is incorporated into functional movement, and compensatory muscle strategies are discouraged. Patients are taught movements such as sit to stand, walking, bending and twisting while maintaining activation of deep muscles. The third and final stage of training is for patients to carry the newly

The 'bare bones' have evolved since 2000 into an evidence-based and integrated approach which is summarised by Hodges et al. [28] and too complex to discuss here. However, it is based on a considerable amount of good evidence, one of which will be summarised in the

Hides et al. [49] assessed LM size in athletes with LBP and determined the effectiveness of a motor control intervention. Ten participants with back pain underwent a 6-week intervention programme of learning to correctly activate TrA and LM. Live biofeedback using ultrasound imaging was used during muscle activation teaching. Abdominal drawing in exercises were used to teach recruitment of TrA while maintaining a normal, relaxed, breathing pattern, followed by participants attempting to swell the LM muscle while holding a breath out and keeping the spine still with a neutral lumbar lordosis. Initially, activation was taught in lying and then progressed to upright sitting and standing, all the while maintaining a neutral lumbar lordosis. Further progression to functional movements was then performed. By the end of the programme, pain scores had dropped from an average of 4.3–2.3 (p < 0.05). Before treatment, asymmetry had been observed in LM cross-sectional area, which also significantly decreased, while overall muscle size increased. This is just one study that provides evidence that motor control exercises including recruitment of deep muscles can improve clinical outcomes.

of neutral lordosis in different postural sets such as sitting and standing.

128 Into Space - A Journey of How Humans Adapt and Live in Microgravity

learned and stable functional movements into their activities of daily life.

**4. In-flight countermeasures for lumbopelvic deconditioning** 

targeted at preventing lumbopelvic muscle loss.

On the International Space Station (ISS), astronauts take part in up to 2.5 h of exercise each day including running, cycling and strength training. These exercise countermeasure programmes are known to be relatively successful at preventing bone loss and loss of muscle mass in some regions of the body. However, as mentioned earlier, they are not specifically

A number of bed rest studies have investigated potential inflight countermeasures, reporting on their effects on lumbopelvic musculoskeletal parameters. However, no studies have tested

following paragraph.

**in space**

Due to the lack of effective in-flight countermeasures targeted at preventing lumbopelvic musculoskeletal adaptations, astronauts require significant rehabilitation to reduce injury risk on their return to Earth. During spaceflight, a general pattern of selective extensor muscle atrophy has been seen throughout the body [51]. Decrease in spinal extensor volume has been reported as being greater than hip flexor (psoas muscle) decline in astronauts [52]. Anecdotal accounts also appear to show selective atrophy of trunk extensor muscles concomitant with improved flexor muscle performance immediately post mission [53]. This muscle imbalance results in temporary loss of lumbopelvic posture, flexion of thoracic spine and hyperextension of cervical spine, the centre of gravity is moved anteriorly and increases the risk of musculoskeletal injury [47]. Hides et al. [40] suggested that deep spinal muscle changes such as atrophy of LM and TrA muscles, along with selective hypertrophy of spinal flexors over extensors [5], may impact on the ability of the spine to distribute loads appropriately shortly after spaceflight simulation via bed rest. The European approach to post-space mission rehabilitation addresses the muscle imbalance and uses motor control training in a way very similar to that described for people with LBP on Earth and based mostly on the existing terrestrial evidence transferred to a post-spaceflight setting. Initially, postural control, muscle control and muscle balance are restored, followed by the use of strategies to normalise muscle recruitment. Astronauts are then supported to redevelop postural alignment in line with the centre of gravity and to develop adequate motor control before they start to exercise with elements of loading and strength training. The latter is only started after the astronauts have regained correct postural alignment and control [54, 55].

No studies have investigated lumbopelvic rehabilitation approaches following actual spaceflight, and only one study has investigated rehabilitation approaches following simulated spaceflight (bed rest) [40]. The study assessed specific motor control (SMC) exercises compared to a control group performing trunk and general strength exercise (TFS) programme in a supine position. Results favoured SMC for restoring spinal length and posterior disc height, suggesting it may reduce the risk of IV disc injury during rehabilitation. However, TFS was favoured for training LM muscle and restoring lordosis angle and overall disc volume. Overall, it was suggested that SMC is favourable over TFS because SMC is expected to place less force on the discs and is associated with the lower rate of change in disc volume and anterior disc height [40]. Lower forces on the discs during rehabilitation—at a time when the discs may be deconditioned and vulnerable to injury—is expected to help restore posture and motor control with reduced risk of damage to the discs in the process. Therefore, in line with current ESA rehabilitation practice, a training programme starting with SMC when disc injury risk is high, then progressing to general trunk strengthening once lumbar postural control is restored would seem to be indicated. Other rehabilitation methods that train the LM muscle and maintain lordosis angle, without high axial loading, would also be worth investigating. Additionally, as noted previously, due to the low sample sizes from the terrestrial evidence on which these methods are based, further studies to improve statistical power would be useful to ensure a robust evidence-based approach. While motor control training has been shown to be useful in LBP rehabilitation, and is already used in the rehabilitation of European astronauts [47, 54, 55], many people have difficulty in recruiting LM, in particular, voluntarily [7]. This presents a challenge to physiotherapists involved in evidencebased practice for LBP. Many of the exercises used early in motor control training also lack functional relevance to activities of daily living, and there is a drive to make rehabilitation more functional [45, 56, 57]. As such, new interventions must be developed to address these challenges.

to recruit them differentially [53]. More recently, FRED exercise has been shown to promote tonic activity of LM, assessed through measurement of superficial muscle activity using surface electromyography [58], as well as the deep lumbopelvic muscles using intramuscular electromyography [59], which is considered the most rigorous way of investigating muscle activity [60]. FRED exercise was shown to result in more selective activation of the LM and TrA muscles than over-ground walking [59], and it was found to reduce lumbopelvic movement when compared to over-ground walking, especially axial rotation of the spine [61]. To date, these studies are all based on normal terrestrial gravity, and the next step will be a clinical trial of the FRED following bed rest as a simulation of space flight. A musculoskeletal modelling study that examined the potential role of the FRED in the recruitment of lumbopelvic muscles in both +1 and 0 Gz environments, Lindenroth et al. [62] predicted that FRED exercise is able to facilitate lumbopelvic muscle recruitment in microgravity similar to how it

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Based on the early research findings relating to the early and current prototypes of FRED, it can be hypothesised that the device uses several mechanisms in combination, to produce rehabilitation effects on several of the problems found in spinal instability simultaneously

These potential mechanisms show how the FRED has already demonstrated the ability to automatically activate both LM and TrA in an asymptomatic population without need for conscious muscle recruitment. This might have potential to solve the LM and TrA conscious recruitment difficulties found in traditional spinal motor control rehabilitation [7]. The exercise is dynamic, functional, weight-bearing, in an upright posture and relevant to common daily activities such as walking. These are all elements of motor control exercises covered in **Section 4**. It appears, therefore, that the device might be a useful intervention to train the LM

recruits the same muscles in Earth gravity.

within one intervention [58], as presented in **Table 1**.

**Figure 3.** Current prototype of the functional readaptive exercise device.

and TrA muscles and segmental spinal stability.

### **6. Developing a new countermeasure for lumbopelvic deconditioning in space**

#### **6.1. Early development of the functional readaptive exercise device (FRED)**

In an attempt to address the challenges discussed earlier relating to motor control training, Debuse et al. [53] investigated the effects of a new exercise device, the functional readaptive exercise device (FRED) (**Figure 3**), that aims to recruit the LM and TrA muscles.

FRED exercise constitutes a combination of weight-bearing, an unstable base of support (at the feet), an upright 'standing' posture with a relatively stable lumbopelvic area, and functional lower limb movement, combined with real-time visual feedback of performance. As the FRED offers no resistance to lower limb movement, it requires good balance and coordination in order to achieve a smooth, controlled cyclical motion. Exercise on the FRED has been shown to recruit LM and TrA automatically (i.e. with no conscious effort by participants) and

**Figure 3.** Current prototype of the functional readaptive exercise device.

No studies have investigated lumbopelvic rehabilitation approaches following actual spaceflight, and only one study has investigated rehabilitation approaches following simulated spaceflight (bed rest) [40]. The study assessed specific motor control (SMC) exercises compared to a control group performing trunk and general strength exercise (TFS) programme in a supine position. Results favoured SMC for restoring spinal length and posterior disc height, suggesting it may reduce the risk of IV disc injury during rehabilitation. However, TFS was favoured for training LM muscle and restoring lordosis angle and overall disc volume. Overall, it was suggested that SMC is favourable over TFS because SMC is expected to place less force on the discs and is associated with the lower rate of change in disc volume and anterior disc height [40]. Lower forces on the discs during rehabilitation—at a time when the discs may be deconditioned and vulnerable to injury—is expected to help restore posture and motor control with reduced risk of damage to the discs in the process. Therefore, in line with current ESA rehabilitation practice, a training programme starting with SMC when disc injury risk is high, then progressing to general trunk strengthening once lumbar postural control is restored would seem to be indicated. Other rehabilitation methods that train the LM muscle and maintain lordosis angle, without high axial loading, would also be worth investigating. Additionally, as noted previously, due to the low sample sizes from the terrestrial evidence on which these methods are based, further studies to improve statistical power would be useful to ensure a robust evidence-based approach. While motor control training has been shown to be useful in LBP rehabilitation, and is already used in the rehabilitation of European astronauts [47, 54, 55], many people have difficulty in recruiting LM, in particular, voluntarily [7]. This presents a challenge to physiotherapists involved in evidencebased practice for LBP. Many of the exercises used early in motor control training also lack functional relevance to activities of daily living, and there is a drive to make rehabilitation more functional [45, 56, 57]. As such, new interventions must be developed to address these

130 Into Space - A Journey of How Humans Adapt and Live in Microgravity

**6. Developing a new countermeasure for lumbopelvic** 

**6.1. Early development of the functional readaptive exercise device (FRED)**

exercise device (FRED) (**Figure 3**), that aims to recruit the LM and TrA muscles.

In an attempt to address the challenges discussed earlier relating to motor control training, Debuse et al. [53] investigated the effects of a new exercise device, the functional readaptive

FRED exercise constitutes a combination of weight-bearing, an unstable base of support (at the feet), an upright 'standing' posture with a relatively stable lumbopelvic area, and functional lower limb movement, combined with real-time visual feedback of performance. As the FRED offers no resistance to lower limb movement, it requires good balance and coordination in order to achieve a smooth, controlled cyclical motion. Exercise on the FRED has been shown to recruit LM and TrA automatically (i.e. with no conscious effort by participants) and

challenges.

**deconditioning in space**

to recruit them differentially [53]. More recently, FRED exercise has been shown to promote tonic activity of LM, assessed through measurement of superficial muscle activity using surface electromyography [58], as well as the deep lumbopelvic muscles using intramuscular electromyography [59], which is considered the most rigorous way of investigating muscle activity [60]. FRED exercise was shown to result in more selective activation of the LM and TrA muscles than over-ground walking [59], and it was found to reduce lumbopelvic movement when compared to over-ground walking, especially axial rotation of the spine [61]. To date, these studies are all based on normal terrestrial gravity, and the next step will be a clinical trial of the FRED following bed rest as a simulation of space flight. A musculoskeletal modelling study that examined the potential role of the FRED in the recruitment of lumbopelvic muscles in both +1 and 0 Gz environments, Lindenroth et al. [62] predicted that FRED exercise is able to facilitate lumbopelvic muscle recruitment in microgravity similar to how it recruits the same muscles in Earth gravity.

Based on the early research findings relating to the early and current prototypes of FRED, it can be hypothesised that the device uses several mechanisms in combination, to produce rehabilitation effects on several of the problems found in spinal instability simultaneously within one intervention [58], as presented in **Table 1**.

These potential mechanisms show how the FRED has already demonstrated the ability to automatically activate both LM and TrA in an asymptomatic population without need for conscious muscle recruitment. This might have potential to solve the LM and TrA conscious recruitment difficulties found in traditional spinal motor control rehabilitation [7]. The exercise is dynamic, functional, weight-bearing, in an upright posture and relevant to common daily activities such as walking. These are all elements of motor control exercises covered in **Section 4**. It appears, therefore, that the device might be a useful intervention to train the LM and TrA muscles and segmental spinal stability.


in terms of their rehabilitation [7]. Therefore, the automatic recruitment of TrA and LM during FRED exercise would appear to offer an advantage over current practice. Increasing crank amplitude was observed to increase movement variability, the range of TrA and LM thickness peaks, as well as mean TrA muscle thickness [63]. These outcomes are all measures of motor

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In a large sample of both symptomatic (LBP) and asymptomatic participants, FRED exercise promoted increased lumbar extension and anterior pelvic tilt compared to over-ground walking [64]. Attaining a lordosis throughout the lumbar spine below the thoracolumbar junction is a common goal of current interventions [48]; it is the sagittal spinal position where LM tends to be most effectively recruited [22, 24, 65]. Although this finding alone does not indicate that the correct lordosis is promoted by FRED exercise, when combined with the finding that FRED exercise appears to recruit key lumbopelvic muscles automatically, there is increased likelihood that the spinal position promoted during FRED exercise is more condu-

In the same sample, the FRED caused increased anteroposterior and mediolateral centre of mass variation compared to walking [64]. This suggests an increased challenge to balance and, therefore, motor control during FRED exercise. This may form part of the motor control mechanism of FRED exercise and adds to the overall evidence that FRED exercise is in line with current motor control interventions and adds weight to the justification for a

Astronauts undergo significant lumbopelvic musculoskeletal deconditioning following their exposure to microgravity. Many experience low back pain, and there is a fourfold increase in the incidence of intervertebral disc injury on their return to Earth as compared to their nonastronaut peers. It is known that the spine lengthens, normal posture is lost, intervertebral discs change their morphology, LM and TrA muscles atrophy and a flexor-extensor lumbopelvic muscle imbalance occur during spaceflight. Current in-flight countermeasures aim to generally prevent physiological adaptations to microgravity. However, they are not specifically targeted enough to do so for the lumbopelvic spaceflight adaptations. Astronauts, therefore, require rehabilitation upon return to Earth's gravity to reduce injury risk. The European rehabilitation interventions follow current evidence-based practice for treating people with LBP in whom dysfunction of TrA and LM is a key contributing factor to their symptoms such

FRED exercise shares many of the characteristics of motor control exercise. The findings of a range of mechanistic studies show that it results in automatic and tonic activation of LM and TrA, promotes normal lumbopelvic positioning against gravity, works trunk and lower limb extensors more than flexors, challenges balance and motor control, and is a functional progressive exercise. On this basis, as well as the results of a very recent clinical study on people with chronic LBP, we are confident that FRED exercise would complement and enhance

control of either global movement or muscle recruitment.

cive to LM recruitment than walking.

clinical trial.

**7. Conclusion**

as motor control training.

**Table 1.** FRED mechanisms.

#### **6.2. Developing FRED for rehabilitation from lumbopelvic deconditioning in space**

Following the early research on the early prototype of the FRED, a range of mechanistic studies were completed on the prototype shown in **Figure 3** in order to develop it for use as a rehabilitation intervention in groups with lumbopelvic muscle deconditioning (e.g. low back pain, astronauts). Winnard et al. [63] compared the thickness of LM and TrA at a range of movement amplitudes in an asymptomatic sample. A large body of evidence has linked dysfunction and/or atrophy of TrA and LM to lumbopelvic deconditioning and LBP [3, 4, 9, 11, 12]. Importantly, these muscles are difficult to recruit voluntarily, this presents a challenge in terms of their rehabilitation [7]. Therefore, the automatic recruitment of TrA and LM during FRED exercise would appear to offer an advantage over current practice. Increasing crank amplitude was observed to increase movement variability, the range of TrA and LM thickness peaks, as well as mean TrA muscle thickness [63]. These outcomes are all measures of motor control of either global movement or muscle recruitment.

