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Programmed cell death, also known as apoptosis, is a biological process occurring in multicellular organisms (Riwaldt et al., 2017). Apoptosis is necessary to maintain the body balance and to remove cells during the organ development phase. It is involved in morphogenesis, the elimination of neoplastic cells or virus-infected cells. Furthermore, it plays a key role in myocardial infarction, cerebral ischemia, infections, neurodegenerative disorders, organ and bone marrow transplant rejection, autoimmune diseases and, among others, in cancer chemotherapy or irradiation (Schoenberger et al., 2008).

In addition, apoptosis is an important process involved in the early phases of wound healing (Greenhalgh, 1998; Wu and Chen, 2014). In normal wound healing, programmed cell death is necessary for removing inflammatory cells and afterwards for scar formation. This removal occurs without tissue damage or inflammation.

Furthermore, conditions of real and simulated microgravity can influence cell survival and increase the amount of apoptosis in different cell types and tissues (Zhao et al., 2018; Jiang et al., 2020; Mao et al., 2020; Pan et al., 2020; Sokolovskaya et al., 2020). The conquest of space is very important to us humans. In the course of the expanded exploration of space, combined with the settlement of the Moon, the installation of new Space Stations as well as planned trips to Mars and other planets, more thought must also be given to the treatment of space travelers (Patel et al., 2020). There will be an increase in crew members and space travelers on board together with more on-board and extravehicular activities. Injuries and burns are a very important focal point, an important aspect in surviving a trauma in space is the sufficient wound healing. Therefore, we need profound knowledge of the healing process and suture behavior in space. Moreover, medical evacuation times to Earth are very lengthy or not possible at all so that medical care in space has to be improved with respect to emergency surgery, treatment of acute wounds and burns on spacecrafts or space stations.

The changes of wound healing in space and the influence of microgravity on wounds need to be clarified, and the influence of apoptosis on wound healing in space is of particular interest. We will discuss the current knowledge about (1) wound healing in space; (2) alterations in healing mechanisms affected by microgravity with respect to apoptosis; and (3) possible therapeutic strategies of wound healing in space.

This review provides an overview of real and simulated microgravity research platforms. Models to study wound repair under microgravity conditions will be discussed. It summarizes the current knowledge of the impact of microgravity on the different cell types involved in wound healing with respect to programmed cell death. We will review recent findings in keratinocytes, lymphocytes, macrophages, endothelial cells, dermal fibroblasts and adipocytes. Finally, this comprehensive review reports the influence of the space environment on the biological process of apoptosis in wound healing and evaluates the new therapies to improve wound repair onboard.

Wounds are a break in the continuity of the skin, a “disruption of normal anatomic structures and functions” (Lazarus et al., 1994; Robson et al., 2001; Chhabra et al., 2017). The skin is a complex, multi-functional organ: it prevents fluid loss, stabilizes body temperature, and relays sensory inputs. In addition, it harbors a highly specialized immunological niche crucial for the maintenance of tissue homeostasis, defense, and repair (Nguyen and Soulika, 2019). Wound healing is a complex and dynamic process that is affected by wound environment and the general health and immune status of the host (Chhabra et al., 2017). Wounds can be classified in a number of different ways, depending on the type of treatment setting. For example, in a trauma setting wounds are defined based on the following factors:

Programmed cell death or apoptosis is a biological process responsible for normal cell turnover in various organs. An example is the apoptosis of lymphocytes in the tonsil. In case of a chronic tonsillitis, the relationship between apoptosis and proliferation of lymphocytes in tonsillar follicles can be disturbed (Avramović et al., 2015). Inappropriate apoptosis is a key factor in various diseases including cancer, ischemia of the heart and brain, autoimmune or neurodegenerative diseases (Elmore, 2007; Schoenberger et al., 2008). In addition, programmed cell death has important functions on growth, differentiation, morphogenesis, organ development and tissue homeostasis. This type of cell death is able to modulate life and death of cells and exhibits a known therapeutic potential in cancer (Schoenberger et al., 2008; Grimm et al., 2011).

Apoptosis shows characteristic morphological cellular changes such as cell shrinkage, chromatin condensation, membrane blebbing and apoptotic bodies without inflammation (Kerr et al., 1972; Böhm and Schild, 2003; Kossmehl et al., 2003). The morphological changes of programmed cell death were first described by Kerr et al. (1972). These changes occur as follows: The first step is cell shrinkage, followed by a stop of adhesion to the neighboring cells. The cell membranes and the organelles of the apoptotic cells remain intact. This step is followed by chromatin condensation, which is also called pyknosis. Membrane blebbing on the cellular membrane occurs and the nucleus ruptures into fragments, a process called karyorrhexis (Kossmehl et al., 2003). Cytochrome c is released after rupture of the mitochondrial membrane, which activates a cascade of degradation reactions resulting in the secretion of lysosomal enzymes. The result of the enzymatic activity is the formation of small cell particles completely enclosed by cell membranes containing cell fragments. These particles are called apoptotic bodies. The cellular fragments are cleared quickly by phagocytosis without an inflammatory reaction (Taylor et al., 2008).

Apoptosis comprises a complex mechanism with specifically interacting pro-apoptotic and anti-apoptotic factors. It can be initiated by external signals via death receptors. This very well-defined pathway is called extrinsic pathway of apoptosis (EPA). These death receptors comprise FAS/CD95 and the receptors for tumor necrosis factors (TNF-R) (Ashkenazi and Dixit, 1998). The ligand-receptor binding results in the initiation of the cell death machinery. The canonical pathway of the EPA starts with the binding of the tumor necrosis factor receptor superfamily (TNFRSF) members to cognate trimeric ligands of the tumor necrosis factor superfamily (TNFSF). These ligands are either soluble or expressed on the cell surface of another cell.

