Validation of human ex-vivo models for studies on wound healing process in microgravity conditions

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1. INTRODUCTION 
1.1 The wound healing process: an overview 
The wound healing process is a complex biological phenomenon involving not only the skin, 
which is the largest organ by surface on human body, but also all biological tissues. Not all 
tissues are composed of cells with a high proliferative activity, which, combined with the 
ability to connect with connective tissue structures, ensures wound repair. In fact, a 
distinction is made between labile, stable and perennial tissues, according to their 
regenerative capacities (Bizzozero, 1989). Labile tissues, consisting of actively proliferating 
cells (high presence of stem cells) are the lining epithelia, mucosecreting epithelia and 
haematopoietic cells; stable tissues, on the other hand, consist of cells that are normally 
quiescent but capable of re-starting proliferation, such as the parenchymal cells of glandular 
organs (liver, kidneys, pancreas), mesenchymal cells (fibroblasts and smooth muscle cells), 
and vascular endothelia; perennial cells are cells that once specialised abandon their cell 
cycle and their proliferative capacity, such as neurons, skeletal and cardiac striated muscle 
cells. In the central nervous system, damaged neurons are replaced by the proliferation of 
glial cells, while in the striated muscle a modest proliferative activity is maintained by 
peripheral satellite cells (stem cells) that provide repair with the formation of a fibrous scar. 
The injury of the soft structures, i.e. the wound, is repaired through scarring, an event 
represented by the neoformation of a connective structure different from the original one, the 
scar, aimed at filling the injury due to loss of substance to restore tissue integrity. 
The wound is always composed of a margin, perilesional skin and a lesional bottom. Healing 
can take place in three different ways which differ not in the healing mechanisms involved, 
but only in the extent of the reparative phenomena: 
- Stab wounds, such as accidental or surgical wounds which are suturated, not infected and 
have sharp margins, heal 'by first intention'. Through the juxtaposition of the wound flaps, 
the loss of substance is minimised. In this way, healing takes place rather quickly, promoting 
the filling of the wound by granulation tissue, i.e. the connective structure representing 
neoangiogenesis. 
- Unsuturated wounds, which have jagged margins, e.g. burns, infected and necrotic wounds, 
heal 'by second intention'. It is a process in which the granulation tissue, which forms at the 
bottom of the wound, needs a rather long time to repair the loss of substance up to the 
surface.
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- Surgical wounds may, in the post-operative course, encounter complications (dehiscence). 
After the wound has been suturated, infections may occur that require reopening of the 
wound, adequate cleansing and re-suturation. In this case the wound heals by 'third 
intention'. 
Wound repair involves different cytotypes (immune cells, stromal cells, keratinocytes, 
fibroblasts, endothelial cells), growth factors (PDGF, EGF, TGF- β , VEGF, FGF), cytokines 
(interleukin 1 α , -1 β , -6, TNF- α ), proteases, extracellular matrix components (collagen, 
glycoproteins, proteoglycans, hyaluronic acid). All these factors characterise different stages 
of the healing process. 
 
1.2 Stages of the wound healing process 
The process of wound repair can only be partially schematised as the phases that represent it 
are closely connected and partially overlapping (Morbidelli et al., 2021). The phases of 
wound repair are: haemostasis, inflammation, proliferation, remodelling. 
The first response to a wound is vasoconstriction of the injured blood vassels and formation 
of a fibrin clot, in order to stop blood flow to prevent exsanguination and to form a 
supporting matrix for invading cells to intervene. Subsequently, inflammation takes place, 
macrophages and granulocytes become active in response to pathogenic insults. Following 
the inflammatory response, granulation tissue production, collagen deposition, angiogenesis 
and re-epithelialisation begin. 
 