In a large sample of both symptomatic (LBP) and asymptomatic participants, FRED exercise promoted increased lumbar extension and anterior pelvic tilt compared to over-ground walking [64]. Attaining a lordosis throughout the lumbar spine below the thoracolumbar junction is a common goal of current interventions [48]; it is the sagittal spinal position where LM tends to be most effectively recruited [22, 24, 65]. Although this finding alone does not indicate that the correct lordosis is promoted by FRED exercise, when combined with the finding that FRED exercise appears to recruit key lumbopelvic muscles automatically, there is increased likelihood that the spinal position promoted during FRED exercise is more conducive to LM recruitment than walking.

In the same sample, the FRED caused increased anteroposterior and mediolateral centre of mass variation compared to walking [64]. This suggests an increased challenge to balance and, therefore, motor control during FRED exercise. This may form part of the motor control mechanism of FRED exercise and adds to the overall evidence that FRED exercise is in line with current motor control interventions and adds weight to the justification for a clinical trial.

### **7. Conclusion**

**6.2. Developing FRED for rehabilitation from lumbopelvic deconditioning in space**

aim of traditional interventions for reducing falls risk in older people [65]

**Problem FRED mechanism**

Poor lumbopelvic motor control of deep spinal muscles

Reduced ability to control spinal posture and balance

Atrophy of spinal extensors

Weakness of lower limb antigravity muscles

**Table 1.** FRED mechanisms.

Following the early research on the early prototype of the FRED, a range of mechanistic studies were completed on the prototype shown in **Figure 3** in order to develop it for use as a rehabilitation intervention in groups with lumbopelvic muscle deconditioning (e.g. low back pain, astronauts). Winnard et al. [63] compared the thickness of LM and TrA at a range of movement amplitudes in an asymptomatic sample. A large body of evidence has linked dysfunction and/or atrophy of TrA and LM to lumbopelvic deconditioning and LBP [3, 4, 9, 11, 12]. Importantly, these muscles are difficult to recruit voluntarily, this presents a challenge

Exercising using a pattern of moving the feet in a quasi-elliptical path in antiphase with minimal

Exercising while maintaining a stable pelvis and upright trunk while having to maintain an even

The abovementioned points create a need for greater control of the lower limbs and pelvis during an unstable dynamic movement. Greater control is particularly needed in resisting a fast descent of the foot in the forward-most position of the cycle. The movement is functional and similar to overground walking. Therefore, muscle activation training is learned in a functional movement, hoped to produce carry over into other functional daily activities. Clinical observations seem to indicate that relatively greater rear foot loading in standing results in greater recruitment of LM, whereas relatively greater front foot loading in standing has a deactivating effect on LM. It is hypothesised that correct exercise on FRED results in reduced front foot loading. FRED provides visual feedback that encourages users to exercise at a constant, controlled speed and frequency ratio, which is hypothetically the most energy efficient movement [63]. Additional feedback encourages users to maintain even movements throughout the exercise, training control of the lumbopelvic area and lower limbs during dynamic functional movements. It is thought that efficient and smooth controlled movement on FRED may improve LM and TrA neuromotor control. The exercise has already been shown to activate LM and TRA without the need to consciously trigger the activation in non-symptomatic populations [53]. In addition to this, LM was shown to have constant tonic activity throughout exercise cycle on the device in an electromyography study. The muscle was

Previous kinematic research has shown FRED exercise promotes an increased degree of anterior pelvic tilt during upright posture [61]. Increased anterior pelvic tilt, within a range where the thoracolumbar junction remains the inflexion point between lumbar lordosis and thoracic kyphosis, has been shown to create a well-balanced sagittal spinal posture [64]. Electromyography data have also shown that this type of posture produces the highest LM and TRA recruitment [22], though this study investigated sitting postures. Additionally, users of the device are required to exercise in an upright posture. It is hoped that these elements together mean FRED exercise promotes a balanced upright sagittal posture, with recruitment of LM and TrA. Having improved control of

EMG data from FRED exercise show that it promotes increased activation of spinal extensors over flexors [58]. This may be relevant to the rehabilitation of astronauts who show increased flexion

Previous kinematic research shows FRED exercise involves constant hip and knee flexion in a dynamic and gravity-loaded exercise, therefore, constantly loading lower limb extensor muscles [61]. This loading is expected to improve strength in the lower limb extensors, which is a common

resistance from the device or support from the upper limbs

active for more time than during over-ground walking [58].

balanced posture is also hoped to improve overall balance.

postures when in space [36]

speed within one revolution.

132 Into Space - A Journey of How Humans Adapt and Live in Microgravity

Astronauts undergo significant lumbopelvic musculoskeletal deconditioning following their exposure to microgravity. Many experience low back pain, and there is a fourfold increase in the incidence of intervertebral disc injury on their return to Earth as compared to their nonastronaut peers. It is known that the spine lengthens, normal posture is lost, intervertebral discs change their morphology, LM and TrA muscles atrophy and a flexor-extensor lumbopelvic muscle imbalance occur during spaceflight. Current in-flight countermeasures aim to generally prevent physiological adaptations to microgravity. However, they are not specifically targeted enough to do so for the lumbopelvic spaceflight adaptations. Astronauts, therefore, require rehabilitation upon return to Earth's gravity to reduce injury risk. The European rehabilitation interventions follow current evidence-based practice for treating people with LBP in whom dysfunction of TrA and LM is a key contributing factor to their symptoms such as motor control training.

FRED exercise shares many of the characteristics of motor control exercise. The findings of a range of mechanistic studies show that it results in automatic and tonic activation of LM and TrA, promotes normal lumbopelvic positioning against gravity, works trunk and lower limb extensors more than flexors, challenges balance and motor control, and is a functional progressive exercise. On this basis, as well as the results of a very recent clinical study on people with chronic LBP, we are confident that FRED exercise would complement and enhance current astronaut rehabilitation practice. Future research should now investigate the effectiveness of the FRED in larger terrestrial populations with low back pain as well as following simulated and actual spaceflight.

[9] Panjabi MM. A hypothesis of chronic back pain: Ligament subfailure injuries lead to

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### **Author details**

Andrew Winnard1 , Dorothee Debuse<sup>2</sup> and Nick Caplan<sup>1</sup> \*

\*Address all correspondence to: nick.caplan@northumbria.ac.uk

1 Northumbria University, Newcastle upon Tyne, UK

2 Lunex University, Differdange, Luxembourg

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\*

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, Dorothee Debuse<sup>2</sup>

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1 Northumbria University, Newcastle upon Tyne, UK

2 Lunex University, Differdange, Luxembourg

\*Address all correspondence to: nick.caplan@northumbria.ac.uk

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**Chapter 9**

**Provisional chapter**

**Tumor Cells in Microgravity**

**Tumor Cells in Microgravity**

http://dx.doi.org/10.5772/intechopen.77214

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

DOI: 10.5772/intechopen.77214

The excessive proliferation and metastasis of tumor cells are due to frequent genetic alterations and subsequent stimulation of abnormal signal transduction pathways. Inventing and improving novel therapeutic strategies are critically needed. However, it remains unknown which of these pathways is essential to tumor initiation and progression. A weightless environment on Earth is a rare phenomenon, achieved using various simulations, but brings about changes of internal cellular structure and interactions among cells not normally seen under normal terrestrial gravitational conditions. For this reason, spaceflight experiments are of great value for cell biology research in general and for cancer research in particular. Many experiments indicate that microgravity, more so actual spaceflight as opposed to simulations, induces changes in the expression and secretion of genes as well as proteins involved in cancer cell proliferation, metastasis, and survival, shifting the cells toward a less aggressive phenotype. Therefore, studies on the biological features and gene expression of tumors cells under microgravity conditions may under-

line new clues to the tumor initiation, process, diagnosis, and therapy.

microgravity simulation studies for growth of cancer cells.

**Keywords:** space, microgravity, morphology, apoptosis, migration, tumor cells

In the past 40 years, the development of the space industry has made people aware of the effects of microgravity on biological life, including cerebrospinal fluid flow change, body fluid electrolyte loss, muscle atrophy, bone demineralization, and immune system function decline [1]. Similarly, microgravity has been shown to alter some properties of cells, including cell morphology, function, and the cellular response to the environment. Observations from cells in the space environment provide inspiration for our research, in particular, the use of

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Jun Chen

Jun Chen

**Abstract**

**1. Introduction**


#### **Chapter 9 Provisional chapter**

#### **Tumor Cells in Microgravity Tumor Cells in Microgravity**

#### Jun Chen Jun Chen

[53] Debuse D, Birch O, Gibson ASC, Caplan N. Low impact weight-bearing exercise in an upright posture increases the activation of two key local muscles of the lumbo-pelvic

[54] Lambrecht G, Petersen N, Weerts G, Pruett C, Evetts S, Stokes M, et al. The role of physiotherapy in the European Space Agency strategy for preparation and reconditioning of astronauts before and after long duration space flight. Musculoskeletal Science and

[55] Petersen N, Lambrecht G, Scott J, Hirsch N, Stokes M, Mester J. Postflight reconditioning for European astronauts – A case report of recovery after six months in space.

[56] Richardson C, Hides J. Open chain segmentral control and progression into function. In: Richardson C, Hodges P, Hides J, editors. Therapeutic Exercise for Lumbopelvic

[57] Richardson C, Hides J. Closed chain segmental control. In: Richardson C, Hodges P, Hides J, editors. Therapeutic Exercise for Lumbopelvic Stabilization. 2nd ed. Edinburgh:

[58] Caplan N, Gibbon KC, Hibbs A, Debuse D. Phasic-to-tonic shift in tunk muscle activity during low-impact weight bearing exercise. Acta Astronautica. 2014;**104**:388-395

[59] Weber T, Debuse D, Salomoni S, Elqueta Cancino E, De Martino E, Caplan N, et al. Trunk muscle activation during movement with a new exercise device for lumbo-pelvic recon-

[60] Richardson CA, Jull GA. Muscle control – Pain control. What exercises would you pre-

[61] Gibbon KC, Debuse D, Caplan N. Low impact weight-bearing exercise in an upright posture achieves greater lumbopelvic stability than overground walking. Journal of

[62] Lindenroth L, Caplan N, Debuse D, Salomoni S, Evetts S, Weber TA. Novel approach to activate deep spinal muscles in space – Results of a biomechanical model. Acta

[63] Winnard A, Debuse D, Wilkinson M, Samson L, Weber T, Movement NC. Amplitude on the functional re-adaptive exercise device: Deep spinal muscle activity and movement

[64] Winnard A, Debuse D, Wilkinson M, Tahmosybayat R, Caplan N. The immediate effects of exercise using the functional re-adaptive exercise device on lumbopelvic kinematics in people with and without low back pain. Musculoskeletal Science and Practice.

[65] O'Sullivan PB, Dankaerts W, Burnett AF, Farrell GT, Jefford E, Naylor CS, et al. Effect of different upright sitting postures on spinal pelvic curvature and trunk muscle activation

control. European Journal of Applied Physiology. 2017;**117**:1597-1606

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in a pain-free population. Spine. 2006;**31**(19):707-712

Practice. 2017;**27**:S15-S22

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77214

#### **Abstract**

The excessive proliferation and metastasis of tumor cells are due to frequent genetic alterations and subsequent stimulation of abnormal signal transduction pathways. Inventing and improving novel therapeutic strategies are critically needed. However, it remains unknown which of these pathways is essential to tumor initiation and progression. A weightless environment on Earth is a rare phenomenon, achieved using various simulations, but brings about changes of internal cellular structure and interactions among cells not normally seen under normal terrestrial gravitational conditions. For this reason, spaceflight experiments are of great value for cell biology research in general and for cancer research in particular. Many experiments indicate that microgravity, more so actual spaceflight as opposed to simulations, induces changes in the expression and secretion of genes as well as proteins involved in cancer cell proliferation, metastasis, and survival, shifting the cells toward a less aggressive phenotype. Therefore, studies on the biological features and gene expression of tumors cells under microgravity conditions may underline new clues to the tumor initiation, process, diagnosis, and therapy.

DOI: 10.5772/intechopen.77214

**Keywords:** space, microgravity, morphology, apoptosis, migration, tumor cells

### **1. Introduction**

In the past 40 years, the development of the space industry has made people aware of the effects of microgravity on biological life, including cerebrospinal fluid flow change, body fluid electrolyte loss, muscle atrophy, bone demineralization, and immune system function decline [1]. Similarly, microgravity has been shown to alter some properties of cells, including cell morphology, function, and the cellular response to the environment. Observations from cells in the space environment provide inspiration for our research, in particular, the use of microgravity simulation studies for growth of cancer cells.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The interest in cellular response to microgravity started early in the 1970s, aboard the US Skylab. Research on Skylab included studying the response of humans as well as their cells to microgravity exposure. Similar research continued on future missions and space stations. However, like all astronaut, biological and physiological system research in space, including studying cells and their growth in space, requires precious resources and astronauts' time, which are scarce and in short supply. As a result, ground analogs were developed to test different physiological systems in simulated microgravity, so space agencies developed ways to replicate the microgravity environment in order to culture cells on Earth.

In 1995, Dr. Jessup supplied and successfully cultured cancer cells aboard STS-70 and a few years later aboard STS-80 using the bioreactor to confirm his results. Jessup and his group showed that their flown samples of colon carcinoma cells grew bigger and aggregated better than ground controls, 30 times the volume of the ground controls, and were more representative of cancer seen when growing in the body. These initial studies showed that microgravity was an environment that is favorable to cell growth and differentiation in addition to being more representative of in vivo growth [2].

Earth-based research groups have used the rotating wall vessel (RWV) bioreactor to support the development of several models of cancer cell lines. These include models of breast cancer, cervical cancer, colon cancer, hepatocellular carcinoma, neuroblastoma, melanoma, ovarian cancer, and prostate cancer. The bioreactor was not only a technological innovation, but was also a unique system benefiting biomedical research by allowing cells to be cultured in a 3-D environment on Earth [3]. Other systems have been developed in an attempt to simulate microgravity, such as the random positioning machine (RPM) and magnetic levitation.

Through ground-based simulations and spaceflight research, we know that simulated microgravity has been emerging as a new tool to develop potential therapy for tumor treatment [4]. With the developments from this research, more interest is being paid to the effects of microgravity on tumor cells. Increasing number of investigations has indicated that microgravity has evident effects on the morphology, proliferation, apoptosis, invasion, and migration, along with inhibiting cancerous cell growth and invasion. However, the details of the exact mechanism still elude us and are still being studied.

### **2. Changes of morphology**

It has been found that after microgravity intervention, mesenchymal stem cell morphology changed significantly from spindle to round. "Spirit composing body," shape change will inevitably lead to changes in their function. Under the guidance of the traditional Chinese medicine "Circumference philosophical," the cells with round change are primitive state. The round bone marrow mesenchymal stem cells have greater differentiation potential in microgravity [5] (**Figure 1**). This study uses ground rotary cultivating device (2-D-clinostat) simulation of cells in space (**Figure 2**), using 1G gravity (normal gravity, NG) as a control with simulated microgravity (SMG) intervention of bone marrow mesenchymal stem cells to observe the differentiation into neural cells and endothelial cells. At the same time, cytoskeleton as well as

the key molecule of RhoA activity is observed, thereby directing the possible differentiation

**Figure 2.** A clinostat or rotating vessel was used to form the weightless environment to prevent the cell from feeling gravity; the machines are based on the theory that sensing no gravity would have similar effects as being microgravity. The clinostat model system (clinorotation) used in this study tends to provide an average of zero vector in the apparent

**Figure 1.** Phase-contrast microscopic analysis of the effect of SMG (b) on the morphology of BMSCs compared to NG (a). (c) The changes of the ratio of the width/length of BMSCs in the different groups. \* denotes P < 0.05, versus the NG group.

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141

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Other than stem cells, real and simulated microgravity induces early alterations of the cytoskeleton in different kinds of human cells, such as thyroid cancer cells [6, 7], endothelial cells

Our previous works have concluded that modeled microgravity causes changes in the cyto-

skeleton and focal adhesions in malignant human MCF-7 cells [10].

mechanism of stem cells.

gravity on the cell culture.

[8], and glial cells [9].