FASL binding to FAS results in the trimerization of FAS. This process is followed by recruiting the initiator caspase-8 via the FAS-associated death domain protein (FADD). Then the plasma membrane-associated death-inducing signaling complex (DISC) converts procaspase-8 into the active form of caspase-8 (Yang, 2015; Siegmund et al., 2017). The next step is the activation of the effector caspase-3 (Siegmund et al., 2017). Caspase-3 is a cysteine–aspartic acid protease, which is known to cleave various targets and to initiate cell death.

Activated caspase-8 induces apoptosis first by the activation of caspase-3 and second by cleavage of BH3 interacting-domain death agonist (BID), a pro-apoptotic Bcl-2 family protein. The cleaved BID now termed truncated Bid (tBID) translocates to the mitochondria. Afterwards it induces the release of cytochrome c, which is a key protein that initiates the intrinsic pathway of apoptosis (IPA). Cytochrome c is normally localized in the compartment between the inner and outer mitochondrial membranes and its release results in the activation of caspase-9 and−3 (Taylor et al., 2008). The apoptotic protease activating factor 1 (Apaf-1) is involved in the cytochrome-c-dependent activation of caspase-3 (IPA). Mitochondrial cytochrome c is released into the cytosol and binds to Apaf-1 and thus forms the apoptosome. Apaf-1 subsequently activates the caspase cascade. Afterwards, the apoptosome recruits and activates the inactive pro-caspase-9. Active caspase-9 is an initiator caspase and activates effector caspases, which results in the activation of several steps leading to apoptosis.

Cysteine aspartases (caspases) are enzymes involved in the process of apoptosis. EPA and IPA merge at the activation of a procaspase. Apaf-1 activates procaspase-9, while Fas-associated protein with death domain (FADD) activates procaspase-8. The caspases-2,−8,−9, and−10 initiate and activate other caspases, while the caspases-3,−6, and−7 execute the process and cleave proteins at the site of an aspartate. Intracellular type I keratin and other intermediate filament destruction by these enzymes results in the characteristic morphological signs of apoptosis (Oshima, 2002).

NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) is a protein complex involved in DNA transcription, cell survival and cytokine production (Taniguchi and Karin, 2018). NF-κB exhibits various transcriptional regulatory functions and is a key player in apoptosis (Ghobrial et al., 2005) and it regulates the immune response to infections (Hayden et al., 2006). Incorrect regulation of NF-κB is among others associated with cancer and infections (Siebenlist et al., 1994), which might be important for wound healing processes in space. NF-κB is inactivated by binding to IκB (inhibitor of NF-κB). Moreover, the IκB degradation induces the translocation of NF-κB into the nucleus, where it changes transcription (Song et al., 2012) and triggers various processes like survival or detachment and aggregation of cells to three-dimensional aggregates (Kopp et al., 2018).

The transcription factor p65 (nuclear factor NF-κB p65 subunit) is a protein which is encoded by the RELA gene. The presence of the transcription factor p65 in the skin (epidermis) inhibits epithelial cell death induced by Fas ligand, tumor necrosis factor-κ, and microbes (Seitz et al., 2000).

The inhibitor of apoptosis protein (IAP) family regulates the activity of caspases, survival, and proliferation by binding to the baculovirus IAP repeat (BIR) domains (Cossu et al., 2019). Moreover, IAPs also control various pathways. The levels of X-linked IAP and other IAPs are often associated with progressive cancer and correlate with prognosis (Cossu et al., 2019). Therefore, compounds targeting the IAPs are of interest in cancer therapy (Saleem, 2013).

Today most of the key players in cellular apoptosis regulation are identified and can be targeted by therapeutic strategies. Examples are drugs targeting the EPA (FAS, tumor necrosis factor-α and tumor necrosis factor related apoptosis-inducing ligand), or compounds binding to factors of the IPA (Bcl-2 family) or Poly (ADP-ribose) polymerase (PARP) inhibitors.

Autophagy is a physiological process, which serves to degrade misfolded or damaged proteins or dysfunctional organelles such as the endoplasmic reticulum or mitochondria in a catabolic fashion and subsequently reuse some of the degradation products (Mizushima, 2007; Glick et al., 2010). Besides its function in cell maintenance, differentiation and survival, autophagy has also been implicated in the development of diseases (reviewed in Levine and Kroemer, 2008). Interestingly, it has also been shown, that elevated autophagy activity in the skin can lead to impaired cutaneous wound healing (Guo et al., 2016).

Only few studies so far have investigated the influence of microgravity on autophagy. Osteoclasts and endothelial cells are the most widely used cells types. When preosteoclasts were exposed to simulated microgravity on an RCCS for 24 h, the cells showed an elevated autophagy activity, which was linked to an increased syncytin-A expression (Ethiraj et al., 2018) and increased differentiation into osteoclasts (Sambandam et al., 2014) reported similar findings. The process of simulated microgravity-induced formation of osteoclasts could be suppressed by melatonin (Yoo et al., 2016) as well as 4-acetylantroquinonol B (Wu et al., 2020) by inhibiting the autophagy pathway. It can be speculated that counteracting autophagy in microgravity might be a suitable countermeasure for bone loss in space.