 
 
 
 
 
 
 
 
 
 
Figure 1: Schematic of the wound repair process in physiological conditions: 
1) Haemostasis and Coagulation; 2) Inflammation; 3) Proliferation and Angiogenesis; 4) Remodeling 
and scar tissue formation. (From: Morbidelli et al., 2021)
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1.2.1 Haemostasis and coagulation 
Constriction of damaged blood vessels occurs in response to a contraction of vascular smooth 
muscle and local release of endothelin (vasoconstrictor) from the damaged endothelium. 
Circulating factors, released by damaged cells, regulate vasoconstriction: catecholamines, 
epinephrine, norepinephrine, and prostaglandins (Godo et al., 2017). Haemostasis can be 
characterized into primary and secondary: in primary haemostasis the activation of platelets 
occurs. Platelets are highly reactive circulating cell fragments, kept inactive precisely by the 
structural and functional integrity of the endothelium, which has anti-thrombotic properties 
producing nitric oxide, prostacyclin and heparin that prevent platelet activation. When, due to 
an injury, the ECM of the endothelium is exposed, platelets are activated : they bind to the 
ECM through their G-protein-coupled surface receptors, integrins, and glycoproteins on their 
surface; activation increases and platelets can adhere to the vessel wall and aggregate with 
each other. Platelet activation continues through remodeling of the actin cytoskeleton, which 
leads to a conformational change. Platelets become flat and develop a series of protrusions 
(filopodia and lamellipodia) that allow stable adhesion to the matrix, and thus the ability to 
seal the vessel. Thanks to these interactions, the platelet plug formation occurs. However, the 
formed plug is only able to stop bleeding for a limited period of time, as it is unstable it can 
be easily removed following trauma. In fact, increasing hypoxia and acidosis of damaged 
tissue lead to muscle relaxation resulting in bleeding. Platelets from the plug produse 
cytokines, PDGF, a growth factor which takes part in vessel wall contraction by activating 
smooth muscles mesenchymal cells, TGF- β , IGF, EGF, and von Willebrand factor (vWF), all 
essential components of the subsequent step: secondary haemostasis. This process is 
intended to permanently stop the bleeding, by the activation of the coagulation cascade 
which involves vasoconstrictor factors such as bradykinin, fibrinopeptides, serotonin, and 
thrombaxane A2. Platelets bind vWF in order to strengthen the plug, and subsequently the 
coagulation cascade by activation of Factor X present in plasma begins. The role of factor X 
(protease), also called prothrombinase, is to activate soluble prothrombin by transforming it 
into active thrombin whose ultimate purpose is to activate and thus break down fibrinogen 
into fibrins to allow the formation of a stable clot or thrombus. Factor X can be activated 
according to two pathways : the extrinsic pathway is the fastest because it involves fewer 
factors. This pathway is activated by the release of thromboplastin (also known as Factor III 
or Tissue Factor) by damaged cells, which activates Factor X. The intrinsic pathway, on the 
other hand, is slower, due to the involvement of numerous factors: it is activated by the
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Hageman Factor (Factor XII) which is activated when the blood gets in contact with the 
collagen of the ECM. Factor XII activates Factor XI, which activates Factor IX. The latter it 
associated with Factor VIII, and this allows the activation of Factor X. After activation of 
Factor X, another factor is involved: Factor XIII binds fibrin leading to the formation of a 
definitive secondary haemostatic plug or thrombus. In addition to permanently stopping 
bleeding, the thrombus also serves as a matrix for the infiltration and adhesion of other cells 
in the subsequent stages of healing. 
 
