State-of-the-Art on Wound Vitality Evaluation: A Systematic Review

The vitality demonstration refers to determining if an injury has been caused ante- or post-mortem, while wound age means to evaluate how long a subject has survived after the infliction of an injury. Histology alone is not enough to prove the vitality of a lesion. Recently, immunohistochemistry, biochemistry, and molecular biology have been introduced in the field of lesions vitality and age demonstration. The study was conducted according to the preferred reporting items for systematic review (PRISMA) protocol. The search terms were “wound”, “lesion”, “vitality”, “evaluation”, “immunohistochemistry”, “proteins”, “electrolytes”, “mRNAs”, and “miRNAs” in the title, abstract, and keywords. This evaluation left 137 scientific papers. This review aimed to collect all the knowledge on vital wound demonstration and provide a temporal distribution of the methods currently available, in order to determine the age of lesions, thus helping forensic pathologists in finding a way through the tangled jungle of wound vitality evaluation.


Introduction
Lesion vitality demonstration is one of the most challenging topics in forensic pathology. It has an undeniable importance in judicial processes, in which it can subvert the reconstruction of an event and influence the judgment. The vitality demonstration refers to determining whether an injury has been caused ante-or post-mortem, while wound age means to evaluate how long a subject has survived after the infliction of an injury [1]. A complex combination of events (acute inflammation, hemorrhage, proliferation, and remodeling) occurs immediately after wounding of tissue to re-establish its integrity and functionality. In the case of skin lesions, the presence of macroscopically evident blood infiltration of soft tissues can reveal the vitality of a bruise, while the change in coloration can indicate the time of survival [2]. However, wound examination with the naked eye is obviously not a reliable method to prove the vitality of a lesion. Histological analysis, based on hematoxylin-eosin, along with other stains (e.g., Prussian blue stain, elastica-van Gieson stain, etc.), helps visualize the vital reactions in human tissues [3]. Red blood cells infiltration, inflammatory reactions, presence of fibroblasts, macrophages, and immigrating granulocytes, as well as tissue alterations, are the main histological findings for wound vitality evaluation. However, this method has some limitations, such as operator dependency and the presence of staining artifacts. Moreover, some studies have shown The papers resulting from our research have been divided into three groups: quantitative analysis in biological fluids and tissues of various markers (24 papers), immunohistochemistry (84 papers), and ribonucleic acids studies (20 papers on mRNAs, 21 papers on miRNAs). Tables 1-3 show a brief description of these three groups of studies, respectively. The papers resulting from our research have been divided into three groups: quantitative analysis in biological fluids and tissues of various markers (24 papers), immunohistochemistry (84 papers), and ribonucleic acids studies (20 papers on mRNAs, 21 papers on miRNAs). Tables 1-3 show a brief description of these three groups of studies, respectively. Table 1. The results of our review on vitality markers in biological fluids and tissues through quantitative analysis (24 articles). * These studies included different kinds of analyses, in order to investigate the differential protein expression in tissues, and they have been included in both the relative tables. α7nAChR indicates α7 nicotine acetylcholine receptor; ATF, activating transcription factors; CaMK II delta, calcium-calmodulin-dependent protein kinase II delta; CORT, corticosterone; CXC, keratinocytes-derived chemokine; CXCR, chemokine receptor; CRP, C-reactive protein; EPO, erythropoietin; HAX-1, HCLS1-associated protein X-1; HMGB1, high-mobility group box-1; IL-6, interleukin-6; LBP, lipopolysaccharide binding; LC3-II, lipid conjugated form II; LTB4, leukotriene B4; Mb, myoglobin; MI, myocardial infarction; MyoD, myoblast determination protein; MPO, myeloperoxidase; MT1-MMP, membrane type-1 matrix metalloproteinase; p62, sequestosome; Pax7, paired-box transcription factor 7; PCT, procalcitonin; RAGE, receptor for advanced glycation end products; sIL-2R, soluble interleukin-2 receptor; sTREM-1, soluble triggering receptors expressed on myeloid cells type 1; TIMP-2, tissue inhibitor of metalloproteinase-2; 1; VEGF, vascular endothelial grow factor; RT-PCR, real-time polymerase chain reaction.

