As the largest organ in the body, the skin is the primary protective barrier between an individual and the surrounding environment. Among the main conditions severely affecting the human skin, large burns are life-threatening situations. One of the therapeutic tools showing promising results in these patients is the UGRSKIN model (16), but further research is still in need to improve the efficiency of this ATMP.
Burn injuries typically destroy the skin integrity, and avascularity, protein rich environment and generalized immune suppression make burn wounds particularly susceptible to infections(17–20). One of the most common bacterial species affecting burn patients is P. aeruginosa(17–20), and its presence may significantly worsen prognosis and survival rate. To overcome these limitations, different innovative approaches have been described, such as biomaterial functionalization with graphene oxide(32) or montmorillonite(33). However, the inability to control severe burn-related infections is still challenging.
In the present study, we generated an improved dermal skin substitute by combining the regenerative properties of the UGRSKIN model with antibiotic-loaded NLCs. These nanoparticles already demonstrated to be an effective drug delivery system suitable for the treatment of P. aeruginosa infections(24), with no associated in vivo side effects(26, 27).
Our results first demonstrated that functionalization with NLCs was very effective, and FSS showed strong antibacterial effectiveness on P. aeruginosa cultures. In the first place, we found that the antibacterial activity of the FSS was dose-dependent with a strong correlation between the concentration of antibiotics and the bacterial inhibition area. This finding was unsurprising, since dose-dependent antibacterial effect is one of the main characteristics of most known antibiotics(34), although this is the first time that this phenomenon is demonstrated for nanoencapsulated antibiotics combined with fibrin-agarose hydrogels. As previously demonstrated, the use of antibiotic loaded NLCs is associated to a significant reduction of the antibiotic doses needed for an efficient ex vivo and in vivo effect, and 2-fold reduction in the inhibition area was obtained as compared to free antibiotics(27).
In the second place, our results showed that both types of antibiotics (AMK and SCM) were very effective against P. aeruginosa as compared to controls, and a global comparison between both antibiotics revealed non-significant differences. In general, these results suggest that both, AMK and SCM loaded into NLCs, could be promising tools that may provide antimicrobial properties to functionalized dermal substitutes in order to treat or prevent bacterial wound infections. However, a detailed comparison of specific concentrations showed that AMK was more efficient than SCM for the highest concentrations. Moreover, considering that P. aeruginosa is one of the bacteria responsible for antimicrobial resistance (AMR), a major global threat facing public health (35), the fact that our improved dermal substitute can be functionalized with AMK and SCM, two of the few antibiotics effective against gram-negative resistant bacteria, provides added value to this ATMP.
In general, these results confirm the ex vivo usefulness of the functionalized models of human dermis described in the present work, and suggest that both antibiotics could be effective for preventing P. aeruginosa infections of the wound bed. The fact that both antibiotics were efficient opens the door to the possibility of using one of them as a first-line treatment for severe skin burns, and reserving the other antibiotic as a second-line option, although a combination of both in a single FSS could also be an option. Future studies should be carried out to determine the most adequate composition of FSS for clinical use.
On the other hand, one of the most important requirements for therapeutic use of biomaterials in tissue engineering is biosafety(36). In this regard, evaluation of the side effects of functionalization is necessary before FSS can be clinically used. For this reason, we analyzed several parameters directly related to biosafety in FSS, including biocompatibility, cell viability, proliferation and ex vivo function. Our results first showed that most cells in the FSS were alive, with no structural membrane damage demonstrated by quantification of free DNA released to the culture medium and no morphological alterations as determined by HE staining. Cell viability was higher than 90% in all conditions, confirming the high biocompatibility of these nanoparticles(27). Although cell proliferation and metabolic function, as determined by WST-1, was partially reduced at 24 h, cells were able to recover their initial condition after 48 h, which is in agreement with our immunohistochemical results for KI67. Previous results demonstrated that cells immersed in hydrogels for tissue engineering use could need some time to adapt to the new culture conditions and may require some days to fully recover the initial cell number and function(37, 38). All these results confirm that FSS fulfill the biocompatibility requirements of bioartificial tissues for future clinical, as previously described(27).
These findings are in agreement with the immunohistochemical analyses of the dermal skin substitutes showing negative reaction for caspase 7, a marker of apoptotic cell death, which reinforces the non-toxic effects of the NLCs used in this work. Similarly, we found a positive immunohistochemical reaction for the cell proliferation markers KI67 and PCNA, confirming the functional status of the cells in all FSS groups. The different behavior found for PCNA and KI67 could be explained by the longer half-life of the PCNA protein (at least 20 h), which implies that nuclei could continue to express PCNA even after completing the cell cycle(39), whereas KI67 is known to be more sensitive and specific(40).