1.2.2 Inflammation 
The inflammatory phase is aimed at wound cleaning, it is a physiological condition often 
associated with erythema, plasma exudation, heat, pain and oedema, but can develop into a 
pathological condition if excessive or of prolonged duration. It begins shortly after the injury, 
it lasts a few days, and it can be induced by pathogens and tissue debris. Following the 
formation of a stable clot, vasodilation occurs mediated by the release of NO and 
inflammatory cells are recruited to the plug, by the signal of several factors: the chemokines 
released by damaged cells, H2O2 production, the increase in ROS and the increase in 
intracellular Ca
2+
, which occurs rapidly following injury. The first immune cells to intervene 
are the neutrophil leucocytes, these have antimicrobic activity and they increase the 
inflammatory response, producing chemokines and cytokines as interleukin IL-1 α , -1 β , -6 
and TNF- α (tumour necrosis factor- α ). Gradually, monocytes are recruited, which turn into 
M1 macrophages with phagocytic activity. M1 cells phagocytose pathogens and synthesise 
MMPs (matrix metalloproteases) that enable them to digest ECM and thrombus to promote 
the migration of inflammatory cells. Another role played by macrophages is efferocytosis, 
i.e. the elimination of exhausted neutrophils at the end of the inflammatory phase, so defects 
in this process can lead to a prolonged inflammatory state and non-specific tissue degradation 
(Bratton et al., 2011). 
 
1.2.3 Proliferation and Angiogenesis 
The proliferation phase of the wound healing is represented by events such as granulation 
tissue formation, collagen deposition, angiogenesis and subsequent re-epithelialisation 
(Morbidelli et al., 2021). Upon resolution of inflammation, the M1 phenotype shifts into M2, 
a pro-vascular phenotype. M2 macrophages release growth factors such as TGF- β , PDGF, 
VEGF and FGF, i.e. pro-angiogenic factors that will activate local endothelial cells to initiate 
the angiogenic process.
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In the proliferation phase, which lasts for several days, connective tissue cells (fibroblasts 
and myofibroblasts), endothelial and epithelial cells begin to proliferate and differentiate. 
This leads to the formation of granulation tissue consisting of fibroblasts, inflammatory 
cells and blood vessel sketches in a collagenous matrix, whose purpose is to replace the fibrin 
clot with a definitive structure. Thus, in the proliferative phase, macrophages on the one hand 
induce pro-angiogenic factors and on the other stimulate local fibroblasts to proliferate and 
invade the wound by synthesising matrix proteins such as collagen, fibronectin, laminin and 
MMPs. An alteration in the number of macrophages may be associated with fibrotic diseases 
(hypertrophic scars). 
Angiogenesis is the formation of new vessels from pre-existing blood vessels that branch out 
in a gemmation phenomenon. This process accompanies physiological developmental events 
such as vasculogenesis (vessel formation in the embryo), adaptation events (wound healing, 
fracture repair, post-ischemic revascularisation), but also pathological events, i.e. tumour 
growth (angiogenesis proves necessary for metastases). Angiogenesis would occur through 
two mechanisms: germinative angiogenesis and intussusception or non-germinative 
angiogenesis. According to the latter mechanism, the protrusions of the endothelium of 
opposing vessel walls extend towards each other until they fuse. The main mechanism of 
neovascularisation, however, is germinative angiogenesis, which occurs by migration and 
proliferation of endothelial cells under the action of pro-angiogenic factors. 
The condition of hypoxia occurring at the site of the wound (due to interruption of blood 
flow) stimulates the production of angiogenic factors as the formation of new vessels is 
necessary to maintain the granulation tissue and provide oxygen and the necessary nutrients. 
In detail, hypoxia leads to up-regulation of the transcription factor HIF1- α which binds to 
HRE (hypoxia regulatory element) leading to up-regulation of the angiogenic factors VEGF, 
FGF and PDGF. These molecules bind to their receptors on the surface of endothelial cells 
of the existing vessels, thus triggering signals for survival, proliferation, migration and 
increased permeability, and the cells migrate (thanks to the production of MMPs that enable 
them to degrade the capillary basement membrane) towards the stimulus (chemotaxis). 
Considering that an essential factor for cell migration is cell adhesion to the substrate, 
migrating endothelial cells are 'tip' cells, polarised and with well-developed protrusions 
essential for matrix, the filopodia. The new vessels are gradually extended and remodelled, 
the endothelial cells connect and cavitation of the gemma occurs, i.e. the formation of a 
hollow tube that allows the blood to flow, pericytes are recruited and with them the 
stabilisation of the mature vessel.