References
Type of Paper Model Fluid Brief Description Zhu et al. 2001 [20] Original research Human Urine The study aimed to investigate differential PM urinary Mb levels for determining the cause of death. PMI < 48 h did not influence urinary Mb levels, while PMI > 48 h showed increased levels (PM/putrefactive changes). Urinary Mb levels were increased when survival time was longer (>12-24 h, no linear correlation), as well as in some cases of vital muscle damage (e.g., fire fatalities, drowning, and head trauma), while they were not in cases of natural death due to MI. In cases of minor muscle damage (e.g., head traumas), the urinary Mb elevation was related to the survival time. The comparison between traumatic and non-traumatic deaths was not performed.
Quan et al. 2008 [11] Original research Human Serum Autoptic samples were analyzed (PMI tested < 48 h). EPO levels were increased in blunts produced 7 days after death, and its increase was higher in non-acute deaths due to wounds.
2018 [21] Original research Animal CSF K+ was significantly higher in TBI than in controls (no traumatized animal) when the samples were collected 12 h after death (no statistical difference when PMI < 12 h). Na+ was significantly higher in controls than TBI, when the samples were collected at the time of death and 6 h after death. Ca 2+ was significantly higher in TBI than controls, when the samples were collected at the time of death and 6 h after death, while it was higher in controls than TBI, when the PMI was 12 h; albumin was higher in TBI than controls only at the time of death (no statistical difference when PMI was 6 or 12 h). The total leucocytic count was significantly higher in TBI than controls, regardless to the PMI (PMI tested 0-12 h).

Plasma
Uric acid and ammonia were significantly higher in TBI than in controls, regardless of the PMI (PMI tested 0-12 h). Lactic acid was significantly higher in TBI than controls only at the time of death and 12 h after death; hypoxanthine was significantly higher in TBI than controls at 6 and 12 h after death.

Serum
TNFα and IL-1β were significantly higher in TBI than in controls, regardless of the PMI (PMI tested 0-12 h). HMGB1 was significantly higher in TBI than in controls at 6 and 12 h after death. Kobeissy et al. 2022 [22] Original research Animal Serum IL1-β, IL-6, and IL-10 were significantly higher in TBI than in controls.

References Type of Paper Model Tissue Brief Description
Njau et al. 1991 [23] Original research Animal Skin In wounded skin, Mg 2+ decreased within 30 min, increased and peaked at the 2nd hour after wounding, then gradually decreased until the 8th hour. Ca 2+ increased within 1 h after wounding, then decreased; however, at the 4th hour, it increased again until the 8th hour. Zn 2+ increased within the first 120 min, then decreased gradually until the 8th hour. No statistical significance was found among different sites of sampling (the lesion, 2 cm from the lesion, and 4 cm from the lesion). Survival time tested in this study: 30 s; 30 min; 1, 2, 4, and 8 h after wounding.
Chen et al. 1995 [24] Original research Human Skin, muscle Fe 2+ concentration in vital wounded skin (different types of lesions) was significantly higher than in controls (not injured skin of the same subjects). Survival time ranged from 5 min to 6 h. PMI ranged from 24 to 72 h.
He et al. 1996 [25] Original research Human Skin LTB4 was only detectable in vital skin lesions and not in wounds inflicted after death. It was also detectable in formalin-fixed injured skin if fixation < 10 days. PMI ranged from 4 h to 1 day. Laiho et al. 1998 [26] Original research Animal Skin MPO was high in vital skin lesions, but no comparison with normal skin or PM injured skin controls was done. MPO activity depended on blood loss (decreased activity with 35% loss of blood), the depth of the lesion (deeper lesions, higher activity), and skin thickness (thicker skin, higher activity).
Grellner et al. 2000 [27] Original research Human Skin The study found great interindividual variability in cytokine levels. In autoptic samples, IL-1β levels were significantly higher in wounded skin than in controls (normal skin) only when the wound age was ≤5 min. IL-6 levels were significantly higher in wounded skin than in controls when the wound age was ≤5 min and >24 h. TNF-α levels were significantly higher in wounded skin than in controls only when the wound age was ≤5 min.
Laiho et al. 2004 [28] Original research Animal Skin Significantly increased albumin levels in vital skin lesions (incision, excoriation, heat, and freezing injuries) with different ages (from 5 min to 15 days for incision, 5 min to 4 weeks for excoriations, and 60 min to 2 weeks for heat and freezing injuries) and sampled soon after death. Still significantly increased in incision wounds aged 15 and 30 min and excoriation aged 30 and 60 min when sampled 3 days after death. Barnes