The interest in cellular response to microgravity started early in the 1970s, aboard the US Skylab. Research on Skylab included studying the response of humans as well as their cells to microgravity exposure. Similar research continued on future missions and space stations. However, like all astronaut, biological and physiological system research in space, including studying cells and their growth in space, requires precious resources and astronauts' time, which are scarce and in short supply. As a result, ground analogs were developed to test different physiological systems in simulated microgravity, so space agencies developed ways to

In 1995, Dr. Jessup supplied and successfully cultured cancer cells aboard STS-70 and a few years later aboard STS-80 using the bioreactor to confirm his results. Jessup and his group showed that their flown samples of colon carcinoma cells grew bigger and aggregated better than ground controls, 30 times the volume of the ground controls, and were more representative of cancer seen when growing in the body. These initial studies showed that microgravity was an environment that is favorable to cell growth and differentiation in addition to being

Earth-based research groups have used the rotating wall vessel (RWV) bioreactor to support the development of several models of cancer cell lines. These include models of breast cancer, cervical cancer, colon cancer, hepatocellular carcinoma, neuroblastoma, melanoma, ovarian cancer, and prostate cancer. The bioreactor was not only a technological innovation, but was also a unique system benefiting biomedical research by allowing cells to be cultured in a 3-D environment on Earth [3]. Other systems have been developed in an attempt to simulate microgravity, such as the random positioning machine (RPM) and magnetic levitation.

Through ground-based simulations and spaceflight research, we know that simulated microgravity has been emerging as a new tool to develop potential therapy for tumor treatment [4]. With the developments from this research, more interest is being paid to the effects of microgravity on tumor cells. Increasing number of investigations has indicated that microgravity has evident effects on the morphology, proliferation, apoptosis, invasion, and migration, along with inhibiting cancerous cell growth and invasion. However, the details of the exact

It has been found that after microgravity intervention, mesenchymal stem cell morphology changed significantly from spindle to round. "Spirit composing body," shape change will inevitably lead to changes in their function. Under the guidance of the traditional Chinese medicine "Circumference philosophical," the cells with round change are primitive state. The round bone marrow mesenchymal stem cells have greater differentiation potential in microgravity [5] (**Figure 1**). This study uses ground rotary cultivating device (2-D-clinostat) simulation of cells in space (**Figure 2**), using 1G gravity (normal gravity, NG) as a control with simulated microgravity (SMG) intervention of bone marrow mesenchymal stem cells to observe the differentiation into neural cells and endothelial cells. At the same time, cytoskeleton as well as

replicate the microgravity environment in order to culture cells on Earth.

more representative of in vivo growth [2].

140 Into Space - A Journey of How Humans Adapt and Live in Microgravity

mechanism still elude us and are still being studied.

**2. Changes of morphology**

**Figure 1.** Phase-contrast microscopic analysis of the effect of SMG (b) on the morphology of BMSCs compared to NG (a). (c) The changes of the ratio of the width/length of BMSCs in the different groups. \* denotes P < 0.05, versus the NG group.

**Figure 2.** A clinostat or rotating vessel was used to form the weightless environment to prevent the cell from feeling gravity; the machines are based on the theory that sensing no gravity would have similar effects as being microgravity. The clinostat model system (clinorotation) used in this study tends to provide an average of zero vector in the apparent gravity on the cell culture.

the key molecule of RhoA activity is observed, thereby directing the possible differentiation mechanism of stem cells.

Other than stem cells, real and simulated microgravity induces early alterations of the cytoskeleton in different kinds of human cells, such as thyroid cancer cells [6, 7], endothelial cells [8], and glial cells [9].

Our previous works have concluded that modeled microgravity causes changes in the cytoskeleton and focal adhesions in malignant human MCF-7 cells [10].

Microtubules, which are components of the cytoskeleton that are involved in maintaining the structure of the cell, were disrupted in MCF-7 cells within 4 h of SMG. Instead of long, strongly labeled microtubules in NG groups, only a few fibers could be distinguished against the strong background (**Figure 3**). Self-organization of microtubules into stationary macroscopic patterns is gravity-dependent, and the patterns correspond to different microtubule orientations [11]. Also, the formation of radial microtubule arrays depends mostly on the activity of centrosome. Lewis et al. reported that microtubule organizing centers (MTOCs) were poorly defined in SMG [12]. It is obvious that the anchoring between microtubule and the centrosome is very complicated; so, many studies targeted the function of cytoplasmic dynein and its cofactor dynactin. Dynein transfers some centrosomal proteins to the centrosome and reorganizes radial microtubule arrays for cell division. Thus, we hypothesize that the disorganization of microtubule fibers and their reestablishment might be associated with MTOCs modification.

7 min [14]. This leads us to conclude that the actin microfilament structure is sensitive to gravity and that rebuilding of cytoskeleton may affect signal transduction [14]. Thyroid cancer cells were flown in a parabolic flight mission and they detected early alterations in the actin microfilaments. After 22-s microgravity stimulation, F-actin changed significantly, and the human beta actin (ACTB) expression was strikingly upregulated after the 1st and 31st parabolas [15].

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Actin monomers polymerize only onto the existing barbed ends of the actin array, so that the actin filaments can be elongated to the cell periphery. In contrast to NG samples, microfilaments did not form preferential orientation with less labeled lamellipodia. Cell microfilament bundles are assembled primarily by bundling of preexisting actin filaments [16]. It has been reported that tension development in the preexisting actin cytoskeleton is critical for the formation of stress fibers [17]. Tension would act on mechanical connections of the actin filaments to reorganize a kind of meshwork. However, spaceflight or stimulated weightlessness would destroy the intracellular prestress and tension balance, and eventually cells show

Glioma is the most common and aggressive form of cancer of the central nervous system with a median survival time of 15 months and a 5-year survival about 5% after initial diagnosis [19]. Despite the standard of therapy available, including maximal safe surgical resection, radiotherapy, and temozolomide (a form of chemotherapy) [20], nearly all patients relapse. The excessive proliferation and metastasis of glioma is due to frequent genetic alterations and subsequent stimulation of abnormal signal transduction pathways [21, 22]. Inventing and improving novel therapeutic strategies are critically needed. However, it remains unknown

To assess the effect of SMG on glioma growth, U251 cells were cultured under either ground condition or SMG (SM-31 random locator applied) for different time periods (0, 12, 16, 20, 24, 36, 48, 72, and 96 h). CCK8 measurements, a chemical kit employed to measure cell proliferation, showed that SMG inhibited U251 cells activity in a time-dependent manner. The more time handled, the less activity the U251 cells had. For 48~96 h, SMG markedly induced cell death of U251, cell activity decreased maximum about 45%. Therefore, we chose 72-h timepoint to use in the following experiments. This was further confirmed by our data derived from fluorescence-activated cell sorter (FACS) analysis via annexin V-FITC and PI double staining to investigate apoptosis of U251 cells exposed to SMG for 72 h, which showed 2.4% of control cells stained positive for both annexin V and PI, representing a minor subpopulation undergoing a spontaneous apoptosis. After being cultured in SMG for 72 h, apoptotic subpopulation increased to 29.3%, indicating that SMG promoted cell death via apoptosis. Western blot (WB) test confirmed that apoptotic promoting proteins cleaved-caspase 3 and cleaved-caspase 9 were markedly upregulated at 48 h by SMG compared with NG condition. Meanwhile, U251 cells' metabolic activity was detected by β-Gal Staining Set, blue-stained cells significantly increased by SMG, about 30% increase compared with NG condition. FACS analysis further demonstrated that SMG treatment led to 14.11 ± 1.73% of U251 cells arrested at G2/M phase (**Figure 4**).

irregular formation of cytoskeletal actin filaments [18].

**3. Changes of cell growth, cycle, and apoptosis**

which of these pathways is essential to glioma initiation and progression.

The cytoskeleton is made up of actin microfilaments, tubulin microtubules, and vimentin intermediate filaments [13]. The F-actin cytoskeleton has been involved in changes in cell morphosis and function as well as signaling path under weightless conditions. It was revealed that the amount of F-actin in A431 epidermoid carcinoma cells increased under real microgravity for

**Figure 3.** Microtubules formation of MCF-7 cells is changed in MMG. Scale bar is 10 μm. Visualization of β-tubulin (TRITC; red) and nuclear chromatin (Hochest; blue). It is shown that microtubules keep radiation from the perinuclear area toward the cell periphery in control cells as time varying. (a–c) (4 h, 48 h, and 7 days, respectively). While in the MMG groups, altered fibers and short microtubules filaments could be observed. (a′–c′) (4 h, 48 h, and 7 days, respectively).

7 min [14]. This leads us to conclude that the actin microfilament structure is sensitive to gravity and that rebuilding of cytoskeleton may affect signal transduction [14]. Thyroid cancer cells were flown in a parabolic flight mission and they detected early alterations in the actin microfilaments. After 22-s microgravity stimulation, F-actin changed significantly, and the human beta actin (ACTB) expression was strikingly upregulated after the 1st and 31st parabolas [15].

Actin monomers polymerize only onto the existing barbed ends of the actin array, so that the actin filaments can be elongated to the cell periphery. In contrast to NG samples, microfilaments did not form preferential orientation with less labeled lamellipodia. Cell microfilament bundles are assembled primarily by bundling of preexisting actin filaments [16]. It has been reported that tension development in the preexisting actin cytoskeleton is critical for the formation of stress fibers [17]. Tension would act on mechanical connections of the actin filaments to reorganize a kind of meshwork. However, spaceflight or stimulated weightlessness would destroy the intracellular prestress and tension balance, and eventually cells show irregular formation of cytoskeletal actin filaments [18].

### **3. Changes of cell growth, cycle, and apoptosis**

Microtubules, which are components of the cytoskeleton that are involved in maintaining the structure of the cell, were disrupted in MCF-7 cells within 4 h of SMG. Instead of long, strongly labeled microtubules in NG groups, only a few fibers could be distinguished against the strong background (**Figure 3**). Self-organization of microtubules into stationary macroscopic patterns is gravity-dependent, and the patterns correspond to different microtubule orientations [11]. Also, the formation of radial microtubule arrays depends mostly on the activity of centrosome. Lewis et al. reported that microtubule organizing centers (MTOCs) were poorly defined in SMG [12]. It is obvious that the anchoring between microtubule and the centrosome is very complicated; so, many studies targeted the function of cytoplasmic dynein and its cofactor dynactin. Dynein transfers some centrosomal proteins to the centrosome and reorganizes radial microtubule arrays for cell division. Thus, we hypothesize that the disorganization of microtubule fibers and their reestablishment might be associated with

The cytoskeleton is made up of actin microfilaments, tubulin microtubules, and vimentin intermediate filaments [13]. The F-actin cytoskeleton has been involved in changes in cell morphosis and function as well as signaling path under weightless conditions. It was revealed that the amount of F-actin in A431 epidermoid carcinoma cells increased under real microgravity for

**Figure 3.** Microtubules formation of MCF-7 cells is changed in MMG. Scale bar is 10 μm. Visualization of β-tubulin (TRITC; red) and nuclear chromatin (Hochest; blue). It is shown that microtubules keep radiation from the perinuclear area toward the cell periphery in control cells as time varying. (a–c) (4 h, 48 h, and 7 days, respectively). While in the MMG groups, altered fibers and short microtubules filaments could be observed. (a′–c′) (4 h, 48 h, and 7 days,

MTOCs modification.

142 Into Space - A Journey of How Humans Adapt and Live in Microgravity

respectively).

Glioma is the most common and aggressive form of cancer of the central nervous system with a median survival time of 15 months and a 5-year survival about 5% after initial diagnosis [19]. Despite the standard of therapy available, including maximal safe surgical resection, radiotherapy, and temozolomide (a form of chemotherapy) [20], nearly all patients relapse. The excessive proliferation and metastasis of glioma is due to frequent genetic alterations and subsequent stimulation of abnormal signal transduction pathways [21, 22]. Inventing and improving novel therapeutic strategies are critically needed. However, it remains unknown which of these pathways is essential to glioma initiation and progression.

To assess the effect of SMG on glioma growth, U251 cells were cultured under either ground condition or SMG (SM-31 random locator applied) for different time periods (0, 12, 16, 20, 24, 36, 48, 72, and 96 h). CCK8 measurements, a chemical kit employed to measure cell proliferation, showed that SMG inhibited U251 cells activity in a time-dependent manner. The more time handled, the less activity the U251 cells had. For 48~96 h, SMG markedly induced cell death of U251, cell activity decreased maximum about 45%. Therefore, we chose 72-h timepoint to use in the following experiments. This was further confirmed by our data derived from fluorescence-activated cell sorter (FACS) analysis via annexin V-FITC and PI double staining to investigate apoptosis of U251 cells exposed to SMG for 72 h, which showed 2.4% of control cells stained positive for both annexin V and PI, representing a minor subpopulation undergoing a spontaneous apoptosis. After being cultured in SMG for 72 h, apoptotic subpopulation increased to 29.3%, indicating that SMG promoted cell death via apoptosis. Western blot (WB) test confirmed that apoptotic promoting proteins cleaved-caspase 3 and cleaved-caspase 9 were markedly upregulated at 48 h by SMG compared with NG condition. Meanwhile, U251 cells' metabolic activity was detected by β-Gal Staining Set, blue-stained cells significantly increased by SMG, about 30% increase compared with NG condition. FACS analysis further demonstrated that SMG treatment led to 14.11 ± 1.73% of U251 cells arrested at G2/M phase (**Figure 4**).

coordinated formation and disassembly of adhesions is crucial for migration. These adhesion sites, named as FAs, serve as traction points to propel the cell forward. Therefore, the important mechanism for creating cell movement includes the maturation of FAs. FAs become mature during the binding and clustering of integrins and function physically as a link of the cytoskeleton to the extracellular matrix (ECM) [28]. This correlation between cytoskeleton and FAs in structure underlines the possibility that FAs formation might be changed during microgravity. In order to explore the possible mechanism, we examined FAs formation by vinculin immunofluorescence. Vinculin is one of the most prevalent elements in FAs. It seems that vinculin facilitates the assembly of FAs by interacting and recruiting its various partners [29]. We demonstrated that microgravity disorganized FAs of MCF-7 cells via quantifying FAs parameters. After 7 days stimulation of SMG, the amount of FAs was still low by detecting topographical adhesion parameters and showed no change as time varied. We therefore could conclude that FAs created in microgravity were less mature than those established in normal gravity. Fewer and smaller FAs can lead to the weaker cell spreading and migrating. Thus, we speculated that abnormal FAs structure in MCF-7 cells under SMG may contribute to the

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Since FAs also contain a lot of growth factor receptors, kinases, and signaling proteins, FAs have been referred as localized sites converging growth factor and adhesion signaling. The integrin family is the major transmembrane ECM receptors in these sites. FAK, PYK2, and ILK are well known as the key effectors in FAs signaling and a potential integrator of inhibition of MCF-7 cell migration by modeled microgravity. Because the changes of morphological and topographical cytoskeletal structures of FAs were observed in MCF-7 cells, we speculated that activities of the kinases would be altered under clinorotation conditions. Here, our hypothesis was confirmed by observing a decrease in FAs kinases phosphorylation level (FAK, PYK2, and ILK) in contrast to NG controls, while there was no significant change in total FAK, ILK, and PYK2 protein expression in both the NG controls and SMG groups. The effects of microgravity on the suppression of FAs kinases activity in our experiments were in accordance with other reports, though they were observed in different cells by different devices, which could suggest that SMG might suppress the FAs kinases activity in various kinds of cells [30].

As we know, integrins play a critical role in cell adhesion and migration. We explored that SMG could decrease the expression of integrins, a downregulation of integrins (integrinβ1 and integrinβ4) at both protein and mRNA levels after SMG compared to controls. Downregulated integrins in SMG are not a new finding, and it has been reported that SMG suppressed integrins (αv, α5,β1) expression in MG-63 cell. Via binding to the ECM, phosphorylation of signaling proteins at FAs was triggered, such as FAK, ILK, and PYK, whereby protein kinase C (PKC) and GTPases pathways were activated. The small G proteins, Rho family regulate the rebuilding of actin fibers and FAs formation, by which determines cell movement. In fact, the previous studies have reported that rho activation in the endothelial cells and MSCs could be affected by simulated microgravity. Also, it has been found repeatedly that microgravity modulates PKC signaling in neurons. Activation of PKC could directly induce cells' motility and migration. Furthermore, it is generally accepted that the releasing from the RhoGDI-1 molecule is required for activation of RhoGTPases. PKCα is confirmed to phosphorylate RhoGDI-1 and then catalyze the release of bound GTPases [31]. No doubt, the

change of cell migration [10].