Several studies conducted on vascular endothelial cells reported an upregulation of autophagy under simulated microgravity. In contrast to the findings in osteoclasts, however, the results hinted toward a protective effect of autophagy against microgravity-induced stress (Wang et al., 2013; Li et al., 2018, 2019). Recently, it was also found that after 10 d of simulated microgravity on a rotating wall vessel, a disorganization of the actin cytoskeleton triggered autophagy/mitophagy and led to a reduction of mitochondrial content, oxygen consumption, and maximal respiratory capacity in human primary endothelial cells (Locatelli et al., 2020).

Similarly, it could be demonstrated, that simulated microgravity induces autophagy in TCam-2 seminoma cells (Ferranti et al., 2014) as well as human Hodgkin's lymphoma cells (Jeong et al., 2018), pointing toward a possible new target for anti-cancer therapy.

The term microgravity in general refers to the still existing residual accelerations. When gravitation is the only force acting on an object then the object is in free fall, and hence, it will experience microgravity. As outlined in Figure 1, there are several opportunities to expose objects to real microgravity. Most importantly, these differ in the duration of microgravity (seconds to months), but several other parameters, like quality of microgravity (10⁻²-10⁻⁶ g), availability, experiment series (daily to 1 per several years), preparation time (month to years), cost, hardware accessibility (hours to months), masses of payload, and degree of automatization change dramatically between the platforms (Sabbatini, 2014; Hemmersbach et al., 2018; Amselem, 2019; Prasad et al., 2020a,b). On this basis, the available microgravity platforms will be described briefly in this chapter.

The most common way to conduct experiments in real microgravity is to use drop towers (or shaft towers or drop wells), which are ground-based research facilities. The duration of the microgravity time provided by operational drop towers is relatively short—3.5–4.7 s depending on the height of the tower and technical facilities. In the case of the 146 m tall ZARM drop tower in Bremen, Germany (see Figure 1a), the experimental time can be extended to 9.3 s due to a clever catapult system. The biggest microgravity drop well facility in the world, JAMIC, is 710 m deep (underground) and located in Japan. It can provide microgravity for the duration of 10 s, although it is currently not operational. The NASA Glenn shaft and Beijing drop tower provide 5.2 s and 3.6 s of microgravity, respectively (Zhang, 2005; Könemann et al., 2015). The experimental setup is installed into an airtight capsule which is released in a tube within the tower. To eliminate the effect of friction forces and drag most drop tower experiments are performed in an air free (vacuum) tube. After the free fall the capsule is exposed to substantial deceleration phases during the dampened impact. To double the microgravity time the capsule can be catapulted upward from the bottom of the tube, by which the experiment is exposed to a high acceleration event, followed by a free fall. Among the available drop towers, the facility at ZARM provides the best microgravity conditions of ~10⁻⁶ g (Selig et al., 2010; Corydon et al., 2016). Drop towers provide several advantages including low-cost access to research in microgravity conditions, high flexibility, high payload masses (up to 265 kg), good availability, hardware accessibility a few hours before and after drop, short experiment planning phase, fast turn-around time, they allow the execution of a series of experiments within a few days with direct intervention by research teams to introduce modifications between drops, and vibrations during microgravity are very low. As a consequence of the short microgravity time and the setup of drop towers, experiment packages must work fully autonomously.

Parabolic flights can be considered as another stepping stone to space (Sabbatini, 2014). Parabolic flights are conducted in a customized aircraft and provide 22 s of microgravity time (Figures 1b,c). To produce a free fall period (microgravity) the aircraft is flying parabolic trajectories consisting of three phases: From steady flight, the aircraft climbs at a gradually steeper angle until 50° is reached. In this first phase, the experiments as well as the personal onboard the aircraft will experience hypergravity in the range of 1.8–2 g (Ma et al., 2014; Pletser et al., 2016). Next, the pilot reduces the thrust to compensate for drag and the aircraft will follow a ballistic trajectory, also known as a parabola. In this second phase, the aircraft is in free fall and the experiments will experience microgravity in the range of ~10⁻² g (Corydon et al., 2016). At the apex of the parabola, the aircraft nosedives until a decline angle of ~42° is reached, thereby entering the third and last phase of the parabola, in which the airplane again experiences hypergravity (1.8–2 g). Finally, the pilot levels out the aircraft for steady flight. The free fall phase persists for 22 s, whereas the two hypergravity phases before and after the free fall phase each last for 20 s. A parabolic flight campaign provided by Novespace, Bordeaux-Merignac, France, has a duration of 3–4 d with 31 parabolas on each flight day. A huge benefit of parabolic flights is that the scientists have the opportunity to be onboard the aircraft during flights, thereby allowing them to direct hardware access during microgravity exposure. Hence, full automatization is not required. In addition, experiments involving microgravity effects on human physiology can be performed. The availability is good, half a dozen flight campaigns are annually conducted by DLR, CNES and ESA in Europe. Typically, the hardware is handed over for installation in the aircraft a few days before the first flight day, and experiments are transferred as soon as a few hours before take-off. High payload masses up to 200 kg can be accommodated by parabolic flights and only few months of experiment planning are needed. The consecutive flight days allow conduction of a series of experiments with modifications between the flights. Hypergravity and vibrations are unavoidable events of parabolic flights, which need to be taken into consideration when evaluating the impact of microgravity (Corydon et al., 2016).