Type of Paper Model Tissue Brief Description
Betz et al. 1992 [43] Original research Human Skin Fibronectin + in the skin from a few seconds to 6 weeks after wounding (with a different distribution pattern than control cases).
Betz et al. 1993 [44] Original research Human Skin Fibronectin + in skin lesions from a few minutes after wounding. A1-ACT provides no information.
Betz et al. 1995 [46] Original research Human Skin Macrophage maturation markers were evaluated. RM 3/1+ cells increase 7 days after wounding, as well as in 7-month-old scar tissue. 25F9+ cells increase 11 days after wounding, as well as in 3-month-old scar tissue.
Kondo et al. 1996 [47] Original research Animal Skin IL-α, IL-ß, IL-6, and TNFα + in 3-6 h-old wounds in neutrophils; by 24 h, they were then substituted by macrophages. TNFα and IL-ß levels increased soon after wounding and peaked at 3 h. IL-α showed a peak at 6 h after wounding, while IL-6 peaked at 12 h after injuring. Dressler et al. 1997 andDressler et al. 2000 [48,49] Original research Human Skin ICAM-1 (CD54) strongly + in vital wounded skin, while only slightly + in undamaged skin. The earliest + was at 15 min after wounding, strong + 4 h after wounding, with still + reaction even 10 days after wounding. The distribution of ICAM-1 expression was different between autopsy (PM sampling) and surgical (AM sampling) cases. In autopsy cases, the PMI was ≤7 days.
Fieguth et al. 1997 [50] Original research Human Skin The study aimed to evaluate the influence of post-mortem clamping and autolysis on the IHC reactions by testing antibodies against αlact, α2m, fibronectin, and lysozyme. Autolysis produced an increase in false + reactions, while post-mortem clamping did not.   TNF-α strongly + on day 3 after surgery, decreasing by day 5-7, TNF-α+ neutrophils on day 1 to 3, and fibroblasts on day 14 post-surgery. TGF-ß1 levels decreased on day 5 after surgery and then increased until day 14.
Guler et al. 2011 [83] Original research Human Skin Tenascin strongly + in all types of wounds investigated, by 24 h after injury. Weak + of ubiquitin since 24 h after wounding, still present in wounds over 40 days old, while tenascin was negative.

Type of Paper Model Tissue Brief Description
Ryu et al. 2011 [84] Original research Animal Oral mucosa TNF-α+ 1-day post-surgery in scalpel wounds and 3 days post-surgery in laser wounds, reaching its peak at day 3 for all groups of surgery. TNF-ß + levels increased 3 days post-surgery, decreased until day 7, and increased further until day 14 both in laser and scalpel wounds. The highest intensity is shown at day 3.

Messenger
Some studies included different kinds of analyses to investigate the differential protein expression in tissues; for example, He et al. applied both a Western blot analysis to quantify the protein levels and the real-time quantitative polymerase chain reaction (RT-qPCR) to evaluate the mRNA expression [40]. In such cases, we included the papers in both the relative tables, adding an asterisk near the reference (*). In the "miRNA" group, we included several papers aimed to investigate the role of specific miRNAs in wound healing and not the differential expression between vital and non-vital injuries. The reason for this choice is that miRNA studies in wound vitality demonstration are a novelty in this field, and only a few of them have been conducted on autoptic human samples. However, we wanted to collect the current knowledge on the role of miRNAs in wound healing, hoping that could be useful for further works in this field.
Considering all the analysis methods, the most investigated tissue is the skin, and only a few studies also evaluated the vitality reaction in other tissues (e.g., the skeletal muscle, brain, liver, kidney, etc.). The most studied method in wound vitality demonstration is immunohistochemistry (84/137 papers), and fibronectin is the most searched protein (15/85 papers).