**Figure 4.** Cell cycle was analyzed by flow cytometry after PI staining. The bar graph represents the number of the cells in different phases.

Caspases are a family of cysteine proteases. Most caspases play a central role in activating cell apoptosis by cleaving selected target substrates in a cysteine-dependent aspartate-directed manner. To further dissect molecular mechanisms of SMG-induced glioma apoptosis, WB analysis was performed to confirm the level of apoptotic protein, Bcl2, Bnip3, pro-caspase 3, cleaved-caspase 3, pro-caspase 9, and cleaved-caspase 9. Zhao et al. found that SMG promotes BL6-10 melanoma cell apoptosis through downregulating Bcl2 and Bnip3 and upregulating caspases 3/7/8 [23]. We showed that two antiapoptotic proteins Bcl2 and Bnip3 were downregulated, while cleaved-caspase 3 and cleaved-caspase 9 were upregulated in cells under microgravity conditions, respectively, leading to enhance glioma cell death.

Never in mitosis gene A (NIMA)-related kinase 2 (Nek2) is one of multiple cell cycle-regulated protein kinases that localizes to the centrosome and is required for mitotic progression and correct bipolar spindle formation [24]. Upregulated Nek2, which is confirmed in a number of neoplastic diseases, including prostate cancer, lung cancers, colorectal cancer [25], and myeloma, also exhibits adverse correlation with overall survival of multiple malignancies [26]. All of this suggests that Nek2 may regulate the metastasis of glioma, although few reports have shown this. In our unpublished study, Nek2 may play a crucial function in SMGtreated U251cells, which arrested at the mitotic phase (chromosome separation).

Nek2 is implicated in centrosome separation and is reported to displace linker proteins from centrosomes through phosphorylation at the beginning of mitosis, and Nek2 phosphorylates the centrosomal linker proteins C-Nap1 and rootletin resulting in linker dissociation. hSav1- Mst2-Nek2 centrosome disjunction pathway becomes essential for bipolar spindle formation, Nek2 kinase to regulate centrosome disjunction. We found SMG suppressed expression of Nek2 and distances between two centrioles by γ-tubulin staining. The experimental results were consistent as Di Agostino et al. [27].

### **4. Changes of migration**

The formation of a protrusion initiates the cell migration cycle process, but the protrusions need to be stabilized to the substratum so as to move forward. The process of continuous coordinated formation and disassembly of adhesions is crucial for migration. These adhesion sites, named as FAs, serve as traction points to propel the cell forward. Therefore, the important mechanism for creating cell movement includes the maturation of FAs. FAs become mature during the binding and clustering of integrins and function physically as a link of the cytoskeleton to the extracellular matrix (ECM) [28]. This correlation between cytoskeleton and FAs in structure underlines the possibility that FAs formation might be changed during microgravity. In order to explore the possible mechanism, we examined FAs formation by vinculin immunofluorescence. Vinculin is one of the most prevalent elements in FAs. It seems that vinculin facilitates the assembly of FAs by interacting and recruiting its various partners [29]. We demonstrated that microgravity disorganized FAs of MCF-7 cells via quantifying FAs parameters. After 7 days stimulation of SMG, the amount of FAs was still low by detecting topographical adhesion parameters and showed no change as time varied. We therefore could conclude that FAs created in microgravity were less mature than those established in normal gravity. Fewer and smaller FAs can lead to the weaker cell spreading and migrating. Thus, we speculated that abnormal FAs structure in MCF-7 cells under SMG may contribute to the change of cell migration [10].

Caspases are a family of cysteine proteases. Most caspases play a central role in activating cell apoptosis by cleaving selected target substrates in a cysteine-dependent aspartate-directed manner. To further dissect molecular mechanisms of SMG-induced glioma apoptosis, WB analysis was performed to confirm the level of apoptotic protein, Bcl2, Bnip3, pro-caspase 3, cleaved-caspase 3, pro-caspase 9, and cleaved-caspase 9. Zhao et al. found that SMG promotes BL6-10 melanoma cell apoptosis through downregulating Bcl2 and Bnip3 and upregulating caspases 3/7/8 [23]. We showed that two antiapoptotic proteins Bcl2 and Bnip3 were downregulated, while cleaved-caspase 3 and cleaved-caspase 9 were upregulated in cells under

**Figure 4.** Cell cycle was analyzed by flow cytometry after PI staining. The bar graph represents the number of the cells

Never in mitosis gene A (NIMA)-related kinase 2 (Nek2) is one of multiple cell cycle-regulated protein kinases that localizes to the centrosome and is required for mitotic progression and correct bipolar spindle formation [24]. Upregulated Nek2, which is confirmed in a number of neoplastic diseases, including prostate cancer, lung cancers, colorectal cancer [25], and myeloma, also exhibits adverse correlation with overall survival of multiple malignancies [26]. All of this suggests that Nek2 may regulate the metastasis of glioma, although few reports have shown this. In our unpublished study, Nek2 may play a crucial function in SMG-

Nek2 is implicated in centrosome separation and is reported to displace linker proteins from centrosomes through phosphorylation at the beginning of mitosis, and Nek2 phosphorylates the centrosomal linker proteins C-Nap1 and rootletin resulting in linker dissociation. hSav1- Mst2-Nek2 centrosome disjunction pathway becomes essential for bipolar spindle formation, Nek2 kinase to regulate centrosome disjunction. We found SMG suppressed expression of Nek2 and distances between two centrioles by γ-tubulin staining. The experimental results

The formation of a protrusion initiates the cell migration cycle process, but the protrusions need to be stabilized to the substratum so as to move forward. The process of continuous

microgravity conditions, respectively, leading to enhance glioma cell death.

treated U251cells, which arrested at the mitotic phase (chromosome separation).

were consistent as Di Agostino et al. [27].

**4. Changes of migration**

in different phases.

144 Into Space - A Journey of How Humans Adapt and Live in Microgravity

Since FAs also contain a lot of growth factor receptors, kinases, and signaling proteins, FAs have been referred as localized sites converging growth factor and adhesion signaling. The integrin family is the major transmembrane ECM receptors in these sites. FAK, PYK2, and ILK are well known as the key effectors in FAs signaling and a potential integrator of inhibition of MCF-7 cell migration by modeled microgravity. Because the changes of morphological and topographical cytoskeletal structures of FAs were observed in MCF-7 cells, we speculated that activities of the kinases would be altered under clinorotation conditions. Here, our hypothesis was confirmed by observing a decrease in FAs kinases phosphorylation level (FAK, PYK2, and ILK) in contrast to NG controls, while there was no significant change in total FAK, ILK, and PYK2 protein expression in both the NG controls and SMG groups. The effects of microgravity on the suppression of FAs kinases activity in our experiments were in accordance with other reports, though they were observed in different cells by different devices, which could suggest that SMG might suppress the FAs kinases activity in various kinds of cells [30].

As we know, integrins play a critical role in cell adhesion and migration. We explored that SMG could decrease the expression of integrins, a downregulation of integrins (integrinβ1 and integrinβ4) at both protein and mRNA levels after SMG compared to controls. Downregulated integrins in SMG are not a new finding, and it has been reported that SMG suppressed integrins (αv, α5,β1) expression in MG-63 cell. Via binding to the ECM, phosphorylation of signaling proteins at FAs was triggered, such as FAK, ILK, and PYK, whereby protein kinase C (PKC) and GTPases pathways were activated. The small G proteins, Rho family regulate the rebuilding of actin fibers and FAs formation, by which determines cell movement. In fact, the previous studies have reported that rho activation in the endothelial cells and MSCs could be affected by simulated microgravity. Also, it has been found repeatedly that microgravity modulates PKC signaling in neurons. Activation of PKC could directly induce cells' motility and migration. Furthermore, it is generally accepted that the releasing from the RhoGDI-1 molecule is required for activation of RhoGTPases. PKCα is confirmed to phosphorylate RhoGDI-1 and then catalyze the release of bound GTPases [31]. No doubt, the pathways regulating cell migration are very complicated and seem to vary in different cells and different species. In general, it seems that decreased integrin expression and downregulated FAs kinase activity are an essential step in suppression of cell migration by SMG.

In this research, VEGFA and VEGFD, which are members of the vascular endothelial growth factor (VEGF) family, were also observed. It is well known that VEGFA is the most important growth factor driving angiogenesis and is also implicated in many processes like tumor vascularization, wound healing, and burn injury. VEGFA expression has been found to increase in papillary thyroid carcinoma in contrast to that in healthy patients [33]. While on the other hand, VEGFD serum levels were found to decrease in patients with thyroid cancer [34]. In this study, an obvious decrease in expression of VEGFA was confirmed in the RPM as well as the spaceflight samples, while no difference was observed between adherent (AD) cells or multicellular spheroids (MCSs). Similarly, VEGFA secretion was also reduced on the RPM, but no influence in real microgravity. Furthermore, VEGFD gene expression upregulated in cells cultivated on the RPM as well as in space. All these results seem to get a conclusion that microgravity exerts a shift of the thyroid tumor cells to a more benign, less metastatic phenotype. Interestingly, the similar tendency was observed with the interleukins in microgravity. IL6 and IL8, encoded by the IL6 and IL8 genes, are involved in tumor cell growth, metastasis, and angiogenesis. IL15, on the contrary, is able to activate several antitumor mechanisms, such as activating CD8 T cells to kill tumor cells [35]. Furthermore, it has been found that IL15 was effective in several tumor therapy experiments [36]. Their results explored a significant downregulation of IL8 gene expression in both simulated and real microgravity cells, which is also in accordance with the IL8 secretion pattern. The weightless effect of reducing IL6 gene expression was only observed in adherent cells, the same to that described for endothelial cells [37]. Of note, both IL6 and IL8 gene expression were strikingly upregulated during a parabolic flight. The study also showed earlier that IL6 and IL8 were most possibly implicated in the gravity-sensitive signaling required for spheroid formation. It seems that this signal pathway is attenuated after exposure to microgravity and that the tumor cells are shifted toward a less aggressive biological behavior. This hypothesis was also confirmed by the observations for IL15. IL15 gene expression increased only slightly on the RPM and during parabolic flight, but they explored a strong increase in the MCSs during spaceflight. Thus, we speculate that microgravity triggers some antitumor pathways involving IL15 and, as MCSs resemble

Tumor Cells in Microgravity

147

http://dx.doi.org/10.5772/intechopen.77214

tumors in their 3-D structure, the cell-cell interactions should be affected.

Taken together, it is no doubt that spaceflight experiments are of great value for cell biology research, especially for cancer research. The previous studies indicate that microgravity, both actual and simulated microgravity, induces changes in cancer cell proliferation, metastasis, and survival, bringing the cells toward a less aggressive phenotype. This effect is greater in actual spaceflight; however, ground-based simulation has been shown to be an essential tool in our understanding of gravity and it effects on a cellular level in cancer cells. The above body shows that not only does the architecture of tumor cells change in microgravity, but the cell function and gene expression also are different. The changes of some gene expression reorganize the cytoskeleton which influences the cell growth, migration, and apoptosis. And some other genes impress tumor cell by regulating the immune antitumor pathway.

**6. Conclusion**

#### **5. Changes of genes**

To investigate the involved mechanisms in more detail, gene level researches are expected to yield novel targets for cancer therapy, which may then be exploited in the form of new chemotherapeutics.

Dr. Xiao Ma cultured thyroid cancer cells in space (Shenzhou 8 space mission) on a random positioning machine (RPM) for 10 days to evaluate differences between real and SMG. About 2881 genes were regulated during 10 days of cell exposure to microgravity [32]. These genes were subdivided into different clusters (**Figure 5**) that allowed us to distinguish the difference between SMG (RPM) and spaceflight effects. However, two clusters of genes expressed similarly under either real or SMG. This research demonstrated that the effects of RPM and spaceflight both exert 3-D growth, but may not change gene expression in the same direction.

For the sake of the very limited amount of cells returned from space, the combination of gene expression and secretion analysis was detected on the cytokine, protease, and kinases factors that may play a pivotal role in the development of metastases. The list was made up of IL6, IL8, IL15 (interleukin family), OPN (osteopontin), VEGFA, VEGFD (vascular endothelial growth factor), and FGF17 (fibroblast growth factor).

**Figure 5.** (A) Gene array analyses on FTC-133 cells cultured for 10 days under both conditions. ANOVA resulted in 2881 twofold-regulated probes. The Davies-Bouldin cluster estimation resulted in a local minimum of seven clusters. (B) RPM-specific effects (clusters 5 and 7), spaceflight-specific AD cell and MCS effects (clusters 1, 2, and 4), and general gravity effects (clusters 3 and 6) were shown in gene map. An outstanding expression level of spaceflight samples was found in contrast to the moderate levels in RPM samples and controls.

In this research, VEGFA and VEGFD, which are members of the vascular endothelial growth factor (VEGF) family, were also observed. It is well known that VEGFA is the most important growth factor driving angiogenesis and is also implicated in many processes like tumor vascularization, wound healing, and burn injury. VEGFA expression has been found to increase in papillary thyroid carcinoma in contrast to that in healthy patients [33]. While on the other hand, VEGFD serum levels were found to decrease in patients with thyroid cancer [34]. In this study, an obvious decrease in expression of VEGFA was confirmed in the RPM as well as the spaceflight samples, while no difference was observed between adherent (AD) cells or multicellular spheroids (MCSs). Similarly, VEGFA secretion was also reduced on the RPM, but no influence in real microgravity. Furthermore, VEGFD gene expression upregulated in cells cultivated on the RPM as well as in space. All these results seem to get a conclusion that microgravity exerts a shift of the thyroid tumor cells to a more benign, less metastatic phenotype.

Interestingly, the similar tendency was observed with the interleukins in microgravity. IL6 and IL8, encoded by the IL6 and IL8 genes, are involved in tumor cell growth, metastasis, and angiogenesis. IL15, on the contrary, is able to activate several antitumor mechanisms, such as activating CD8 T cells to kill tumor cells [35]. Furthermore, it has been found that IL15 was effective in several tumor therapy experiments [36]. Their results explored a significant downregulation of IL8 gene expression in both simulated and real microgravity cells, which is also in accordance with the IL8 secretion pattern. The weightless effect of reducing IL6 gene expression was only observed in adherent cells, the same to that described for endothelial cells [37].

Of note, both IL6 and IL8 gene expression were strikingly upregulated during a parabolic flight. The study also showed earlier that IL6 and IL8 were most possibly implicated in the gravity-sensitive signaling required for spheroid formation. It seems that this signal pathway is attenuated after exposure to microgravity and that the tumor cells are shifted toward a less aggressive biological behavior. This hypothesis was also confirmed by the observations for IL15. IL15 gene expression increased only slightly on the RPM and during parabolic flight, but they explored a strong increase in the MCSs during spaceflight. Thus, we speculate that microgravity triggers some antitumor pathways involving IL15 and, as MCSs resemble tumors in their 3-D structure, the cell-cell interactions should be affected.

### **6. Conclusion**

pathways regulating cell migration are very complicated and seem to vary in different cells and different species. In general, it seems that decreased integrin expression and downregulated FAs kinase activity are an essential step in suppression of cell migration by SMG.