A longer microgravity-exposure demands a visit to space, and suborbital spaceflights in terms of sounding rockets can be an option. For the TEXUS-type sounding rocket (DLR) the payload is in free fall for a period of 6 min (Figures 1e,f), and time spans as long as 14 min of microgravity-exposure are delivered by the MAXUS platform (ESA/DLR) (Sabbatini, 2014). In both cases microgravity below 10⁻⁴ g is obtained. The sounding rockets follow a parabolic trajectory with an apex of ~250 km (TEXUS) or 750 km (MAXUS) above the Earth's surface. The hardware is typically installed in the rocket a few days before lift-off, whereas the experiments (e.g., late access modules) are handed over 1–2 h prior to launch. Inherently, experiment packages must be fully autonomous. The payload will be subjected to hypergravity and vibrations during lift-off and landing which need to be taken into consideration (Corydon et al., 2016). Otherwise, residual accelerations are very low. Recovery of the payload after landing takes a few hours, bad weather conditions might double or triple this period. Operating costs are considerably higher compared to the previously discussed platforms.

Building a road to space is not only an endeavor for public institutions but has also, step by step, become a strategy for private companies. Blue Origin and Virgin Galactic are noticeable examples of suborbital spaceflight options delivering up to 4–6 min of microgravity (Vanderploeg et al., 2011). Key to these companies is to make access to space less expensive and more reliable through reusable launch vehicles, including development of the so-called VTVL technology (vertical take-off and vertical landing). All experiments must be fully autonomous.

Exposure that lasts up to 5 days can be obtained onboard a spacecraft during a resupply mission to the International Space Station (ISS). However, only a few studies have been reported using this rare option (Horn et al., 2007). An unmanned spacecraft can increase the duration of microgravity time. These are usually satellites, equipped with return vehicles designed to descent back to Earth (e.g., Russian Bion-M- and Foton-M 4-type or Chinese FSW-type) which can provide exposure that lasts a few weeks. These options are likewise very rare: The first Bion-M and Foton-M4 missions were launched in 2013 and 2014, respectively, and relaunches are scheduled in 2023. Experiment packages must be fully autonomous, and years of preparation time should be anticipated. Payload masses are typically low.

As China became the third country sending humans to space in 2003, scientific experiments were conducted on board the Shenzhou spaceships also. Shenzhou missions are launched on the Long March rocket 2F from the Jiuquan Satellite Launch Center.

In order to gain long-term access to real microgravity, we took advantage of the opportunity to participate in the Shenzhou-8 Spaceflight mission, accomplished by a joint endeavor of the German Space Agency and the Chinese Manned Space Engineering Office (CMSEO). Shenzhou-8 was launched by means of a Long March rocket 2F in the Cosmodrome Jiuquan on November 1, 2011 (Pietsch et al., 2013). SIMBOX (Science in Microgravity BOX) contained 17 experiments (https://www.dlr.de/rd/en/desktopdefault.aspx/tabid-2283/3420_read-32536/). Currently, manned and unmanned Chinese spacecrafts are available for scientific endeavors.

The ISS is currently the only option that provides exposure to microgravity for months or longer. It is however, also the most expensive flight option. On the other hand, there are many flight opportunities (e.g., NASA/ESA missions operated by SpaceX or Russian Soyuz missions, Figure 1g). A microgravity quality of 10⁻⁴ g is obtained. In general, experiment packages must be fully autonomous as crew members have a tight schedule. The hardware is typically handed over 1–2 d before lift-off. Experiments should be designed in a way that allows for a 3–5 day delay of the experiment start due to the flight time to the ISS. Accelerations are low during lift-off and the flight. Access to experimental data is possible during or after the experiment has been completed. Hand-over of the hardware is possible several days after return to the Earth.

Moreover, China is presently constructing a Chinese Space Station (CSS) named Tiangong, which will be approximately one-fifth the mass of the ISS. The CSS should consist of multiple modules allowing scientific experiments to be carry out onboard, and it is anticipated to be operational by 2022.

Access to space is expensive, limited and a logistic challenge. On top of this, repetitions of the experiments as well as the number of samples are severely limited (Brungs et al., 2016). To simulate microgravity on Earth a variety of ground-based alternatives have therefore been developed (Herranz et al., 2013; Brungs et al., 2016). One of the simplest devices used for small samples, like cells, is the rotating wall vessel (RWV). In this device simulated microgravity is obtained by placing the cells in a horizontal vessel rotating with a speed that cancels sedimentation (Schwarz et al., 1992). Ideally, the RWV device produces a low shear fluid environment that is optimized for tissue growth and suspension cultures (Klaus, 2001).

In an attempt to reproduce the quiescent, unstirred fluid conditions achievable in orbit, clinostats have been developed for cell culture systems. The so-called clinorotation provided by the device also seeks to simulate microgravity by canceling sedimentation. In clinostats this is accomplished by subjecting the cells to rotations, either constantly or through directional alterations. In the former case the classical two-dimensional (2D)-clinostat rotates a sample perpendicular to the gravity vector (Hemmersbach et al., 2006; Herranz et al., 2013). If samples do not exceed a radius of 1.5 mm around the rotation axis, and the speed of rotation is limited to <60 rpm, a theoretical g-force in the range of 10⁻²-10⁻³ g can be obtained (Hemmersbach et al., 2006). Even though the sample size of the 2D-clinostat platform is limited, shear forces and fluid disturbances are low (Hemmersbach et al., 2006). The 3D-clinostat uses a comparable principle but can add another dimension. Hence, the sample rotates around two axes in order to provide a status of “vector-averaged gravity,” and the generated multidirectional g-force thereby cancels the cumulative gravity vector at the center of the device (Hoson et al., 1997; Schwarzenberg et al., 1999). The quality of g-force provided by a 2D-clinostat is comparable to that of 3D-clinostats. In the latter case, randomized directional changes are delivered by a random positioning machine (RPM) (Figure 1d). During operation, the movement of the sample describes a sphere, and studies have demonstrated that at 70 mm around the rotation point the g-force is in the range of 10⁻³-10⁻⁴ g (Van Loon, 2007). Compared to the 2D clinostat the RPM clearly provides a larger sample size. However, shear forces arise, especially on the edges of the culture flasks, which need to be taken into consideration (Wuest et al., 2015, 2017).