Discussion
To determine if a lesion has been produced in life or post-mortem, as well as how much time has passed between the lesion's production and death, is often crucial to understanding if there was a causal relationship between the wound and death. However, wound vitality evaluation is one of the most challenging fields for the forensic pathologist because there is not a standardized and internationally accepted method to prove the vitality of a lesion and its age. We performed a comprehensive review on wound vitality demonstration, in order to collect the current knowledge in this field. We found 137 papers that are heterogeneous and used different investigation methods; this is the greatest limitation of our work, which did not allow us to perform a proper quantitative synthesis. As the results of our review show, the most studied method for evaluating the vitality of a lesion in the literature is immunohistochemistry. It has been mainly used to highlight the presence of a healing and inflammatory response, which should be absent in post-mortem lesions. Nowadays, immunohistochemistry could be considered the "gold standard" in distinguishing between ante-and post-mortem lesions [113]. It is a morphological technique and could show the different distribution of the molecules of interest in the harmed tissue alongside their quantification. It is easily applicable, it does not require expensive or sophisticated machinery to be done, and it could also be applied in formalin-fixed paraffin embedded tissues, which means it is also possible to evaluate unsolved "cold cases" [157]. On the other hand, the quantification of protein expressions through immunohistochemical staining is highly operator-dependent, and the quality of staining can be influenced by many variables, so it could lead to errors or scarcely reproducible results [158,159]. Moreover, robust immunohistochemical protocols and automated measure procedures have not been developed yet in forensic pathology, as has been accomplished in clinical disciplines [160][161][162]. Besides, several immunohistochemical vitality markers have been tested. In Figure 2 and Table 4, we collected the most promising immunohistochemical vitality markers in skin, concerning the timing of positivity, as described in the collected papers.
immunohistochemical vitality markers in skin, concerning the timing of positivity, as described in the collected papers.  Another field in wound vitality determination is represented by blood coagulation and hemostasis. Molecules involved in these processes are potentially valuable due to their early production in wounding and healing. Studies on FXIII, which regulates the cross-linking process of fibrin monomers and therefore clots stabilization, and its positive effects against MPPs action on fibroblast cultures could be encouraging. Moreover fibrin organization in the blood clot seems to be promising as a potential timing technique. It has been observed that, when using the Picro-Mallory staining method, fibrin stained either red, violet, or blue according to clot maturation timing. At 30 minutes to 6 h, fibrin stained red, from 6 to 12 h appeared purple or violet while in clots older than 24 h fibrin stained blue [122]. Fibrin deposits could also be found in vital bone fractures from 34 minutes to 26 days after wounding [81].
Some researchers tried to apply quantitative techniques in lesion vitality evaluation, such as protein or ions quantification. In Figure 3, the main vitality markers that are evaluable with quantitative analysis of the skin are presented in a timeline. However, as our results show, there is only a minority of papers about this topic (24/137); therefore, these methodologies have not been sufficiently tested to be used in lesion vitality evaluation, especially when it is done for forensic purposes and these data need to be used in courts.
In Table 5, we collected both electrolytes and biomarkers as their concentration and expression variates in time after wounding, as described in the gathered papers. Electrolytes, such as nagnesium and calcium, are deeply involved in enzyme regulation and cell metabolism at the injured site, as well as in muscular contractility. Moreover, elevated levels of zinc can be found in later phases of reparation and seems to be involved in various inflammatory processes [23]. Sodium and potassium level variations could be related to membrane disruption, due to trauma, which causes a change in membrane potentials [21]. Table 4. This table shows the timing of positivity of immunohistoichemical vitality markers in different tissues after wounding. IL, interleukin; α7nAChR, α7 nicotine acetylcholine receptor; A1-ACT, alpha1-antichymotrypsin; RM3/1, anti-CD163 marker; 25F9, mature macrophages marker; GM-CSF, granulocyte macrophage-colony stimulating factor; GPA, glycophorin A; HSP, heat-shock protein; ICAM-1, intercellular adhesion molecule 1; iNOS, inducible nitric oxide synthase; mAb, monoclonal antibody; MCP, monocyte chemoattractant protein; MHC-II, major histocompatibility complex II; MMP, matrix metallopeptidase; RAGE, receptor for advanced glycation endproducts; TNF-α, tumor necrosis factor α; VEGF, vascular endothelial growth factor; VCAM-1, vascular cell adhesion molecule-1; ATP, adenosine triphosphate; MRP, myeloid-related protein; M-CSF, macrophage colony-stimulating factor; MIG, monokine inducible by interferon gamma; ORP, oxygen regulated protein; HLA, human leukocyte antigen; TGF, transforming growth factor; MPO, myeloperoxidases; COX, cyclooxygenase; CB2R, cannabinoid receptor type 2; SP, surfactant protein; HIF, hypoxia inducible factor; AQ, aquaporin; MIP, macrophage inflammatory protein; CML, carboxymethyllysine; Flk, receptor for vascular growth factor; EPC, endothelial progenitor cell; TIMP, metallopeptidase inhibitor; Chil, chitinase-like; INF, interferon; Ub, ubiquitin. We included all the biomarkers that showed a variation (increasing or decreasing) before 21 days. Regarding the biomarkers that showed an expression during an interval, only the upper limit has been considered. *IL 6 has been considered in different studies; it shows a different expression only in two studies. In recent years, the great development of genetic techniques has allowed for studying ribonucleic acid variations as vitality markers. One of the main problems in this field is represented by the immediate post-mortem period, a phase characterized by the persistence of vital reactions (so-called "residual-life phenomena") within a very short time after death [163,164]. In fact, metabolic processes and vital activities do not cease in all the cells and tissues at the same time; so, immediately after death, they could mimic vital reactions, making it hard to differentiate vital from early post-mortem findings [165]. This problem involves a wide range of techniques used in wound vitality evaluation, from immunohistochemistry to miRNAs and proteomics. This limit of vitality markers could be addressed, when solving the single case, with careful evaluation of circumstantial data, case investigations, and medical history review. When confronted with an autopsy, forensic pathologists should always adopt a comprehensive approach, collaborating with the investigators.