To investigate the involved mechanisms in more detail, gene level researches are expected to yield novel targets for cancer therapy, which may then be exploited in the form of new

Dr. Xiao Ma cultured thyroid cancer cells in space (Shenzhou 8 space mission) on a random positioning machine (RPM) for 10 days to evaluate differences between real and SMG. About 2881 genes were regulated during 10 days of cell exposure to microgravity [32]. These genes were subdivided into different clusters (**Figure 5**) that allowed us to distinguish the difference between SMG (RPM) and spaceflight effects. However, two clusters of genes expressed similarly under either real or SMG. This research demonstrated that the effects of RPM and spaceflight both exert 3-D growth, but may not change gene expression in the same direction. For the sake of the very limited amount of cells returned from space, the combination of gene expression and secretion analysis was detected on the cytokine, protease, and kinases factors that may play a pivotal role in the development of metastases. The list was made up of IL6, IL8, IL15 (interleukin family), OPN (osteopontin), VEGFA, VEGFD (vascular endothelial

**Figure 5.** (A) Gene array analyses on FTC-133 cells cultured for 10 days under both conditions. ANOVA resulted in 2881 twofold-regulated probes. The Davies-Bouldin cluster estimation resulted in a local minimum of seven clusters. (B) RPM-specific effects (clusters 5 and 7), spaceflight-specific AD cell and MCS effects (clusters 1, 2, and 4), and general gravity effects (clusters 3 and 6) were shown in gene map. An outstanding expression level of spaceflight samples was

**5. Changes of genes**

chemotherapeutics.

growth factor), and FGF17 (fibroblast growth factor).

146 Into Space - A Journey of How Humans Adapt and Live in Microgravity

found in contrast to the moderate levels in RPM samples and controls.

Taken together, it is no doubt that spaceflight experiments are of great value for cell biology research, especially for cancer research. The previous studies indicate that microgravity, both actual and simulated microgravity, induces changes in cancer cell proliferation, metastasis, and survival, bringing the cells toward a less aggressive phenotype. This effect is greater in actual spaceflight; however, ground-based simulation has been shown to be an essential tool in our understanding of gravity and it effects on a cellular level in cancer cells. The above body shows that not only does the architecture of tumor cells change in microgravity, but the cell function and gene expression also are different. The changes of some gene expression reorganize the cytoskeleton which influences the cell growth, migration, and apoptosis. And some other genes impress tumor cell by regulating the immune antitumor pathway. However, most of the studies are in vitro, and the evidence from human or animals is much rare. Is the microgravity strategy safe for human? How to create microgravity environment in body? Those are the thorny tissue for clinical translation. With the recent discoveries of nanomagnetic fluids, it suggests an innovative method of treating tumors using magnetic fluid-modeled microgravity. Magnetic fluids are delivered by outside magnetic field to tumor tissue either intravenously or through direct injection, and this is followed by application of a uniform external magnetic field that causes microgravity. The concept of magnetic fluid-modeled microgravity to treat tumor is novel, and the technology involved is simple, economical, and might be suitable for clinical applications in future.

[5] Wang ND, Wang H, Chen J, Zhang XF, Xie J, Ma J, Wang W, Wang ZR. The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells. Cytotechnology. 2014;**66**:119-131. DOI: 10.1007/s10616-013-9544-8 [6] Grimm D, Kossmehl P, Shakibaei M, Schulze-Tanzil G, Pickenhahn H. Effects of simulated microgravity on thyroid carcinoma cells. Journal of Gravitational Physiology.

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http://dx.doi.org/10.5772/intechopen.77214

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### **Acknowledgements**

The present chapter was funded by the National Natural Science Foundation of China (grant no. 81402055), and Youth Science and Technology Innovation Personnel Training Project of Shaanxi Province (grant no.2017KJXX-84).

### **Author details**

Jun Chen

Address all correspondence to: chj2002819@163.com

Department of Encephalopathy, Traditional Chinese Medicine Hospital of Shaanxi Province, Xi'an, Shaanxi, China

### **References**


[5] Wang ND, Wang H, Chen J, Zhang XF, Xie J, Ma J, Wang W, Wang ZR. The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells. Cytotechnology. 2014;**66**:119-131. DOI: 10.1007/s10616-013-9544-8

However, most of the studies are in vitro, and the evidence from human or animals is much rare. Is the microgravity strategy safe for human? How to create microgravity environment in body? Those are the thorny tissue for clinical translation. With the recent discoveries of nanomagnetic fluids, it suggests an innovative method of treating tumors using magnetic fluid-modeled microgravity. Magnetic fluids are delivered by outside magnetic field to tumor tissue either intravenously or through direct injection, and this is followed by application of a uniform external magnetic field that causes microgravity. The concept of magnetic fluid-modeled microgravity to treat tumor is novel, and the technology involved is simple, economical,

All in all, as new information about the biology of cancer emerges, treatments will be developed and modified to increase effectiveness, precision, survivability, and quality of life. It is

The present chapter was funded by the National Natural Science Foundation of China (grant no. 81402055), and Youth Science and Technology Innovation Personnel Training Project of

Department of Encephalopathy, Traditional Chinese Medicine Hospital of Shaanxi Province,

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and might be suitable for clinical applications in future.

148 Into Space - A Journey of How Humans Adapt and Live in Microgravity

**Acknowledgements**

**Author details**

Xi'an, Shaanxi, China

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Jun Chen

Shaanxi Province (grant no.2017KJXX-84).

Address all correspondence to: chj2002819@163.com

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[28] Zamir E, Geiger B. Molecular complexity and dynamics of cell-matrix adhesions. Journal

[29] Cox EA, Huttenlocher A. Regulation of integrin-mediated adhesion during cell migra-

[30] Meyers VE, Zayzafoon M, Gonda SR, Gathings WE, McDonald JM. Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells. Journal of Cellular Biochemistry. 2004;**93**:697-707. DOI:

ment. Cell Cycle. 2016;**15**:895-907. DOI: 10.1080/15384101.2016.1152430

Journal of Cancer. 2017;**116**:1572-1584. DOI: 10.1038/bjc.2017.126

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150 Into Space - A Journey of How Humans Adapt and Live in Microgravity

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of Cell Science. 2001;**114**:3583-3590


**Chapter 10**

Provisional chapter

**Plants in Space**

Plants in Space

Bratislav Stankovic

Abstract

Bratislav Stankovic

Additional information is available at the end of the chapter

Plants will play a critical role in the survival of human beings on long-duration space missions, probably beginning pretty soon with a mission to Mars. Plants can adapt to extreme environments on Earth, and model plants have been shown to grow and develop through a full life cycle in microgravity. In space, long-term human space exploration missions require a life support system in which higher plants play a vital role. Growing crops in space is as much about developing the humans' technological capacity to provide plants with adequate growth conditions in the unique microgravity environment, as is about the symbiotic relationship between plants and space travelers. After several decades of research, we have learned a lot about the impediments to growing plants in microgravity, in outer space, and on other planets. As human space exploration advances, we should feel confident about our ability to grow plants on board spacecraft during long-term space missions, on the Moon, and on other planets. Plants will require specialized environments for growth and development in microgravity, but – at least on a small scale – we already

DOI: 10.5772/intechopen.74230

know how to produce such growth chambers and greenhouses.

Keywords: space biology, gravitational biology, microgravity, plants, spaceflight,

The phrase "plants in space" refers to plants that are grown in the physical universe known as outer space, a region beyond the Kármán line in the Earth's atmosphere, at an altitude of approximately 200–450 km above sea level, which is the typical orbit range of the Space Shuttle missions and of the International Space Station, where most of human spaceflight and research has taken place [1]. Outer space represents a challenging environment for human exploration for a number of reasons, including the lethal hazards of extreme temperatures, high vacuum, electromagnetic radiation, particle radiation, and magnetism. A deep understanding of the

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74230

international Space Station

1. Introduction

#### **Chapter 10** Provisional chapter

#### **Plants in Space** Plants in Space

#### Bratislav Stankovic Bratislav Stankovic

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74230

#### Abstract

Plants will play a critical role in the survival of human beings on long-duration space missions, probably beginning pretty soon with a mission to Mars. Plants can adapt to extreme environments on Earth, and model plants have been shown to grow and develop through a full life cycle in microgravity. In space, long-term human space exploration missions require a life support system in which higher plants play a vital role. Growing crops in space is as much about developing the humans' technological capacity to provide plants with adequate growth conditions in the unique microgravity environment, as is about the symbiotic relationship between plants and space travelers. After several decades of research, we have learned a lot about the impediments to growing plants in microgravity, in outer space, and on other planets. As human space exploration advances, we should feel confident about our ability to grow plants on board spacecraft during long-term space missions, on the Moon, and on other planets. Plants will require specialized environments for growth and development in microgravity, but – at least on a small scale – we already know how to produce such growth chambers and greenhouses.

DOI: 10.5772/intechopen.74230

Keywords: space biology, gravitational biology, microgravity, plants, spaceflight, international Space Station

#### 1. Introduction

The phrase "plants in space" refers to plants that are grown in the physical universe known as outer space, a region beyond the Kármán line in the Earth's atmosphere, at an altitude of approximately 200–450 km above sea level, which is the typical orbit range of the Space Shuttle missions and of the International Space Station, where most of human spaceflight and research has taken place [1]. Outer space represents a challenging environment for human exploration for a number of reasons, including the lethal hazards of extreme temperatures, high vacuum, electromagnetic radiation, particle radiation, and magnetism. A deep understanding of the

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

biological consequences of exposure to the space environment is required to design efficient countermeasures to minimize their negative impact on living organisms, humans and plants alike. In addition, the economic cost of sending anything into space is very high. In outer space, plants are typically grown in a microgravity (often referred to as weightlessness) controlled environment, in specific space plant growth chambers.

recovered. The first seeds launched into space and successfully recovered were "ordinary corn seeds", launched on July 30, 1946; see [8] for a descriptive chronology of the early biological experiments in rockets. These early and very brief biological experiments were primarily concerned with the effects of radiation exposure on living tissue, including seeds. Some years later, the first plant materials taken into a microgravity environment for a longer ride in orbit were seeds of wheat, pea, maize, and onion, flown on board of Sputnik 4 in 1960 [2]. The first full life cycle of a plant (Arabidopsis thaliana) in space was completed on Salyut-7, resulting in clearly observable developmental alterations and in some viable seed, but mostly in seed

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155

http://dx.doi.org/10.5772/intechopen.74230

Opportunities for space experiments greatly increased by the initiation of scientific operations in orbital laboratories, the latest being the International Space Station (ISS). Building upon accumulated knowledge, researchers took advantage of well-developed plant growth chambers for microgravity, which in general provided a very good environment for growing plants on the ISS [10]. These proved that it is possible to have plants pass the full cycle of ontogenesis in space (on the ISS), and to produce plants and viable seed similar to the ground controls. The first example of seed-to-seed-to-seed (i.e., two consecutive life cycles) of a plant (Arabidopsis thaliana) in space was completed in 2000–2001 [11]. With advanced plant growth chambers that in general provided a well-regulated environment for growing plants in microgravity on the ISS, most of the problems seen in previous plant spaceflight experiments were successfully eliminated. It turns out that gravity is not necessary for seed-to-seed growth of plants, though

The effort and resources allocated to plant cultivation in space have revealed many answers, while at the same time raising new research questions. Periodic literature updates on the status of plant space biology have reviewed the documented influence of gravity on both plant growth and development, and specifically on a myriad of cellular and molecular responses, including cell cycle, embryogenesis and seed development, photosynthesis and gas exchange, gravitropic sensing and response, phototropism, cell wall development, and gene expression changes [12–15]. More recent and also more sophisticated plant experiments during the Space Shuttle and the ISS era produced key science insights on the molecular and cellular mechanisms underlying biological adaptation to spaceflight, and especially to plant growth, devel-

The first experiments with higher plants grown in space were intended to assess whether plants could grow outside Earth and to determine what differences there were between spaceflight-grown and Earth-grown plants. As plant-growth hardware started to adapt to spaceflight, opportunities were created for more sophisticated plant experiments. Direct microgravity effects started being differentiated from confinement effects, and Earth orbit became a laboratory where plants could be grown without the influence of Earth gravity.

it plays a direct role in plant form, and may influence seed reserves [11].

opment, tropisms, and stress responses in microgravity [16–19].

having nonviable embryos [9].

3. Particulars

Plant space biology has been closely associated with human space exploration in that plants are considered as key parts of biologically based life support. Learning to grow plants in space is an essential goal for long duration space missions since crop growth in space will be beneficial in a variety of ways, aiding with air regeneration, food production, and water recycling [2–6]. The logistical challenges of the long-term human space exploration missions require a selfsustainable life support system. Traveling in a spacecraft to other worlds will put constraints on the quantity and weight of commodities that could be brought along. In that context, higher plants are of paramount importance for providing in situ resource utilization through a continuous supply of fresh food, atmosphere revitalization, and clean water for humans.

The many challenges of spaceflight research have logistical and resource constraints, including significant limitations on available space, power, crew time, cold stowage, and data downlinks. Additional issues are related to hardware development, safety concerns, and the engineering versus science culture in space agencies. There is not much space for growing plants in space. Concerning research, the difficulties of publishing the results from spaceflight research stem from the lack of adequate controls, limited sample size, the frequent impossibility for verification of the obtained data, and the indirect effects of the spaceflight environment.

The concept of growing crops in space is as much about developing the humans' technological capacity to provide plants with adequate growth conditions in the unique microgravity environment, as it is about the symbiotic relationship between plants and space travelers. Plants in space provide numerous benefits to the humans that accompany them. They improve the quality of indoor air by helping control humidity levels, and by removing and converting the carbon dioxide from air into essential oxygen that humans can breathe. Central to the concept of regenerative life support systems for space exploration is the use of photosynthetic organisms and light to generate oxygen and food. It is also axiomatic that plants can be consumed as food, providing a nutritive value to organisms throughout the food chain. Growing plants in space may also provide psychological and neurocognitive benefits to the human spaceflight crews, in the form of therapeutic people-plant interactions [7].

### 2. Fundamentals

Humans began the physical exploration of space during the 20th century with the advent of high-altitude balloon flights, followed by rocket launches. Plants have been used in space experiments since the early days of the space program. The early suborbital launches saw the first organisms in space, as unspecified "specially developed strains of seeds", sent to 134 km altitude above Earth on July 9, 1946, on a U.S. launched V-2 rocket; these samples were not recovered. The first seeds launched into space and successfully recovered were "ordinary corn seeds", launched on July 30, 1946; see [8] for a descriptive chronology of the early biological experiments in rockets. These early and very brief biological experiments were primarily concerned with the effects of radiation exposure on living tissue, including seeds. Some years later, the first plant materials taken into a microgravity environment for a longer ride in orbit were seeds of wheat, pea, maize, and onion, flown on board of Sputnik 4 in 1960 [2]. The first full life cycle of a plant (Arabidopsis thaliana) in space was completed on Salyut-7, resulting in clearly observable developmental alterations and in some viable seed, but mostly in seed having nonviable embryos [9].

Opportunities for space experiments greatly increased by the initiation of scientific operations in orbital laboratories, the latest being the International Space Station (ISS). Building upon accumulated knowledge, researchers took advantage of well-developed plant growth chambers for microgravity, which in general provided a very good environment for growing plants on the ISS [10]. These proved that it is possible to have plants pass the full cycle of ontogenesis in space (on the ISS), and to produce plants and viable seed similar to the ground controls. The first example of seed-to-seed-to-seed (i.e., two consecutive life cycles) of a plant (Arabidopsis thaliana) in space was completed in 2000–2001 [11]. With advanced plant growth chambers that in general provided a well-regulated environment for growing plants in microgravity on the ISS, most of the problems seen in previous plant spaceflight experiments were successfully eliminated. It turns out that gravity is not necessary for seed-to-seed growth of plants, though it plays a direct role in plant form, and may influence seed reserves [11].

The effort and resources allocated to plant cultivation in space have revealed many answers, while at the same time raising new research questions. Periodic literature updates on the status of plant space biology have reviewed the documented influence of gravity on both plant growth and development, and specifically on a myriad of cellular and molecular responses, including cell cycle, embryogenesis and seed development, photosynthesis and gas exchange, gravitropic sensing and response, phototropism, cell wall development, and gene expression changes [12–15]. More recent and also more sophisticated plant experiments during the Space Shuttle and the ISS era produced key science insights on the molecular and cellular mechanisms underlying biological adaptation to spaceflight, and especially to plant growth, development, tropisms, and stress responses in microgravity [16–19].