For larger samples, e.g., mice or rats, the rodent hindlimb suspension (HS) model has been used to study various aspects of musculoskeletal loading (Simske et al., 1994; Morey-Holton and Globus, 2002). In this model the hindlimbs are elevated, unloading them from any force, to produce a 30 degrees head-down tilt. Besides simulating reduced forces on the skeleton and muscles of the hindquarters the model also results in a cephalad fluid shift (Morey-Holton and Globus, 2002). The typical duration of a HS experiments is in the range of days or a few weeks.

To simulate microgravity effects in humans the head-down bed rest (HDBR) model can be applied. In this model the subjects adhered to a strict 6° head-down tilt bed rest, and several studies have demonstrated that it is replicating the effects of spaceflights (Pavy-Le Traon et al., 2007). The HDBR model induces several physiological changes, including a cephalad fluid shift, promotes movement of abdominal organs toward the chest, reduces the hydrostatic gradient in the cardiovascular system, and results in reduced loading on the musculoskeletal system (Fortney et al., 2011). Notably, in all cases it is important to compare the results from experiments conducted under simulated microgravity with data obtained from experiments performed under real microgravity.

Several dermal cell types had been investigated in space and under conditions of simulated microgravity. Various studies focused on the influence of microgravity on programmed cell death (Figure 2). A detailed summary of these publications in respect to apoptosis detection in different dermal cell types is given in Table 1.

Soon, increased commercial spaceflights and new space exploration programs to, among others, Moon and Mars will expand the amount and duration of space missions, the number of humans in space together with on board activities and elevated extravehicular work. These activities will increase the possibility to be injured and will heighten the amount of emergency surgeries in orbit. Therefore, to improve wound healing in space is currently a hot topic.

Drudi et al. reported in 2012 about the “state of the art” in-space surgery including wound healing and sutures in space (Drudi et al., 2012). As already reviewed and discussed earlier, wound healing is a well-known long-term process starting with the injury, together with tissue damage and can last months. Effective wound repair ensures the integrity of the human body and our survival.

A journey to the Mars comprises about 50 million km and thus, a cargo resupply from Earth is not possible (Thirsk, 2020). Therefore, it is mandatory to organize the onboard facilities and supplies for wound surgery in case of accidents, burns or injuries of the space crew from a diagnostic and therapeutic point of view. Additionally, long-term exposure of space travelers to cosmic radiation can negatively influence wound repair and result in degenerative tissue diseases (Thirsk, 2020).

Onboard the spacecraft medical resources such as laboratory analysis equipment, imaging, surgery, and emergency care is mandatory (Thirsk, 2020). It is of fundamental importance, that a well-trained physician with experience in remote medicine is on board as crew member, due to the communication lag it is currently not possible to guide the medical doctor remotely. In respect to the planned Mars mission, training countermeasures need to be developed in the near future. Astronaut training courses to manage in-flight accidents on long-term missions are absolutely necessary (Robertson et al., 2020).

A study investigated wound healing and mucosal immunity during a short MARS analog mission and found that stress can have significant consequences for wound healing (Rai et al., 2012). The authors concluded that the effects of stress on wound repair can influence the recovery from surgery (Rai et al., 2012).

In general, little is known about wound healing in space, because stays on the ISS or in the past on Space Shuttles and MIR were not problematic for wound repair and could be easily managed.

Interestingly, endothelial cell culture experiments performed in real microgravity revealed alterations in endothelial cell function, changes in ECM production, and 3D growth (Versari et al., 2013; Pietsch et al., 2017; Krüger et al., 2019).

Activated T cells investigated in space on board the ISS showed an activation of genes (cell cycle check-point, oxidative stress response, heat shock proteins) or by repressing genes involved in antigen recognition (Pippia et al., 2011). Battista et al. demonstrated 5-Lipoxygenase-dependent programmed cell death of human lymphocytes in space onboard the ISS (ROALD experiment) (Battista et al., 2012). This space experiment confirmed earlier data obtained with the RPM (Maccarrone et al., 2003).

Astronauts staying for a prolonged time in space exhibit an impaired immune function. The development of acquired immunity and immune responses are disturbed (Akiyama et al., 2020). Acquired immune responses are also influenced by gravitational fluctuation, as well as stressors together with cosmic radiation (Akiyama et al., 2020). A long-term spaceflight triggered a sustained stress-dependent release of endocannabinoids. Stress is stimulating inflammation-related diseases in people at risk (Buchheim et al., 2019). These health problems are resembling some features of systemic diseases which can impair wound healing on Earth. Stress, diabetes and others can negatively influence the body's response to injury and wound healing. Therefore, it is important to increase our knowledge in this field, and to design studies focusing on wound healing in space.

The international experiment of principal investigator Professor Monica Monici and co-investigators entitled “Wound Healing and Sutures in Unloading Conditions,” selected by ESA (ILSRA-2014-0043), has the objective to investigate the behavior of in vitro sutured wound models in real microgravity on the ISS (Figure 4). Preparatory investigations and a detailed characterization of the wound healing model had been published earlier (Riwaldt et al., 2017). Importantly, no signs of apoptosis were found in the skin samples after different culture conditions and culture duration times. The findings of the project ILSRA-2014-0043 are expected to increase our knowledge on wound healing and sutures in space. The project will help to develop novel strategies for tissue healing and management of defective healing in space and on Earth. The performed techniques and newly defined analytical protocols demonstrated to be applicable for post-flight studies on skin samples after return of the future space mission (Riwaldt et al., 2017). The principal aim of this project is to enlarge our current knowledge in wound healing in space leading to novel strategies for wound repair, tissue regeneration and engineering not only for humans in space but also for patients on Earth.