Timing of Markers' Positivity
Another issue in vitality demonstration is represented by post-mortem manipulations. It could happen that certain circumstances or post-mortem activities could artificially induce the findings that appear vital [166]. For example, ventilation could reproduce emphysema, and resuscitation maneuvers, which keep blood circulating, could induce post-mortem red blood extravasation in tissues. We included all the biomarkers that showed a variation (increasing or decreasing) before 21 days. Regarding the biomarkers that showed an expression during an interval, only the upper limit has been considered. *IL 6 has been considered in different studies; it shows a different expression only in two studies. In recent years, the great development of genetic techniques has allowed for studying ribonucleic acid variations as vitality markers. One of the main problems in this field is represented by the immediate post-mortem period, a phase characterized by the persistence of vital reactions (so-called "residual-life phenomena") within a very short time after death [163,164]. In fact, metabolic processes and vital activities do not cease in all the cells and tissues at the same time; so, immediately after death, they could mimic vital reactions, making it hard to differentiate vital from early post-mortem findings [165]. This problem involves a wide range of techniques used in wound vitality evaluation, from immunohistochemistry to miRNAs and proteomics. This limit of vitality markers could be addressed, when solving the single case, with careful evaluation of circumstantial data, case investigations, and medical history review. When confronted with an autopsy, forensic pathologists should always adopt a comprehensive approach, collaborating with the investigators.
Another issue in vitality demonstration is represented by post-mortem manipulations. It could happen that certain circumstances or post-mortem activities could artificially induce the findings that appear vital [166]. For example, ventilation could reproduce emphysema, and resuscitation maneuvers, which keep blood circulating, could induce post-mortem red blood extravasation in tissues.
Eventually, a limitation of the analyses described in this paper could be the decomposition and other post-mortem alterations (e.g., maceration or adipocere formation). Biomarkers and electrolytes, especially in fluids, are certainly influenced by the putrefactive phenomena and the reliability of their determination for wound vitality demonstration decreases when the post-mortem interval (PMI) increases. Immunohistochemistry seems reliable even when the corpse is putrefied, as some Authors demonstrated [53,67,85]. On the other hand, it is possible that decomposition, altering the tissues' architecture, could influence not only the intensity of the immunohistochemical response, but also its localization. mRNAs and miRNAs are stable molecules and theoretically they could be less influenced by the putrefaction. However, there are not a lot of studies investigating the trustworthiness of these analyses in such conditions (we found only three papers evaluating immunohistochemistry in putrefied corpses) [53,67,85]. Therefore, more studies are needed to deepen the role of decomposition in the applicability of such techniques.
Since mRNAs and miRNAs have a role in protein production at a very early step, they could hypothetically be used as really precocious markers of an inflammatory response, differentiating vital and post-mortem lesions, even when the survival time is very short or such influencing factors occurred. Table 6 and Figure 4 show the timeline of detectability of some mRNAs and miRNAs implicated in wound healing, which may be used in lesion aging. Table 6. This table shows the progressive expression of different mRNAs in time after wounding. Each element depicted in this table is an mRNA. If two or more articles provided contrasting results for the same mRNA, it has not been included. Only mRNAs whose expressions vary, according to a specific timeframe, have been considered. α7nAChR, α7 nicotine acetylcholine receptor; IL, interleukin; MMP9, matrix metallopeptidase; TNF-α, tumor necrosis factor α; TIMP-2, tissue inhibitor of metalloproteinases; MCP-1, monocyte chemoattractant protein 1; MT1-MMP, membrane type-1 matrix metalloproteinases; COX, cyclooxygenase; SNAT2, amino acid transporter 2; CCL, C-C motif chemokine ligand; Fosl1, FOS-like 1; MyOD, myoblast determination protein; FDZ4, frizzledclass receptor 4; SFRP5, secreted frizzled-related protein 5; CSF, colony stimulating factor; PAI1, plasminogen activator inhibitor 1; Pax7, paired-box protein 7; TGF; transforming growth factor; FGF, fibroblast growth factor.  