### 3. Particulars

biological consequences of exposure to the space environment is required to design efficient countermeasures to minimize their negative impact on living organisms, humans and plants alike. In addition, the economic cost of sending anything into space is very high. In outer space, plants are typically grown in a microgravity (often referred to as weightlessness)

Plant space biology has been closely associated with human space exploration in that plants are considered as key parts of biologically based life support. Learning to grow plants in space is an essential goal for long duration space missions since crop growth in space will be beneficial in a variety of ways, aiding with air regeneration, food production, and water recycling [2–6]. The logistical challenges of the long-term human space exploration missions require a selfsustainable life support system. Traveling in a spacecraft to other worlds will put constraints on the quantity and weight of commodities that could be brought along. In that context, higher plants are of paramount importance for providing in situ resource utilization through a contin-

The many challenges of spaceflight research have logistical and resource constraints, including significant limitations on available space, power, crew time, cold stowage, and data downlinks. Additional issues are related to hardware development, safety concerns, and the engineering versus science culture in space agencies. There is not much space for growing plants in space. Concerning research, the difficulties of publishing the results from spaceflight research stem from the lack of adequate controls, limited sample size, the frequent impossibility for verification of the obtained data, and the indirect effects of the spaceflight environment.

The concept of growing crops in space is as much about developing the humans' technological capacity to provide plants with adequate growth conditions in the unique microgravity environment, as it is about the symbiotic relationship between plants and space travelers. Plants in space provide numerous benefits to the humans that accompany them. They improve the quality of indoor air by helping control humidity levels, and by removing and converting the carbon dioxide from air into essential oxygen that humans can breathe. Central to the concept of regenerative life support systems for space exploration is the use of photosynthetic organisms and light to generate oxygen and food. It is also axiomatic that plants can be consumed as food, providing a nutritive value to organisms throughout the food chain. Growing plants in space may also provide psychological and neurocognitive benefits to the human spaceflight

Humans began the physical exploration of space during the 20th century with the advent of high-altitude balloon flights, followed by rocket launches. Plants have been used in space experiments since the early days of the space program. The early suborbital launches saw the first organisms in space, as unspecified "specially developed strains of seeds", sent to 134 km altitude above Earth on July 9, 1946, on a U.S. launched V-2 rocket; these samples were not

uous supply of fresh food, atmosphere revitalization, and clean water for humans.

controlled environment, in specific space plant growth chambers.

154 Into Space - A Journey of How Humans Adapt and Live in Microgravity

crews, in the form of therapeutic people-plant interactions [7].

2. Fundamentals

The first experiments with higher plants grown in space were intended to assess whether plants could grow outside Earth and to determine what differences there were between spaceflight-grown and Earth-grown plants. As plant-growth hardware started to adapt to spaceflight, opportunities were created for more sophisticated plant experiments. Direct microgravity effects started being differentiated from confinement effects, and Earth orbit became a laboratory where plants could be grown without the influence of Earth gravity.

The physiological effects of gravity range from subtle to substantial, and influence numerous molecular and cellular events in addition to those solely associated with gravitropism. Many of the early plant space biology experiments resulted in morphological and physiological changes, manifested as cellular and phenotypic abnormalities. These include chromosomal breakage [20], failure to produce seed [21], altered or nonviable embryos [9], alterations in cell wall composition and properties [22], increased breakdown of xyloglucans [23], changes in polar auxin transport [24], or other morphological abnormalities [25]. Indeed, spaceflight appears to initiate both molecular and cellular remodeling throughout the plant. For example, spaceflight can induce significant genomic and epigenomic mutations [26]. In the absence of gravity plants rely on other environmental cues to initiate the morphological responses essential to successful growth and development, and the basis for that engagement lies in the differential expression of genes in an organ-specific manner [11, 16, 27], which is followed by a microgravity-driven remodeling of the proteome [28].

ISS provide oxygen via water electrolysis, absorb and remove carbon dioxide, and manage vaporous emissions (e.g., ammonia, acetone) from the astronauts themselves; water is

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157

http://dx.doi.org/10.5772/intechopen.74230

Central to the concept of bio-regenerative life support systems is the use of photosynthetic organisms and light to generate oxygen and food. Learning to grow plants in space is thus an essential goal for long duration space missions since crop growth in space will aid with air regeneration, food production and water recycling for astronauts during long-term space missions [2, 40]. Research on plants in space, in addition to producing key scientific insights into specific plant gravitropic and abiotic stress responses, fosters the overall development of bioregenerative life support systems for the production of oxygen, food, and nutrients [41].

The cultivation of higher plants occupies an essential role within bioregenerative life support systems (BLSS), which are designed to provide a habitation environment similar to the Earth's biosphere for space missions with extended durations and in deep space. It contributes to all key functional aspects by closing the different loops in a habitat like oxygen production, carbon dioxide reduction, food production, water management, and metabolic waste recycling. Fresh crops are also expected to have a positive impact on crew psychological

Different designs and technological solutions have been implemented in higher plant flight experiments. Continuous subsystem improvements and increasing knowledge of plant response to the spaceflight environment has led to the design of current plant growth systems, the latest being the Vegetable Production System (Veggie) [42, 43] and the Advanced Plant Habitat [44]. Plants can adapt to extreme environments on Earth, and model plants have been shown to grow and develop through a full life cycle in microgravity. Adequate environmental control, including forced ventilation, trace gas control, and a well-functioning system for water

To put this issue in perspective, the planned early Martian missions (around 500 days overall duration) will primarily focus on water recycling, atmosphere regeneration, and stockpiling of food. Due to the different orbits between Earth and Mars, the launch/return window for the trip is limited either to 30 days, or longer than 2 years (about 780 days). These relatively long space missions can only be sustained with a bioregenerative life support system. Due to the long permanence of the crew and the difficulty to transport and store a large quantity of food, it is estimated that bioregenerative life support system should provide around 80–90% of the nourishment and oxygen needed, which translates to about 40–50 m2 of plant growing area needed per crew member [45]. To satisfy this requirement, permanent greenhouses and/or

Growing plants in space helps solve one of the biggest issues in space travel: the supply and the price of food. Space food has evolved since 1961, when the cosmonaut German Titov became the first human to eat in space. The first foods were highly engineered, thermostabilized and packaged, capable of meeting the rigid requirements imposed by spacecraft

and nutrient delivery are required for long-term plant growth in space [3, 45].

sizeable agricultural modules for space would need to be developed.

3.2. Plants for food in space

recycled.

health.

Reflecting on the early spaceflight experiments, we now know that a number of the early obtained results were more likely due to the rigors of the microgravity environment than to the lack of gravity itself. For example, altered starch content has been reported for different species of space grown plants: pepper [29], lepidium [30], maize [31], and Arabidopsis [32]. However, just improving plant ventilation during space flight was found to eliminate carbohydrate differences [33]. In addition, ethylene, a plant stress hormone, is a common problem in microgravity experiments. Plant ethylene production increases in space [34]. Elevated ethylene levels (1100–1600 ppb on a Shuttle) caused anomalous seedling growth of Arabidopsis in spaceflight studies, although they had no effect on relative graviresponsiveness [35]. Furthermore, ethylene levels on the MIR space station were very high (800–1200 ppb) during a Brassica spaceflight growth study [36]. While Brassica plants were capable of producing seed at this ethylene level, the same environment stopped a wheat crop from producing seed on board MIR [37]. Novel plant growth spaceflight hardware uses ethylene scrubbers to mitigate the negative effects of elevated ethylene levels in spacecraft [10, 11].

The absence of natural convection in space makes it easy for plants to become oxygen starved [38]. Hypoxia symptoms in seed include reduction in size of the protein bodies, failure of the protein bodies to fill, free floating lipid droplets in the cytoplasm, abnormally vacuolated cells, and degeneration of portions of the embryo. In a full life-cycle spaceflight experiment with Brassica, the protein bodies that were found to be 44% smaller, starch grains were aberrantly deposited in the seed, and the cotyledon cell number was reduced by 80% [36]. This study concluded that alterations in the oxygen and ethylene concentrations within developing siliques were problematic in the experiment [36]. While the Svet greenhouses used to grow Brassica on MIR used a fan to circulate air, the circulation rate was insufficient (below 0.5 m/s) to prevent hypoxia [38]. Control of the gaseous environment appears to be a key factor for plant reproduction in microgravity [39].

#### 3.1. Plants for bio-regenerative life support systems

The logistical challenges of long-term human space exploration missions require a life support system capable of regenerating all the essentials for survival. The life support systems on the ISS provide oxygen via water electrolysis, absorb and remove carbon dioxide, and manage vaporous emissions (e.g., ammonia, acetone) from the astronauts themselves; water is recycled.

Central to the concept of bio-regenerative life support systems is the use of photosynthetic organisms and light to generate oxygen and food. Learning to grow plants in space is thus an essential goal for long duration space missions since crop growth in space will aid with air regeneration, food production and water recycling for astronauts during long-term space missions [2, 40]. Research on plants in space, in addition to producing key scientific insights into specific plant gravitropic and abiotic stress responses, fosters the overall development of bioregenerative life support systems for the production of oxygen, food, and nutrients [41].

The cultivation of higher plants occupies an essential role within bioregenerative life support systems (BLSS), which are designed to provide a habitation environment similar to the Earth's biosphere for space missions with extended durations and in deep space. It contributes to all key functional aspects by closing the different loops in a habitat like oxygen production, carbon dioxide reduction, food production, water management, and metabolic waste recycling. Fresh crops are also expected to have a positive impact on crew psychological health.

Different designs and technological solutions have been implemented in higher plant flight experiments. Continuous subsystem improvements and increasing knowledge of plant response to the spaceflight environment has led to the design of current plant growth systems, the latest being the Vegetable Production System (Veggie) [42, 43] and the Advanced Plant Habitat [44]. Plants can adapt to extreme environments on Earth, and model plants have been shown to grow and develop through a full life cycle in microgravity. Adequate environmental control, including forced ventilation, trace gas control, and a well-functioning system for water and nutrient delivery are required for long-term plant growth in space [3, 45].

To put this issue in perspective, the planned early Martian missions (around 500 days overall duration) will primarily focus on water recycling, atmosphere regeneration, and stockpiling of food. Due to the different orbits between Earth and Mars, the launch/return window for the trip is limited either to 30 days, or longer than 2 years (about 780 days). These relatively long space missions can only be sustained with a bioregenerative life support system. Due to the long permanence of the crew and the difficulty to transport and store a large quantity of food, it is estimated that bioregenerative life support system should provide around 80–90% of the nourishment and oxygen needed, which translates to about 40–50 m2 of plant growing area needed per crew member [45]. To satisfy this requirement, permanent greenhouses and/or sizeable agricultural modules for space would need to be developed.

#### 3.2. Plants for food in space

The physiological effects of gravity range from subtle to substantial, and influence numerous molecular and cellular events in addition to those solely associated with gravitropism. Many of the early plant space biology experiments resulted in morphological and physiological changes, manifested as cellular and phenotypic abnormalities. These include chromosomal breakage [20], failure to produce seed [21], altered or nonviable embryos [9], alterations in cell wall composition and properties [22], increased breakdown of xyloglucans [23], changes in polar auxin transport [24], or other morphological abnormalities [25]. Indeed, spaceflight appears to initiate both molecular and cellular remodeling throughout the plant. For example, spaceflight can induce significant genomic and epigenomic mutations [26]. In the absence of gravity plants rely on other environmental cues to initiate the morphological responses essential to successful growth and development, and the basis for that engagement lies in the differential expression of genes in an organ-specific manner [11, 16, 27], which is followed by

Reflecting on the early spaceflight experiments, we now know that a number of the early obtained results were more likely due to the rigors of the microgravity environment than to the lack of gravity itself. For example, altered starch content has been reported for different species of space grown plants: pepper [29], lepidium [30], maize [31], and Arabidopsis [32]. However, just improving plant ventilation during space flight was found to eliminate carbohydrate differences [33]. In addition, ethylene, a plant stress hormone, is a common problem in microgravity experiments. Plant ethylene production increases in space [34]. Elevated ethylene levels (1100–1600 ppb on a Shuttle) caused anomalous seedling growth of Arabidopsis in spaceflight studies, although they had no effect on relative graviresponsiveness [35]. Furthermore, ethylene levels on the MIR space station were very high (800–1200 ppb) during a Brassica spaceflight growth study [36]. While Brassica plants were capable of producing seed at this ethylene level, the same environment stopped a wheat crop from producing seed on board MIR [37]. Novel plant growth spaceflight hardware uses ethylene scrubbers to mitigate

The absence of natural convection in space makes it easy for plants to become oxygen starved [38]. Hypoxia symptoms in seed include reduction in size of the protein bodies, failure of the protein bodies to fill, free floating lipid droplets in the cytoplasm, abnormally vacuolated cells, and degeneration of portions of the embryo. In a full life-cycle spaceflight experiment with Brassica, the protein bodies that were found to be 44% smaller, starch grains were aberrantly deposited in the seed, and the cotyledon cell number was reduced by 80% [36]. This study concluded that alterations in the oxygen and ethylene concentrations within developing siliques were problematic in the experiment [36]. While the Svet greenhouses used to grow Brassica on MIR used a fan to circulate air, the circulation rate was insufficient (below 0.5 m/s) to prevent hypoxia [38]. Control of the gaseous environment appears to be a key factor for

The logistical challenges of long-term human space exploration missions require a life support system capable of regenerating all the essentials for survival. The life support systems on the

a microgravity-driven remodeling of the proteome [28].

156 Into Space - A Journey of How Humans Adapt and Live in Microgravity

the negative effects of elevated ethylene levels in spacecraft [10, 11].

plant reproduction in microgravity [39].

3.1. Plants for bio-regenerative life support systems

Growing plants in space helps solve one of the biggest issues in space travel: the supply and the price of food. Space food has evolved since 1961, when the cosmonaut German Titov became the first human to eat in space. The first foods were highly engineered, thermostabilized and packaged, capable of meeting the rigid requirements imposed by spacecraft design. However, both the Apollo and the Shuttle missions demonstrated that astronauts did not consume sufficient nutrients, and determined that adequate nutrition begins with appropriate food presented to the consumer in a familiar form [46]. Accordingly, much progress has been made from the first tubed food. Today, the food for the astronauts on the ISS includes a variety of individually packaged, thermostabilized, irradiated, intermediate moisture, and natural form foods [46].

farm their own food using local dirt [50]. Additional research is needed to improve our understanding of the water holding capacity and other physical characteristics of the extraterrestrial soils, the availability of reactive nitrogen and other (essential) nutrients, further combined with the addition of nutrients and creating a balanced nutrient availability, and the influence of gravity, light and other conditions [50]. Further efforts should include mechanistic modeling of plant growth for better understanding of the intricate and combined physical, biochemical, and morphological phenomena involved, necessary to accurately control and

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Plant in space also provide a substantial non-nutritive value; they are not just for eating or producing oxygen. Plants generally act as a form of emotional sustenance sometimes called horticultural therapy, and can mitigate the negative psychological consequences of space travel. Humans have a preference for nature scenery. Humans (especially humans in a confined space) positively react to plants, and they derive a variety of physiological benefits from exposure to plants. These include human well-being, sense of mastery of the environment, social development, health support, overcoming boredom and mental fatigue, and stress reduction and recovery [51, 52]. Studies of the potential psychological consequences of longterm exposure to conditions common to long-term isolated environments indicated that humans are less stressed and perform better in conditions that include plants and natural

The spaceflight environment induces a host of physiological, biomedical, and environmental stressors to flight crews. Long duration spaceflight has revealed a group of stressors that impact crew performance and health: hypochondria, diminished motivation and performance, impaired cognitive ability, withdrawal, impulsive behavior, hallucinations, mood swings, helplessness, depression, and anger [53]. These have spurred the emergence of areas of specialty within the behavioral sciences, including space psychology, space human factors, space habitability, space performance, and space sociology [53]. In that context, the benefit of plants as a countermeasure for difficulties experienced by humans living in isolated or extreme environments, including space travel [54]. A symbiotic relationship between plants and space

travelers, including a plant garden for Major Tom, is probably a very good idea.

