Wound Healing: A Case Study

Professional tattoos causes the breakage of the skin, triggering the automatic response of wound healing. Otherwise the standard removal of ink using laser can produce scars depending on the depth of the colour. Scarring tissue heavily involves fibroblasts and the specialised myofibroblast role is to replace the ECM components. The scarring and aging of the skin results in increase friction and declined mechanical properties-compression, tension and elasticity (Richard Wong). Wound healing can be described as a ‘sophistically regulated process that involves inflammation, new tissue formation and remodelling’ (Nyman 2015).
Similarly to Ketova, (Ref: Grant 2012) made usage of the indentation function of the AFM, but this time comparing normal
to scarred tissue. The scarred tissue derived from a male aged 59 was stored at -80 degrees; it should be mentioned that the scar was from a wound endured several years ago. The specimens with a 10µm thickness was obtained from the centre of the scar and 1cm of skin was removed from nearby. Images was scanned under aqueous conditions to produce conditions close to the original environment. Using a silicon nitride cantilever with a spring constant of 0.32N/m and intermittent contact mode, several indentations at a load of Fmax=20 nN to analyse the mechanical traits of the skin.
The importance of looking in to the mechanical properties of the skin provided by the collagen affects the level of force required to be applied by the armature bar. A hole penetrated deeper than the dermis will require external assistance or secondary intention repair i.e. sutures as the dermis layer is not regenerative.
The results demonstrated that the dermal region is stiffer in the scarred tissue than the healthy skin, moreover the scarred tissue developed with low viscoelasticity creep which subdued the dissipation of energy reducing the skins elasticity which became evident in figure 5.0. The graph portrays the scarred tissue had half the creep ability to return back to original shape unlike the healthy skin which had a mean indentation creep of 80nm. The study also showed the morphological changes occurred, there was an increase collagen density and a strong alignment with regards to the wound, a characteristic seen in scarred tissue. Majority of the fibrils were orientated in the same direction unlike the normal skin which showed randomness and entwined fibres. This is the main structural difference visible between the normal and scarred tissue. How do collagen grow back?

The properties explored in figure 9.0 are elasticity and the hardness of the two tissues of the dermis. Graph 9a demonstrates the normal skin has a large elasticity range of 141 nm compared scarred peak value of 32 nm; the normal skin had areas that was able to deform over a 3 second period of up to 160nm, the graph fits the normal distribution model proving the skin has an even spread of elastic property whereas the scar lacks greatly as shown in the positive skewed graph, this is important as the change in elasticity behaviour is proportional to the skins ability to perform its function. As the two graphs are complimentary, the same goes for the graph representing the modulus figure 9b with the normal tissue being less stiff (ref: Grant, 2012). Conclusively the graph suggests the normal skin has a viscous response to the indents made. An increase in stiffness results in the skin unable to dissipate the energy thus tearing occurs.

2.3 Tattoo particles/Nano-particles
Individual tattoo ink are classed as nanoparticles with carbon black ink confined to 41-165 nm in range and spherical in shape (ref: Hosberg 2011). This can be confirmed as (grant, 2015) was able to retrieve a single particle with a diameter of 30nm. Tattoos are constituted from stable and insoluble pigments (extraction ref). Commercially available ink was measured to have a diameter of 17nm and 68 nm in width. This produces a larger risk as the biological and chemical activity is increased with the high volume to surface ratio. (find ref- principles of characterising the potential human health effects from exposure to nanomaterials).
Although tattoo pigment can reach the papillary dermis layer, yet unable to penetrate the skin fully. This is vital as inclusion of exogenous particles into the blood stream and lymph vessels can be dangerous. This is very rare but can occur when tattoos fade and enter the circulatory system, it has been stated that ink are transported to some extent in the human body by means of the lymphatic system (ref Karin lehner). The nano-scale of the particles means there is a higher chance of the particles penetrating the cells.
A study included 3 female aged 30, 35 and 39 with tattoos undergoing a non-evasive method of attaining cross-sectional scans from the usage of an optical coherence tomography (OCT). Similar to that of a confocal microscope, the OCT measures the reflected light intensity to obtain the images. The tattoo particles are deposed in the dermal through the epidermis as it was observed that the tattoo clusters (100 – 600 µm) appear as columns in the papillary dermis, tattoo deep into the dermis was also observed (hanan).. This was also evaluated by (grant, 2015) who also noticed several particles gathering in clumps rather than dispersing which suggests the reactivity of the nanoparticles are reduced due to the agglomerates formed. An interesting factor brought to attention was that the images consisted of uneven distribution of pigment and few clusters were seen in the non-tattoo section
The investigation into the risk of tattoo ink was carried out that examined tattoo skin of a 62 year old male as well as the consequence from the contact between the ink and cells (ref-Grant et al. 2015). After filtration and using the light scattering method, the peak mode for particle size was 151; within the range specified by (ref-Hosberg 2011). However it was noticed several were agglomerates larger than the dermal cells, hence eliminating the reactivity of nanoparticles, this can be viewed in figure 10a. More clusters of tattoo nanoparticles were apparent than single particles creating uneven surface with a roughness Ra of 30nm. These particles are located in proximity to collagen fibrils, the orientation of the collagen fibres were very alike to scarring tissue (grant 2015 – nano scale observation of tattoo).
The uneven distribution observed was thought to be due to the ink in the fibroblast ref Lea PJ (4), as the mobility of the fibroblast has caused the spread of the tattoo pigments (ref Hanan Morsy, printed paper). (Grant 2015) also mentioned that tattoos are not stationary. This proposes ink penetrating in the fibroblast can have adverse consequences.