al.'s results validate the involvement of this miRNA in both collagen deposition and wound contraction [139]. Table 7 shows a summary of some of the main molecular pathways influenced by miRNAs.   In the past few years, miRNAs have attracted researchers' attention because they are involved in various inflammation and healing processes. Among the molecular pathways highlighted in this work, we found that miR-19a/b and miR-20a suppress poly(I:C)-induced expression of CXCL8, CXCL5, TNF-α, and IL-1A, proinflammatory chemokines, and cytokines at the mRNA level by regulating the nuclear factor kappa-light-chain-enhancer of the activated B cell (NF-кB) signaling pathway [156]. This pathway is activated by the tumor necrosis factor (TNF-α), which phosphorylates the inhibitor of nuclear factor kappa B (IкB). Similarly, miR-92a-3p, inhibits the intracellular transduction of the toll-like receptors (TLR), hence its role as a pro-inflammatory stimulus [149]. Moreover, miR-19b, targeting C-C motif chemokine ligand 1 (CCL1), seems to be involved in the regulation of the transforming growth factor-ß (TGF-ß) signaling pathway. This pathway, which is involved in all the phases of the repair process, is also targeted by miR-26a, miR-149, and miR-21 [153]. MiR-149, in particular, could be able to contain the inflammation process by downregulating the expression of IL-1α, IL-1ß, and IL-6 and is believed to act as a positive regulator of the skin healing process [145]. In corneal epithelial cells, miR-205 stimulates the healing process by inhibiting the inwardly rectifying K+ channel, KCNJ10 [141]. Furthermore, miR-205 is a positive regulator of keratinocytes migration, actively altering the organization of F-actin, decreasing cell-substrate adhesion, and, along with miR-184, regulating the SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) levels and phosphorous protein kinase B (AKT) signaling [134]. These miRNAs act in a very peculiar way; it seems that miR-184 can suppress the expression of miR205, therefore maintaining elevated SHIP2 levels. Additionally, SHIP2 s influence on keratinocyte migration could be both positive and negative, depending on the local levels of phosphoinositide pools, which are also involved in the cell adhesion process. Similarly, miR-205 downregulation determines the enhancement of cell migration by suppressing F-actin in HEKs and altering the levels of p-Akt, thus activating Rho, p-cofilin, and ERM. All these proteins are strictly associated with processes of remodeling and migration [167,168]. Members of the miR-99 family act as regulators in cell proliferation, apoptosis, and migration, through the PIK3/AKT pathway, therefore influencing the mTOR signaling pathway [140]. The mTOR pathway is also associated with the re-epithelialization of skin wounds [169]. Among this family, for example, miR-100 acts to reduce the phosphorylation of signaling molecules, such as p70 S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), which is involved in the pathway previously described. Another important pathway targeted by miRNAs, such as miR-149 and miR-222, is the MAPK pathway. MAPKs direct different cellular responses to stimuli including heat shock and gene expression regulation, cell proliferation, differentiation, and survival [142]. Furthermore miR-222 targets different genes, such as axin-like protein 2 (AXIN2), Dickkopf-related protein 2 (DDK2), and FRAT regulator of the Wnt signaling pathway 2 (FRAT2), therefore influencing the winglessrelated integration site (Wnt) signal [169,170]. Pastar et al. found that miR-21 and miR-130a overexpression leads to inhibition of the epithelial growth factor (EGF) pathway by suppressing the early growth response 3 (EGR3) gene [137]. Suppression of EGR3 and vinculin could also be linked to the inhibition of keratinocyte migration in chronic wounds. Another important target of miR-21 is the leptin receptor (LepR) gene in the epidermis; this signaling is well-renowned as a pleiotropic stimulus on wound healing. MiR-203, as highlighted by Viticchiè et al., could be crucial in the regulation of different pathways, such as p63, LIM, and SH3 domain protein 1 (Lasp1), as well as the Ras-related nuclear protein (Ran) and Ras-associated and Pleckstrin homology domains 1 (Raph1), which are implicated in the reepithelialization process [138]. The downregulation of this miRNA could probably mediate the switch to activated keratinocytes in wound closure; on the contrary, its overexpression could be the stimuli to commitment to differentiation in the healthy epidermis, as well as in injured ones. Furthermore, when miR-203 is upregulated, we can assist in an alteration of the Wnt/B-catenin signaling pathway, thus determining the decreased levels of MAPK8, MAPK9, Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), and protein kinase C alpha (PRKCA). In addition, miR-203, through the IL8/AKT pathway, is implicated in the epithelial-mesenchymal transition (EMT) process [150]. MiRNAs are also involved in the angiogenesis process, namely miR-26a regulates the bone morphogenetic protein (BMP)/small mother against the decapentaplegic 1 protein (SMAD1) signaling pathway. The angiogenic impulse is carried out by BMP, as well as by the vascular endothelial growth factor (VEGF), which has a crucial role in cell migration and proliferation, in order to form new blood vessels [143]. Icli et al. found that miR-26a plays a part in promoting fibroblast migration in wound sites. miR-21 has multiple targets, such as Smad and Smad7, involved in promoting collagen deposition in granulation tissue [143]. Wang et al.'s results validate the involvement of this miRNA in both collagen deposition and wound contraction [139]. Table 7 shows a summary of some of the main molecular pathways influenced by miRNAs. Table 7. This table summarizes the various miRNAs and their genes and/or proteins target, as studied in the papers included in this review, if available. CCL, chemokine ligand; TGF, transforming growth factor; LepR, leptin receptor; EGR, early growth response; TIMP, metallopeptidase inhibitor; TIAM1, T-cell lymphoma invasion and metastasis inducing protein 1; TP, tumor protein; ITGA, integrin subunit alpha; PI3K, phosphoinositide 3 kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; BMP, bone morphogenetic protein; SMAD, small mother against decapentaplegic; GSK, glycogen synthase kinase; IGR1R, insulin-like growth factor 1 receptor; IL, interleukin, PTPRC, protein tyrosine phosphatase receptor type C; CD, differentiation cluster; SHIP2, SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase; RNU6B, RNA U6 small nuclear; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor kappa-light-chain-enhancer of activated b cells; TCF-4, transcription factor 4; ID-2, inhibitor DNA-binding 2 protein HLH; VEGFA, vascular endothelial growth factor A; NRCAM, neuronal cell adhesion molecule; C-MET, tyrosine-protein kinase Met; LASP1, LIM and SH3 protein 1; RAN, RAs-related nuclear protein; RAPH1, Ras-associated and pleckstrin homology domains-containing protein 1; ERM, ezrin/radixin/moesin; DDK2, dickkopfrelated protein 2; FRAT2, FRAT regulator of Wnt signaling pathway 2; MK2, mitogen-activated protein kinase-activated protein kinase 2; YAP1, yes-associated protein 1; MKI67, marker of proliferation Ki-67.

Conclusions
In the literature, several studies investigated the potential use of several markers in wound vitality demonstration [171][172][173][174][175][176][177]. However, a standardized method is not available yet, and these data could not be reliably used in forensic practice [178]. There are still several unknown factors that may influence the protein expression and molecular pathways involved in inflammation and wound healing, thus inducing misinterpretation [179][180][181]. In this review, we collected the current knowledge in wound vitality demonstration, through different fields of research; however, more evidence is certainly required. The need for an internationally accepted and interdisciplinary approach is urgent; we hope that, through this review, the readers find inspiration for further research, in order to deepen this topic.

Conflicts of Interest:
The authors declare no conflict of interest.