Wound healing is a continuous process over an extended stretch of time, a complicated and multifaceted repair procedure involving various changing cell types throughout the progression of the process. To recap from chapter 2.3, wound healing is categorized into four phases: the homeostasis and coagulation phase starts immediately after injury, followed promptly by the inflammation phase. Thereafter, the migration and proliferation phase sets in until lastly, new skin is developed during the remodeling phase (Hunt et al., 2000; Guo and Dipietro, 2010; Reinke and Sorg, 2012).

During the first phase, the wound healing cascades are instigated, with vascular constriction and fibrin clotting to stop bleeding and form a provisional wound matrix triggered by platelets, with support from leukocytes, keratinocytes, fibroblasts and endothelial cells. During this process, platelets and leukocytes deposit pro-inflammatory cytokines and growth factors (Reinke and Sorg, 2012).

While these pro-inflammatory factors attract immune cells as neutrophils and macrophages, growth factors TGF-β1, platelet derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) stimulate tissue growth (Gosain and DiPietro, 2004; Broughton et al., 2006). Neutrophils amplify the inflammatory response, degrade necrotic cells and prevent bacterial infection (Eming et al., 2007). Macrophages are responsible for the host defense by clearing the site of pathogens and apoptotic cells within the wound, additionally they contribute to the initiation of proliferation and angiogenesis (Mosser and Edwards, 2008; Koh and DiPietro, 2011).

The most prominent cell types during the proliferative phase are fibroblasts and epithelial cells. Within the wound, fibroblasts immigrate along the fibrin network and produce extracellular matrix components like collagen, proteoglycans, glycosamyloglycans, to name a few, and so promote vascularization and filling of the wound cavity (DiPietro and Polverini, 1993; Gosain and DiPietro, 2004). The formation of new vessel in the wound filling is a complex order of events, initiated by growth factors like VEGF, PDGF, and then serine protease thrombin. These factors activate the endothelial cells of existing vessels and initiate the re-vascularization via sprouting (Reinke and Sorg, 2012).

During the last process of wound healing, the proliferation and remodeling phase, a flurry of various cell types are recruited to the close the wound. The main drivers of this stage are fibroblasts who produce the majority of materials necessary such as e.g., collagen end extracellular matrix components (hyaluronic acid, proteoglycans, glycosaminoglycans, fibronectin). In this way they form the scaffolding for cell adhesion during the tissue repair which organizes the growth and movement of the involved cells (Eckes et al., 2010; Barker, 2011). However, it should be mentioned that during the transition between the different repair phases and in order to finalize the process, cells from previous stages that are not required any longer are consistently eliminated via apoptosis throughout the entire process (Greenhalgh, 1998; Cialdai et al., 2017). An investigation in preparation to wound healing experiments during spaceflight revealed that in skin tissue cultures the healing process did not result in excessive apoptosis or necrosis after 10 d of incubation under 1 g conditions (Riwaldt et al., 2017).

The space environment subjects the human organism to drastic gravitational changes compared to terrestrial conditions. The lack of mechanical forces in microgravity settings causes widespread impact on the human body. Spaceflight instigates not only muscle and bone loss, immune system impairment and cardiac problems, but also negatively affects normal regenerative tissue repair and wound healing (Blaber et al., 2014; Paulsen et al., 2015; Cialdai et al., 2017). The underlying physiological changes can be described on an organic level as well as changes in protein and gene expression in the respective cell types (Blaber et al., 2014).

Cialdai et al. showed in detail how cell behavior adapts to simulated microgravity, particularly in would healing (Cialdai et al., 2017). Since fibroblasts are drivers of tissue regeneration, the focus on this study was on put on the changes of these cells after exposure to modeled microgravity. Due to a thorough rearrangement in the cytoskeleton, the adherence and migration ability of fibroblasts in wounds was significantly impaired. These findings are supported by the investigation of Davidson et al. who examined wound healing in rats sent in orbit. The animals exposed to real microgravity form 60% less collagen in an injury site compared to ground controls, hinting that wound healing in orbit is delayed (Davidson et al., 1999). Similarly, Infanger et al. showed that simulated microgravity resulted in an increase in ECM components and alteration of cytoskeletal structures in human EA.hy926 cells, moreover apoptosis increased after only 4 h of microgravity conditions (Infanger et al., 2006). In addition to the effects of the change in mechanical forces in microgravity, space radiation is another influencing factor. DNA damage due to ionizing radiation leads to increased DNA repair, cell cycle arrest and apoptosis in various cell types (Prasad et al., 2020a).

Under normal gravity, the deposition of ECM is mainly found during the proliferation phase of wound healing when fibroblast and endothelial cell proliferation peaks. Endothelial cells though were found to exhibit increased apoptosis during simulated as well as real microgravity conditions. After only 72 h of simulated microgravity conditions downregulation of the PI3K/Akt pathway and increased expression of NF-κB could be demonstrated, which may explain another aspect of the delay in healing (Kang et al., 2011).

In another in vivo experiment Fisher 344 rats were investigated to test wound healing responses in the orbiting Space Shuttle for 10 d (Davidson et al., 1999). The results of this study suggested that the spaceflight retarded the wound healing process and the response to exogenous stimuli.