3.4. Case study: growing Arabidopsis thaliana on the International Space Station

As the International Space Station was being assembled, we designed and custom-built a novel advanced plant growth chamber for microgravity experiments [55]. The ADVanced AStroCulture (ADVASC) was the first plant growth chamber flown on the ISS [56]. We used this chamber to grow Arabidopsis thaliana from seed-to-seed-to-seed (i.e., two consecutive full cycles of ontogenesis) wholly in microgravity, on the ISS. Arabidopsis plants were germinated, grown and maintained on the ISS prior to returning to Earth [10]. Some of these seeds were used in a subsequent experiment, to successfully produce a second (back-to-back) generation

predict plant growth in space.

settings [51, 52].

of microgravity-grown Arabidopsis [11].

3.3. Plants for the mental well-being of major tom

The ultimate goal of growing plants for food in space is to create a self-sustainable regenerative growth system, so plants for food could be continually grown in orbit, in Moon colonies, or on other planets. Challenges to growing plants in space are primarily in the areas of nutrient delivery, lighting, and ventilation (gas exchange). With adapted growth chambers, plant growth in space is similar to plant growth on Earth, except for some morphological traits. However, only small-scale experiments on plant growth have been performed in Earth orbit. These have not provided sufficient data on crop yield for space environments. [47]. Microgravity can reduce cell growth, alter gene expression and protein synthesis, and influence plant morphology – all aspects which critically affect plant cultivation in space. Seeds produced in space also seem to have different composition compared to seeds grown on Earth. As well as affecting the performance and nutritional content of space seeds, this could influence the flavor of plants produced in space, which might become a problem for crews reliant on plant-based diets during long space missions [47].

The theme of agriculture for space has contributed to, and benefited from, terrestrial, controlled environment agriculture; and will continue to do so into the future. For a comprehensive historical review of agricultural systems that have been developed for outer space see [4]. What started with studies on algal production in controlled environment agriculture in the 1950s in the USA and in the USSR has undergone significant improvements via NASA's Controlled Ecological Life Support Systems (CELSS) Program, Japan's Controlled Ecological Experiment Facility (CEEF), the European Space Agency's MELiSSA Project, and most recently, the Chinese Lunar Palace 1 plant factory [4, 48].

The innovative studies for space agriculture have resulted in the development of novel technologies, for both space and Earth applications. These include the use of light emitting diodes for growing crops, the demonstrations of vertical agriculture, use of hydroponic approaches for subterranean crops, crop yields that surpassed reported record field yields, the ability to quantify volatile organic compound production from whole crop stands, innovative approaches for controlling water delivery, and approaches for processing and recycling wastes back to crop production systems [4]. In addition, application of the space environment for mutagenesis and crop breeding has been suggested [26].

Recent research has focused on the possible growth of plants on the Moon and on Mars [49, 50]. In principle, it is possible to grow crops and other plant species in Martian and Lunar soil simulants, even without addition of nutrients. For the record, the Mars simulant can be obtained from a volcanic cone in Hawaii, and has a chemical composition similar to the Mars dirt that the Viking 1 lander analyzed; the Moon simulant comes from volcanic ash deposits near Flagstaff, Arizona. Beyond a Hollywood movie, experiments with 14 plant species in soils that simulate the Martian and lunar regolith suggest that future space colonizers may be able to farm their own food using local dirt [50]. Additional research is needed to improve our understanding of the water holding capacity and other physical characteristics of the extraterrestrial soils, the availability of reactive nitrogen and other (essential) nutrients, further combined with the addition of nutrients and creating a balanced nutrient availability, and the influence of gravity, light and other conditions [50]. Further efforts should include mechanistic modeling of plant growth for better understanding of the intricate and combined physical, biochemical, and morphological phenomena involved, necessary to accurately control and predict plant growth in space.

#### 3.3. Plants for the mental well-being of major tom

design. However, both the Apollo and the Shuttle missions demonstrated that astronauts did not consume sufficient nutrients, and determined that adequate nutrition begins with appropriate food presented to the consumer in a familiar form [46]. Accordingly, much progress has been made from the first tubed food. Today, the food for the astronauts on the ISS includes a variety of individually packaged, thermostabilized, irradiated, intermediate moisture, and

The ultimate goal of growing plants for food in space is to create a self-sustainable regenerative growth system, so plants for food could be continually grown in orbit, in Moon colonies, or on other planets. Challenges to growing plants in space are primarily in the areas of nutrient delivery, lighting, and ventilation (gas exchange). With adapted growth chambers, plant growth in space is similar to plant growth on Earth, except for some morphological traits. However, only small-scale experiments on plant growth have been performed in Earth orbit. These have not provided sufficient data on crop yield for space environments. [47]. Microgravity can reduce cell growth, alter gene expression and protein synthesis, and influence plant morphology – all aspects which critically affect plant cultivation in space. Seeds produced in space also seem to have different composition compared to seeds grown on Earth. As well as affecting the performance and nutritional content of space seeds, this could influence the flavor of plants produced in space, which might become a problem for crews reliant on plant-based

The theme of agriculture for space has contributed to, and benefited from, terrestrial, controlled environment agriculture; and will continue to do so into the future. For a comprehensive historical review of agricultural systems that have been developed for outer space see [4]. What started with studies on algal production in controlled environment agriculture in the 1950s in the USA and in the USSR has undergone significant improvements via NASA's Controlled Ecological Life Support Systems (CELSS) Program, Japan's Controlled Ecological Experiment Facility (CEEF), the European Space Agency's MELiSSA Project, and most

The innovative studies for space agriculture have resulted in the development of novel technologies, for both space and Earth applications. These include the use of light emitting diodes for growing crops, the demonstrations of vertical agriculture, use of hydroponic approaches for subterranean crops, crop yields that surpassed reported record field yields, the ability to quantify volatile organic compound production from whole crop stands, innovative approaches for controlling water delivery, and approaches for processing and recycling wastes back to crop production systems [4]. In addition, application of the space environment for

Recent research has focused on the possible growth of plants on the Moon and on Mars [49, 50]. In principle, it is possible to grow crops and other plant species in Martian and Lunar soil simulants, even without addition of nutrients. For the record, the Mars simulant can be obtained from a volcanic cone in Hawaii, and has a chemical composition similar to the Mars dirt that the Viking 1 lander analyzed; the Moon simulant comes from volcanic ash deposits near Flagstaff, Arizona. Beyond a Hollywood movie, experiments with 14 plant species in soils that simulate the Martian and lunar regolith suggest that future space colonizers may be able to

natural form foods [46].

158 Into Space - A Journey of How Humans Adapt and Live in Microgravity

diets during long space missions [47].

recently, the Chinese Lunar Palace 1 plant factory [4, 48].

mutagenesis and crop breeding has been suggested [26].

Plant in space also provide a substantial non-nutritive value; they are not just for eating or producing oxygen. Plants generally act as a form of emotional sustenance sometimes called horticultural therapy, and can mitigate the negative psychological consequences of space travel. Humans have a preference for nature scenery. Humans (especially humans in a confined space) positively react to plants, and they derive a variety of physiological benefits from exposure to plants. These include human well-being, sense of mastery of the environment, social development, health support, overcoming boredom and mental fatigue, and stress reduction and recovery [51, 52]. Studies of the potential psychological consequences of longterm exposure to conditions common to long-term isolated environments indicated that humans are less stressed and perform better in conditions that include plants and natural settings [51, 52].

The spaceflight environment induces a host of physiological, biomedical, and environmental stressors to flight crews. Long duration spaceflight has revealed a group of stressors that impact crew performance and health: hypochondria, diminished motivation and performance, impaired cognitive ability, withdrawal, impulsive behavior, hallucinations, mood swings, helplessness, depression, and anger [53]. These have spurred the emergence of areas of specialty within the behavioral sciences, including space psychology, space human factors, space habitability, space performance, and space sociology [53]. In that context, the benefit of plants as a countermeasure for difficulties experienced by humans living in isolated or extreme environments, including space travel [54]. A symbiotic relationship between plants and space travelers, including a plant garden for Major Tom, is probably a very good idea.

#### 3.4. Case study: growing Arabidopsis thaliana on the International Space Station

As the International Space Station was being assembled, we designed and custom-built a novel advanced plant growth chamber for microgravity experiments [55]. The ADVanced AStroCulture (ADVASC) was the first plant growth chamber flown on the ISS [56]. We used this chamber to grow Arabidopsis thaliana from seed-to-seed-to-seed (i.e., two consecutive full cycles of ontogenesis) wholly in microgravity, on the ISS. Arabidopsis plants were germinated, grown and maintained on the ISS prior to returning to Earth [10]. Some of these seeds were used in a subsequent experiment, to successfully produce a second (back-to-back) generation of microgravity-grown Arabidopsis [11].

The ADVASC plant growth unit was designed to control environment parameters including temperature, relative humidity, lighting, fluid nutrient and water delivery, and CO2 and ethylene concentrations. Advanced control software provided control of each environmental parameter in the plant chamber, creating environmental conditions suitable for growing a wide variety of plant species. Auto-prime technologies eliminated the need for power during Space Shuttle ascent/descent, greatly relieving the shortage of Shuttle resources and the ISS crew time. Fault tolerance and recovery algorithm significantly increased overall system robustness and efficiency. Tele-science features allowed engineers and scientists to receive telemetry data, to send remote commands, and to monitor plant development status via the video images and other data (Figure 1).

The first ADVASC payload with 91 Arabidopsis thaliana seeds planted in the root module was launched on STS-100 (ISS-6A), and returned to Earth on STS-104 (ISS-7A). During approximately 70 days in space, the experiment went through seed germination, plant development, seed formation, and seed maturity, which formed a complete life cycle. The experiment was designed to perform autonomously through the entire life cycle [10]. Post-mission analysis data shows that fully 90% of seeds germinated in space, which was similar to the 1 g–grown control plants (grown in a separate ADVASC growth chamber on Earth). Approximately 70% of seeds grew to produce siliques which contained mature seeds in space; An average of 24 siliques per plant were produced, each one containing an average of 36 seeds per silique; plants were healthy and growing normally with the exceptions of the roots and the inflorescent branches from the main stem of flowers. The directions that these organs grew were different in comparison to ground-controlled experiment, and were consistent with an apparent micro-

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Plant growth and development in microgravity proceeded similarly to the ground controls that were grown under 1 g in an identical chamber [10, 11]. Morphologically, the most striking feature of space-grown Arabidopsis was that the secondary inflorescence branches and siliques formed nearly perpendicular angles to the inflorescence stems. The branches grew out perpendicularly to the main inflorescence stem, indicating that gravity is the key determinant of branch and silique angle, and that light has either no role or a secondary role in branch and silique orientation [10, 11]. Seed protein bodies were 55% smaller in space seed than in controls, but protein assays showed only a 9% reduction in seed protein content. Germination rates for space-produced seed were 92% indicating that the seed developed in microgravity were healthy and viable. We determined that gravity is not necessary for seedto-seed growth of plants, though it plays a direct role in plant form, and may influence seed reserves [10, 11]. Indeed, it appears that plants undergo somewhat different growth and morphogenesis under space conditions; plant organs show automorphogenesis in space, which may be masked by gravimorphogenesis on earth, except when growing on a clinostat

Upon return of the plants to Earth, we conducted biochemical, cellular and molecular analyses. We observed a 55% reduction in protein body size; however, since the protein bodies in spacedeveloped seed were filled and we did not observe any other signs of hypoxia such as degeneration of the embryos, deposition of starch grains or alterations in cell structures or cell numbers, we conclude that the aerial portions of the plant were not starved for oxygen. The high forced airflow rates (2–3 m/s) and accompanying ethylene removal provided by the growth chamber improved growing conditions for the aerial part of the plants when compared

Root zone hypoxia could explain the reduced seed protein content. ADVASC uses passive airflow to move air through the root tray. Root zone hypoxia has been prevalent in space flight experiments [58, 59]. We used our own mix of porous arcillite matrix that is one of the favored rooting systems for space [60]. Arcillite reduces root zone hypoxia by allowing air to penetrate between the arcillite grains. Nonetheless, air movement through arcillite is restricted, especially if the spaces between arcillite grains are filled with roots, water, or both. If passive

gravity impact [10].

(Figures 2 and 3) [57].

to the previous studies [9, 33, 39].

The first flight of ADVASC provided an opportunity to study the patterns of plant growth and development, as well as seed and plant morphology in microgravity (first seed-to-seed Arabidopsis experiment on the ISS) [10]. The subsequent flight of ADVASC was used to obtain a second generation of microgravity-grown Arabidopsis plants (second seed-to-seed Arabidopsis experiment on the ISS), and to obtain fresh plant tissue for DNA microarray analysis (gene expression profiling) [11]. Since previous investigators found abnormalities in seed produced on long duration missions, we wanted to see if ADVASC's improvements in remote plant care had translated into improved seed quality. We were also interested to learn if microgravity would alter plant form and cause biochemical, cellular, and molecular changes.

Figure 1. Advanced Astroculture (ADVASC) environmentally controlled plant growth chamber, designed for experiments on the ISS, able to support plant research for a maximum of 6 months in microgravity environment.

The first ADVASC payload with 91 Arabidopsis thaliana seeds planted in the root module was launched on STS-100 (ISS-6A), and returned to Earth on STS-104 (ISS-7A). During approximately 70 days in space, the experiment went through seed germination, plant development, seed formation, and seed maturity, which formed a complete life cycle. The experiment was designed to perform autonomously through the entire life cycle [10]. Post-mission analysis data shows that fully 90% of seeds germinated in space, which was similar to the 1 g–grown control plants (grown in a separate ADVASC growth chamber on Earth). Approximately 70% of seeds grew to produce siliques which contained mature seeds in space; An average of 24 siliques per plant were produced, each one containing an average of 36 seeds per silique; plants were healthy and growing normally with the exceptions of the roots and the inflorescent branches from the main stem of flowers. The directions that these organs grew were different in comparison to ground-controlled experiment, and were consistent with an apparent microgravity impact [10].

The ADVASC plant growth unit was designed to control environment parameters including temperature, relative humidity, lighting, fluid nutrient and water delivery, and CO2 and ethylene concentrations. Advanced control software provided control of each environmental parameter in the plant chamber, creating environmental conditions suitable for growing a wide variety of plant species. Auto-prime technologies eliminated the need for power during Space Shuttle ascent/descent, greatly relieving the shortage of Shuttle resources and the ISS crew time. Fault tolerance and recovery algorithm significantly increased overall system robustness and efficiency. Tele-science features allowed engineers and scientists to receive telemetry data, to send remote commands, and to monitor plant development status via the

The first flight of ADVASC provided an opportunity to study the patterns of plant growth and development, as well as seed and plant morphology in microgravity (first seed-to-seed Arabidopsis experiment on the ISS) [10]. The subsequent flight of ADVASC was used to obtain a second generation of microgravity-grown Arabidopsis plants (second seed-to-seed Arabidopsis experiment on the ISS), and to obtain fresh plant tissue for DNA microarray analysis (gene expression profiling) [11]. Since previous investigators found abnormalities in seed produced on long duration missions, we wanted to see if ADVASC's improvements in remote plant care had translated into improved seed quality. We were also interested to learn if microgravity would alter plant form and cause biochemical, cellular, and molecular changes.

Figure 1. Advanced Astroculture (ADVASC) environmentally controlled plant growth chamber, designed for experi-

ments on the ISS, able to support plant research for a maximum of 6 months in microgravity environment.

video images and other data (Figure 1).

160 Into Space - A Journey of How Humans Adapt and Live in Microgravity

Plant growth and development in microgravity proceeded similarly to the ground controls that were grown under 1 g in an identical chamber [10, 11]. Morphologically, the most striking feature of space-grown Arabidopsis was that the secondary inflorescence branches and siliques formed nearly perpendicular angles to the inflorescence stems. The branches grew out perpendicularly to the main inflorescence stem, indicating that gravity is the key determinant of branch and silique angle, and that light has either no role or a secondary role in branch and silique orientation [10, 11]. Seed protein bodies were 55% smaller in space seed than in controls, but protein assays showed only a 9% reduction in seed protein content. Germination rates for space-produced seed were 92% indicating that the seed developed in microgravity were healthy and viable. We determined that gravity is not necessary for seedto-seed growth of plants, though it plays a direct role in plant form, and may influence seed reserves [10, 11]. Indeed, it appears that plants undergo somewhat different growth and morphogenesis under space conditions; plant organs show automorphogenesis in space, which may be masked by gravimorphogenesis on earth, except when growing on a clinostat (Figures 2 and 3) [57].