Grant (2015) used the AFO to analyse the first point of contact with the fibroblast cells situated in the dermal layer to witness the interaction.
The ink attached to the surface in clumps of 200-500nm. A MTT test also undertook to test the cytotoxicity on cells, figure 10b shows the cell population decreased for both filtered with particle size ranging 30-600 nm and unfiltered ink 40-970nm, a larger decrease in cell numbers for filtered ink signifying singular tattoo nanoparticles has a negative effect on the cells. Similar test on fibroblast was taken by Biolip 27 ink also showed a similar trend, it showed a reduction of fibroblast by 47% after a MTT test was taken ruminating the ink as toxic. After 72 hours treatment the surface was covered in particles and remained firmly attached after 96 hours, the background free of particles portraying specific adhesion to fibroblast. (mirella). Both results showed that the insertion of ink could lead to fibroblast death (Grant 2015, mirella falconi). The size of the particle had an effect on the death as it was noted that higher number of fibroblast with filtered ink inserted died compared to
unfiltered.
Although death was recorded as a decrease in cell viability, the morphology of the fibroblast never changed. However it was observed that particles were intervening with the production of procollagens α 1, the precursor of collagen type 1. This was proven by using immunofluorescence experiment which consists of immunolabelling for procollagen, a fibroblast exposed for 72 hours and 2 weeks showed staining around the nucleus. However fibroblast diluted in Biolip 27 ink revealed a decrease in fluorescence surrounding the nucleus while after 2 weeks no clusters surrounded the nucleus; this is related to the ink particles scattered on the cell surface, possibly due to covering the receptor channel exhibiting the essential cell-reception binding required to initiate collagen production (Mirella).
The results of tattoo ink leading to fibroblast death may be deemed as a hazard by the medical professions however no serious disease has been linked to tattoos (ref - Hosberg). On the other hand, it was not always the circumstance, Jana and nikolai report numerous cases of individual with tattoos contracting tuberculosis cutis, pyoderma, Leprosy, granulomas. Allergic reaction, Lichenoid and granulomatous are the most common, also infection resulting after a tattoo is very likely as it has been reported that infectious microorganism live within the ink but due to the sterilization of the equipment it is no longer a problem. One case in mentioned was a male age 23 who recalled symptoms of fever, malaise, nodules and plaques on his leg after one week in receipt of a tattoo on his left knee. A biopsy revealed early stages of granulomatous panniculitis. There was also an increase in C-reactive protein therefore the body triggered the natural inflammation phase. Allergic reaction is majorly related to the colour pigments attributable to the presence of metal ions i.e. mercury, chromium, cadmium, cobalt and sulphur (ref- Tang, M.M; Beltraminelli, H A tattoo complicated by allergic contact dermatitis and panniculitis.)
The tattoo pigments come in various constituents unknown, the physical structure influences the extent at which it transports in the dermis, some metabolize in skin which can have an adverse effect.
One should be careful which pigments are inserted in to their body. Pigment Red 22 is banned in cosmetics in Europe because of carcinogenic amides produced as side-products and due to the solubility the risks of cancer are high (ref extraction of tattoo pigments). Polycyclic aromatic hydrocarbons PAH are organic bonded with benzene rings (ref Ki-Hyun Kim), has been reported to be found in black ink which is concerning since they are related to carcinogenicity and mutagenicity (End note Karin Lehner, Wolfgang B). this is important to review as an animal experiment had proven that ink is a third of 1.5 mg of tattoo pigment penetrated in to the skin disappeared within a few weeks (wolfgang
endnote).
Health problems associated with PAH molecules not only effect the skin but also in any other organs reached via the circulatory system and lymph nodes.
It is therefore obligatory to carry out studies toxicological and epidemiological to clarify the possible risks of tattooing.