This chapter reports about the current knowledge about wound healing studies using microgravity simulators like the RPM, 3D clinostat or the RCCS.

Microgravity may influence the process of wound healing and its progression both by changing the tissue response, apoptosis or the suture behavior. In the past, several publications showed that conditions of microgravity impaired and delayed wound repair. Until today the underlying mechanisms for an imperfect wound repair with scarring in space are not clear.

In chapter 6, we had summarized the current knowledge about various cell types active in wound repair but when exposed to microgravity several of them can become apoptotic which might result in a delay and an imperfect wound repair.

Human lymphocytes were activated and proliferate in space and on the 3D clinostat (Cogoli and Cogoli-Greuter, 1997). In free-floating cells (lymphocytes) the mitogenic response is depressed by 90% in microgravity. Maccarrone et al. showed that human lymphocytes exposed to a clinostat exhibited apoptosis by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release (Maccarrone et al., 2003). These findings might influence the early phases of wound healing in space.

Another cell type of the dermis which is affected by microgravity are endothelial cells. There are several reports that endothelial cells exposed to simulated microgravity can become apoptotic and show upregulations of apoptosis signaling, downregulation of the PI3K/Akt pathway, increased expression of NF-κB, depolymerization of F-actin and that miR-503-5p can induce apoptosis of human pulmonary microvascular endothelial cells through, at least in part, inhibiting the expression of Bcl-2 (Morbidelli et al., 2005; Infanger et al., 2006; Kang et al., 2011; Maier et al., 2015; Xu et al., 2018; Tang et al., 2019; Zhao et al., 2021). In addition, clinorotation for 48 h activated autophagy in vascular endothelial cells (Wang et al., 2013).

Fibroblasts are key players in wound repair and are mechanosensitive cells (Gabbiani, 2003) and are able to produce extracellular matrix proteins (Buken et al., 2019). It is well-known that cells exposed to simulated microgravity created by an RPM show changes in the cytoskeleton and the extracellular matrix (Buken et al., 2019). Earlier studies (Morbidelli et al., 2005; Monici et al., 2011) showed that cells exposed to simulated microgravity reveal changes in extracellular matrix proteins and endothelial function, which might affect the edema behavior and tissue stiffness in wound healing. Fibroblasts exposed to a RCCS showed changes in the cytoskeleton, a reduced VEGF and elevation of COX2, responsible for an inflammatory response (Cialdai et al., 2017). Fibroblasts exposed to both, real and simulated microgravity revealed changes in their gene expression profile (Liu and Wang, 2008; Beck et al., 2012), thus suggesting the hypothesis that these alterations in fibroblast behavior, increased stress, inflammatory signals and altered gene expression pattern may be involved in disturbed wound healing in space. The findings are supported by Liu and Wang who studied normal human WI-38 fibroblasts flown on the STS-93 space shuttle mission. They reported altered key genes related to oxidative stress, DNA repair, and fatty acid oxidation in spaceflight samples (Liu and Wang, 2008). An important finding was the up-regulation of pro-apoptotic genes (Liu and Wang, 2008). The data show spaceflight induced changes in gene expression associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence, which might impact wound repair and promote scarring.

Little is known about skin tissue samples exposed to simulated microgravity. One group cultured living skin equivalents (LSEs) in a microgravity environment (NASA-designed bioreactor) for 3 days (Doolin et al., 1999). Microgravity-exposed LSEs showed nuclear and cellular hypertrophy. Finally, a study of three mice living for 91 days on the ISS reported about abnormalities in the animals' skin. The spaceflight induced skin atrophy, deregulation of their hair follicle cycle, and markedly affected the transcriptomic repertoire of the cutaneous striated muscle panniculus carnosus (Neutelings et al., 2015).

The rat HS model of microgravity mimics physical inactivity by removing weight-bearing loads from the hindlimbs and producing a systemic cephalic fluid shift. Using this in vivo microgravity model Radek and team (Radek et al., 2008) demonstrated a delay in wound healing and wound closure in cutaneous tissue with retarded epithelial cell migration across the wound bed in the tail-suspended hindlimb-unloaded rats. The authors suggested an impaired keratinocyte and endothelial cell function during the wound healing process under simulated microgravity in the rat (Radek et al., 2008).

The principal goal is to develop novel strategies (including drugs and biochemical factors, devices, protocols and techniques) which allow a long-term stay in orbit or on other planets. This approach should promote wound repair and tissue regeneration in space. Furthermore, these strategies should be transferable to clinical applications for regenerative medicine and tissue engineering.

In this chapter, we focus on drugs and growth factors as well as biofabrication to improve wound repair.

Examples for countermeasures toward a defective neoangiogenesis are proangiogenic factors like VEGF, PDGF, and other endothelial protectives which can favor healing by modulating the neoangiogenesis process (Öhnstedt et al., 2019). Several papers reported about a reduction of VEGF in skin cells exposed to microgravity. Application of VEGF164 in rat wound healing models on Earth has demonstrated cell-protective beneficial effects on reendothelialization and vascular healing after microsurgery (Infanger et al., 2005). In addition, VEGF164 application to endothelial cells exposed to modeled microgravity exerted anti-apoptotic and cell-protective on the cells (Infanger et al., 2007).

Therefore, VEGF seems to be a promising candidate to be tested in skin wound healing models. Another option to be tested is PDGF which is approved by the FDA for non-healing wounds. Becaplerim, a gel containing recombinant PDGF (the BB isoform) is a treatment availability. Furthermore, granulocyte macrophage colony stimulating factor (GM-CSF) as injection, cream or gel can be tested.