Upon return of the plants to Earth, we conducted biochemical, cellular and molecular analyses. We observed a 55% reduction in protein body size; however, since the protein bodies in spacedeveloped seed were filled and we did not observe any other signs of hypoxia such as degeneration of the embryos, deposition of starch grains or alterations in cell structures or cell numbers, we conclude that the aerial portions of the plant were not starved for oxygen. The high forced airflow rates (2–3 m/s) and accompanying ethylene removal provided by the growth chamber improved growing conditions for the aerial part of the plants when compared to the previous studies [9, 33, 39].

Root zone hypoxia could explain the reduced seed protein content. ADVASC uses passive airflow to move air through the root tray. Root zone hypoxia has been prevalent in space flight experiments [58, 59]. We used our own mix of porous arcillite matrix that is one of the favored rooting systems for space [60]. Arcillite reduces root zone hypoxia by allowing air to penetrate between the arcillite grains. Nonetheless, air movement through arcillite is restricted, especially if the spaces between arcillite grains are filled with roots, water, or both. If passive

Approximately 80% of the roots formed a dense mat in the top 13 mm of arcillite, while the roots of the ground control plants penetrated deeply throughout the root tray. Evapotranspiration data showed that the porous tubes in the growth chamber delivered an average of 110 mL/d of aerated water during the major growth portion of the experiment. There was not enough oxygen in this amount of water to meet the physiological needs of the roots [58]. In the absence of moisture sensor in the root tray, we had no way of knowing the relative moisture level in the root tray. An anoxic root zone in space resembles an environment similar to flooded soil on earth. Anoxia reduces nitrogen uptake by the roots therefore seed protein content is reduced. On Earth, applying fertilizer to flooded plants improves seed protein content. Because our growth chamber used an artificial soil with no native nutrient value, the plants were fertilized four times during the experiment. This may explain how the plants

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This was the first report of altered branch and silique angles for space-grown plants. The reduced branch angles and perpendicular growth of the siliques in space appear to be true microgravity phenotypes. The branching pattern seen in the first spaceflight experiment [10] was replicated during the second spaceflight experiment [11], indicating that this phenotype is persistent in Arabidopsis development on long duration space flights. Light plays a principle role in the "upright" or light-seeking growth habit of the primary axis of many plants, and is responsible for houseplants curving towards the nearest window. On Earth, this response interacts with negative gravitropism in the shoot and requires that shoot gravitropism experiments be conducted in the dark [61]. In our spaceflight experiments the primary axis of Arabidopsis always grew towards the light source, supporting a central role for light in the orientation of the primary axis. The reduced branch angles and tendency of the branches to ignore or curve away from the light source in space shows that gravity plays the key role in signaling branches to curve upwards on Earth. The reduced angles that the siliques made with the stems also show that gravity has a direct role in determining the silique angles. Since Arabidopsis branches do not naturally curve towards the light in microgravity, light plays either a negative or a secondary role in the branch form. Spaceflight appears to initiate cellular remodeling throughout the plant, yet specific strategies of the response are distinct among specific organs of the plant. In the absence of gravity plants rely on other environmental cues to initiate the morphological responses essential to successful growth and development; the basis for that engagement lies in the differential expression of genes in an organ-specific manner [27]. We also conducted the first ever transcriptional profiling of higher plants fully grown in microgravity [11]. The gene expression data were suggestive of the presence of an abiotic stress response. However, we cautioned with respect to deriving conclusions from our gene expression profiling study, because the observed expression patterns may be at least in part induced by other interacting suboptimal environmental conditions, e.g., an anoxic root zone in space. During the second seed-to-seed experiment on the ISS (that provided plants used for transcriptional profiling), technical issues interfered with the priming of the growth chamber and its transition into steady state [11]. These may have contributed to the observed gene expression patterns.

While Arabidopsis plants grown in microgravity may have shown some signs of root zone hypoxia, the ADVASC growth chamber in general provided a very good environment for

achieved only 82% of the normal protein content in the seed [10, 11].

Figure 2. First plant life cycle experiment on-board the International Space Station, showing Arabidopsis thaliana retrieved from the ISS; these were grown in the period between ISS 6A–ISS 7A missions.

Figure 3. Expedite the Processing of Experiments to the Space Station (EXPRESS) rack 1 on the ISS is pictured on-orbit May 14, 2001, with astronaut James Voss checking ADVASC functioning. Image credit: NASA/JSC. The EXPRESS rack is a multipurpose rack system that houses and supports research aboard the space station.

airflow through the arcillite is cut off then oxygen can only reach the roots by diffusion from the air above the soil, or by the arrival of oxygenated water. Diffusion rates are negligible when the diffusion distances are more than a few millimeters [60].

Approximately 80% of the roots formed a dense mat in the top 13 mm of arcillite, while the roots of the ground control plants penetrated deeply throughout the root tray. Evapotranspiration data showed that the porous tubes in the growth chamber delivered an average of 110 mL/d of aerated water during the major growth portion of the experiment. There was not enough oxygen in this amount of water to meet the physiological needs of the roots [58]. In the absence of moisture sensor in the root tray, we had no way of knowing the relative moisture level in the root tray. An anoxic root zone in space resembles an environment similar to flooded soil on earth. Anoxia reduces nitrogen uptake by the roots therefore seed protein content is reduced. On Earth, applying fertilizer to flooded plants improves seed protein content. Because our growth chamber used an artificial soil with no native nutrient value, the plants were fertilized four times during the experiment. This may explain how the plants achieved only 82% of the normal protein content in the seed [10, 11].

This was the first report of altered branch and silique angles for space-grown plants. The reduced branch angles and perpendicular growth of the siliques in space appear to be true microgravity phenotypes. The branching pattern seen in the first spaceflight experiment [10] was replicated during the second spaceflight experiment [11], indicating that this phenotype is persistent in Arabidopsis development on long duration space flights. Light plays a principle role in the "upright" or light-seeking growth habit of the primary axis of many plants, and is responsible for houseplants curving towards the nearest window. On Earth, this response interacts with negative gravitropism in the shoot and requires that shoot gravitropism experiments be conducted in the dark [61]. In our spaceflight experiments the primary axis of Arabidopsis always grew towards the light source, supporting a central role for light in the orientation of the primary axis. The reduced branch angles and tendency of the branches to ignore or curve away from the light source in space shows that gravity plays the key role in signaling branches to curve upwards on Earth. The reduced angles that the siliques made with the stems also show that gravity has a direct role in determining the silique angles. Since Arabidopsis branches do not naturally curve towards the light in microgravity, light plays either a negative or a secondary role in the branch form. Spaceflight appears to initiate cellular remodeling throughout the plant, yet specific strategies of the response are distinct among specific organs of the plant. In the absence of gravity plants rely on other environmental cues to initiate the morphological responses essential to successful growth and development; the basis for that engagement lies in the differential expression of genes in an organ-specific manner [27].

We also conducted the first ever transcriptional profiling of higher plants fully grown in microgravity [11]. The gene expression data were suggestive of the presence of an abiotic stress response. However, we cautioned with respect to deriving conclusions from our gene expression profiling study, because the observed expression patterns may be at least in part induced by other interacting suboptimal environmental conditions, e.g., an anoxic root zone in space. During the second seed-to-seed experiment on the ISS (that provided plants used for transcriptional profiling), technical issues interfered with the priming of the growth chamber and its transition into steady state [11]. These may have contributed to the observed gene expression patterns.

While Arabidopsis plants grown in microgravity may have shown some signs of root zone hypoxia, the ADVASC growth chamber in general provided a very good environment for

airflow through the arcillite is cut off then oxygen can only reach the roots by diffusion from the air above the soil, or by the arrival of oxygenated water. Diffusion rates are negligible when

Figure 3. Expedite the Processing of Experiments to the Space Station (EXPRESS) rack 1 on the ISS is pictured on-orbit May 14, 2001, with astronaut James Voss checking ADVASC functioning. Image credit: NASA/JSC. The EXPRESS rack is a

Figure 2. First plant life cycle experiment on-board the International Space Station, showing Arabidopsis thaliana retrieved

the diffusion distances are more than a few millimeters [60].

multipurpose rack system that houses and supports research aboard the space station.

from the ISS; these were grown in the period between ISS 6A–ISS 7A missions.

162 Into Space - A Journey of How Humans Adapt and Live in Microgravity

growing plants on the ISS, and successfully eliminated most of the problems seen in previous plant spaceflight experiments, allowing us to discover alterations in plant form and architecture. We were thus able to successfully grow two consecutive generations of Arabidopsis thaliana in space, i.e., seed-to-seed-to-seed. Future experiments should be conducted to see if these alterations can be generalized across different species of plants. As well, future designs of space growth chambers (e.g., the Vegetable Production System [43] and the Advanced Plant Habitat [45]) should consider improving the root zone aeration to prevent root zone hypoxia.

Finally, it is not far beyond the realms of possibility that selected plant species can be genetically engineered and remotely controlled to provide food, clean air, and potable water, while at the same time acting as a source of raw materials and as small pharmaceutical factories, many miles away from Earth. Such "programmable plants" could uniquely support human missions in space by receiving and responding to remote signals for the synthesis of compounds needed

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I would like to pay special thanks to those who risk their lives to advance our understanding of both Earth and space. In particular, thanks to the several astronaut crews for taking care of our experiments while onboard the ISS and on the Space Shuttle. In addition, I would like to thank the team of dedicated engineers and scientists at the Wisconsin Center for Space Automation and Robotics, University of Wisconsin-Madison, who devoted their time, energy, and creativity to spaceflight experiments on the Space Shuttle and the ISS. Funding was generously

Bratislav Stankovic contributed to this article in his personal capacity; the views expressed are

[1] Darrin A, O'Leary BL, editors. Handbook of Space Engineering, Archaeology, and Heri-

[2] Stankovic B. 2001: A plant space odyssey. Trends in Plant Science. 2002;6:591-593. DOI:

his own and do not necessarily represent the views of the USPTO or the United States

yet unavailable off-the-shelf in deep space [6].

Acknowledgements

provided by NASA.

Conflict of interest

Author details

Bratislav Stankovic†

†

Government

References

The author declares no conflict of interest.

Address all correspondence to: bratislav.stankovic@fulbrightmail.org

United States Patent and Trademark Office, Alexandria, VA, USA

tage. Boca Raton: CRC Press; 2009. 1035 p

http://dx.doi.org/10.1016/S1360-1385(01)02158-6

#### 4. Prospects

This is a very exciting time for space science, as the search for extraterrestrial life is one of the great intellectual enterprises of our species. At the same time, better understanding of the profound biodiversity and adaptability of life on Earth is part of the same continuum. Results from the performed space experiments were previously plagues by inconclusiveness due to the small number of experiments, small number of replicates, use of diverse flight hardware, growth conditions, limited possibilities for tissue preservation and subsequent analysis, etc. Future space experiments should therefore have standardized conditions for plant growth [3, 62]. Plus, it is the one area of space science in which you get to eat your experiment.

The theme of agriculture for space has contributed to, and benefited from, terrestrial, controlled environment agriculture; it will continue to do so into the future. The ISS ability to provide an opportunity for direct comparison of microgravity vs. 1 g (in on-board centrifuge) conditions, and for on-the-spot modification to the experiment conditions, create unprecedented advantages for plant space biology investigators. This is particularly helpful when investigators are surprised after taking a well-understood experiment on Earth and attempting to reproduce it on the ISS.

Understanding gene and protein expression is the key to unlocking the mechanisms behind microgravity-induced problems, and to finding effective countermeasures to spaceflightinduced phenotype alterations. Even though large-scale tests on growing crops for food production in microgravity are lacking, the body of acquired knowledge that there is little impediment to growing plants in microgravity, in outer space, and on other planets; even if the plants do experience some level of genotoxic stress and anatomic changes [49]. As human space exploration continues to advance, we should feel confident about our ability to grow plants on the Moon, on other planets, and on board spacecraft during long-term space missions. We still need to investigate how plants deal long-term with cosmic radiation and with the soils of other planets. We do, however, know that plants require specialized environments for growth and development in microgravity, including efficient watering and nutrient-delivery systems, precise environmental controls for temperature, humidity and air composition, and lowenergy lighting. We already known how to produce such specialized growth chambers and greenhouses; we could design light absorption systems that take advantage of sunlight on the surface of planets and moons, to help us more efficiently grow plants in them.

Finally, it is not far beyond the realms of possibility that selected plant species can be genetically engineered and remotely controlled to provide food, clean air, and potable water, while at the same time acting as a source of raw materials and as small pharmaceutical factories, many miles away from Earth. Such "programmable plants" could uniquely support human missions in space by receiving and responding to remote signals for the synthesis of compounds needed yet unavailable off-the-shelf in deep space [6].

### Acknowledgements

growing plants on the ISS, and successfully eliminated most of the problems seen in previous plant spaceflight experiments, allowing us to discover alterations in plant form and architecture. We were thus able to successfully grow two consecutive generations of Arabidopsis thaliana in space, i.e., seed-to-seed-to-seed. Future experiments should be conducted to see if these alterations can be generalized across different species of plants. As well, future designs of space growth chambers (e.g., the Vegetable Production System [43] and the Advanced Plant Habitat [45]) should consider improving the root zone aeration to prevent root zone hypoxia.

164 Into Space - A Journey of How Humans Adapt and Live in Microgravity

This is a very exciting time for space science, as the search for extraterrestrial life is one of the great intellectual enterprises of our species. At the same time, better understanding of the profound biodiversity and adaptability of life on Earth is part of the same continuum. Results from the performed space experiments were previously plagues by inconclusiveness due to the small number of experiments, small number of replicates, use of diverse flight hardware, growth conditions, limited possibilities for tissue preservation and subsequent analysis, etc. Future space experiments should therefore have standardized conditions for plant growth [3, 62]. Plus, it is the one area of space science in which you get to

The theme of agriculture for space has contributed to, and benefited from, terrestrial, controlled environment agriculture; it will continue to do so into the future. The ISS ability to provide an opportunity for direct comparison of microgravity vs. 1 g (in on-board centrifuge) conditions, and for on-the-spot modification to the experiment conditions, create unprecedented advantages for plant space biology investigators. This is particularly helpful when investigators are surprised after taking a well-understood experiment on Earth and attempting

Understanding gene and protein expression is the key to unlocking the mechanisms behind microgravity-induced problems, and to finding effective countermeasures to spaceflightinduced phenotype alterations. Even though large-scale tests on growing crops for food production in microgravity are lacking, the body of acquired knowledge that there is little impediment to growing plants in microgravity, in outer space, and on other planets; even if the plants do experience some level of genotoxic stress and anatomic changes [49]. As human space exploration continues to advance, we should feel confident about our ability to grow plants on the Moon, on other planets, and on board spacecraft during long-term space missions. We still need to investigate how plants deal long-term with cosmic radiation and with the soils of other planets. We do, however, know that plants require specialized environments for growth and development in microgravity, including efficient watering and nutrient-delivery systems, precise environmental controls for temperature, humidity and air composition, and lowenergy lighting. We already known how to produce such specialized growth chambers and greenhouses; we could design light absorption systems that take advantage of sunlight on the

surface of planets and moons, to help us more efficiently grow plants in them.

4. Prospects

eat your experiment.

to reproduce it on the ISS.

I would like to pay special thanks to those who risk their lives to advance our understanding of both Earth and space. In particular, thanks to the several astronaut crews for taking care of our experiments while onboard the ISS and on the Space Shuttle. In addition, I would like to thank the team of dedicated engineers and scientists at the Wisconsin Center for Space Automation and Robotics, University of Wisconsin-Madison, who devoted their time, energy, and creativity to spaceflight experiments on the Space Shuttle and the ISS. Funding was generously provided by NASA.