After demonstrating that the nanoparticles used in the present work were safe for the cells immersed in the dermal substitute, we analyzed the influence of these NLCs on the biosynthetic capability of the FSS cells. Results showed that the synthesis of collagen and proteoglycans, the most important extracellular matrix components synthetized by the dermal substitute of a bioartificial skin(15), were actively synthetized by the FSS cells, with no differences with controls. Although longer follow-up studies should be carried out, these preliminary results confirm that functionalization of the human dermal skin substitute did not affect negatively the human dermal cells. Fibroblasts remained viable, morphologically normal and biosynthetically functional as they were in non-functionalized human dermal skin substitutes.
In addition, bioengineered tissues should resemble the native tissues not only at histological level, but also from a biomechanical standpoint. For this reason, in the present work we performed a biomechanical characterization of the FSS in order to determine if functionalization may have modified the main properties of these bioartificial tissues, and to assess if the incorporation of the different types of NLCs could have changed the three-dimensional structure of the biomaterial and, therefore, its biomechanical properties. A proper biomechanical characterization of the novel FSS would contribute to future clinical translation, since a comprehensive characterization of novel tissue substitutes is a requirement of all National Medicines Agencies before clinical use(3, 36, 41, 42). Thus, once effectivity and safety of the functionalized human dermal skin substitutes were proven, we decided to test the biomechanical properties of the FSS. In general, our results showed that both the type and concentration of NLCs could influence the biomechanical properties of the human dermal skin substitutes.
Interestingly, we found that functionalization was able to modify the Young modulus of the FSS in a dose and type-dependent manner. In this regard, we found a statistically significant increase of this parameter in FSS containing the lowest concentrations of AMK and SCM nanoparticles as compared to the SS group, whereas the highest concentrations showed the opposite effect. Strikingly, the use of CTR-NLCs showed a different behavior. As the Young modulus is directly linked to the biomaterial stiffness(6), these results suggest that antibiotic-loaded NLCs could be used not only for tissue functionalization, but also to improve the biomechanical properties and stiffness of human tissue substitutes. Related to the Young modulus, we analyzed the stress at fracture break and break load of each type of bioengineered human skin. Both parameters are associated to the maximum stress force that the bioartificial tissues can resist before irreversible damage(6). As for the Young modulus, we found that functionalization with the lowest concentrations of nanoparticles, especially in the case of AMK, were able to improve the stiffness of the FSS, although a high concentration of particles resulted in the opposite effect.
Other critical biomechanical parameters of bioengineered human tissues are the strain at fracture break and the traction deformation. Both parameters are related to the deformation that the bioartificial tissue can sustain before irreversible damage, and are strictly dependent on the tissue elasticity(6). Remarkably, our results showed that these parameters were also dependent on the type and concentration of NLCs. Whereas low concentrations of nanoparticles were associated to a loss of elasticity, we found a significant increase of both parameters for the highest concentration of SCM-NLCs and in FSS-CTR300, suggesting that the plastic deformation capability of FSS can be modified upon functionalization.
Altogether, our biomechanical results show that functionalization of the dermal skin substitute can significantly tune the biomechanical behavior of this bioartificial tissue. In this line, not only the concentration, but also the type of encapsulated antibiotic, are associated to these biomechanical changes. This new generation of functionalized dermal skin substitutes could improve the biomechanical properties of the current UGRSKIN model by combining an optimum type and concentration of antibiotic-loaded nanoparticles.
The reasons why functionalization is able to increase or decrease the biomechanical properties of FSS requires further research. However, we may hypothesize that NLCs could associate to fibrin-agarose monomers during polymerization of the hydrogel and increase the biomechanical stability of the 3D fiber mesh, thus contributing to hold the incoming forces with higher efficiency. In fact, we previously found that different types of nanoparticles can be adsorbed on the fibrin fibers and increase the attraction between adjacent fibers, resulting in a significant improvement of the biomechanical properties of the hydrogel(43). On the opposite, it is likely that an excess of particles may interfere the fibrin-agarose polymerization process and alter the mesh polymerization. Regarding differences among the types of nanoparticles, previous reports showed that AMK and SCM have different hydrophobicity(27). While AMK has lower water solubility and a higher affinity for the lipid phase, SCM typically has higher encapsulation efficacy and slower drug released profile. If this factor may influence linkage of NLCs to the fibrin-agarose fibers should be determined in future studies.