Another growth factor with beneficial effects is epidermal growth factor (EGF). rhEGF is available as a spray and was tested in a phase III double-blind, randomized, placebo-controlled trial (Park et al., 2018). As published by Park et al. “167 adult patients with chronic diabetic foot ulcers at six medical centers were randomized to receive routine wound care plus either topical spray treatment with 0.005% rhEGF (n = 82) or an equivalent volume of saline spray (n = 85) twice a day until ulcer healing or for up to 12 weeks.” The study showed that application of spray-applied rhEGF in the patients induced a faster healing velocity and higher complete healing rate regardless of HbA1c levels (Park et al., 2018).

Recently, Cialdai et al. studied the influence of gravitational unloading on wound healing and the effectiveness of platelet rich plasma (PRP) as a countermeasure (Cialdai et al., 2020).

They used a new in vivo sutured wound healing model in the leech (Hirudo medicinalis) to evaluate the effect of microgravity on the healing process and the effects of PRP. The authors found a healing delay and structural alterations in the repair tissue, which were prevented by PRP treatment. In addition, PRP was able to counteract the microgravity-induced impairment in fibroblast migration to the wound site. In addition, it contains various growth factors. This would explain the beneficial effects of PRP in wound healing in vivo. Therefore, it can be assumed that PRP is also effective to promote healing in space. In respect to PRP application in space, on the ISS, its effectiveness, stability and storage conditions in microgravity have to be evaluated. Moreover, requirements to automate direct PRP preparation during spaceflight will be evaluated.

3D bioprinting technology or biofabrication of skin using endothelial cell, dermal fibroblast, and multilayered keratinocyte layers for skin tissue are currently a hot topic (Haldar et al., 2019; Barros et al., 2020).

Furthermore, biofabrication of new tissues in space on the ISS is possible, which will help humans traveling to the Moon or Mars and prosper there for years. In collaboration with ESA, OHB System AG and Blue Horizon, a research team from Dresden Technical University has developed a 3D bioprinting method for use in space, creating new skin and bone tissue from resources that might be available to astronauts, taikonauts and cosmonauts as well as space tourists traveling to the outer universe (https://www.esa.int/Enabling_Support/Space_Engineering_Technology/Upside-down_3D-printed_skin_and_bone_for_humans_to_Mars).

Taken together, several therapeutic strategies are available for humans in space on a deep exploration mission.

In the near future, increased commercial spaceflights and space exploration programs will elevate the number of space missions. With the Artemis program, NASA will land the first woman and next man on the Moon in 2024. In addition, Russia and PR China will build a Moon station together. It is envisioned as “a complex of experimental research facilities created on the surface and/or in the orbit of the moon.” Innovative technologies for the exploration of the lunar surface will start. Afterwards, the next goal is to send astronauts to Mars.

These new activities also mean an increased number of crew members and space tourists in space and on the Moon, which is accompanied by a higher risk of traumatic events or unexpected emergencies.

In case of emergency, ISS crew members will be transported to Earth, which is not possible in outer space. There is a continuous resupply with resources and medical equipment to the ISS. In outer space, guidance of the crew members remotely is not possible. Therefore, in this respect the spacecraft has to be self-sufficient. The crew members must be capable to manage severe situations like emergency surgery, acute trauma, wounds and burns. For these reasons, the spacecraft, space stations, Moon or Mars bases should host cutting-edge medical technology like 3D bioprinting equipment, regenerative medicine capacities, surgical instruments, functioning laboratories as well as amongst other medical equipment, freezers for blood samples, cells and tissues.

An important aspect is the behavior of the wound and/or suture and its healing. In the microgravity environment in space on the ISS are wound/suture behavior and repair processes completely unknown. Apoptosis plays an important role in normal wound healing without pathologic scarring. Several cells involved in wound repair have demonstrated an increase in apoptosis, when exposed to simulated or real microgravity. Especially lymphocytes and endothelial cells cultured in space and on microgravity simulators exhibited an increased number of apoptotic cells (Maccarrone et al., 2003; Infanger et al., 2006). In contrast, dermal fibroblasts exposed to the RPM did not reveal increases in programmed cell death (Buken et al., 2019), which implicates that mainly the early phases of wound repair might be influenced by microgravity.

In vitro experiments focusing on processes involved in wound repair demonstrated abnormal migration behavior, collagen formation, enhanced inflammation and reduced cellular organization (Morbidelli et al., 2005; Monici et al., 2006; Cialdai et al., 2017). Cells exposed to real microgravity revealed changes in the gene expression pattern with pro-apoptotic signaling (Liu and Wang, 2008). Mice flown for 91 d in space exhibited signs of skin senescence with skin atrophy (Neutelings et al., 2015).

Taken these findings together, it can be assumed that wound healing in space might be delayed and defective, which needs to be proven. Therefore, space missions to the ISS with focus on wound healing models are necessary.

The spaceflight proposal to the ISS with the number ILSRA-2014-0043 (PI Professor Monica Monici, Florence, Italy) with the title “Wound healing and sutures in unloading conditions” was selected by ESA. The spaceflight experiment focuses on the influence of gravity on the behavior of sutures and wound healing on the ISS. It will clarify how suturing materials and techniques can be adapted in space and it will develop strategies to improve wound healing in space. Furthermore, the project aims to improve suturing methods and wound healing avoiding scarring on Earth.

TC compiled the Table 1 and Figure 1. DM performed the NHDF RPM experiment and the TUNEL assay. MK provided Figures 2–4. All authors reviewed and evaluated the literature, wrote the article, contributed to the article, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.