Treatment of severe or chronic skin wounds is an important challenge facing medicine and a significant health care burden. Proper wound healing is often affected by bacterial infection; where biofilm formation is one of the main risks and particularly problematic because it confers protection to microorganisms against antibiotics. One avenue to prevent bacterial colonization of wounds is the use of silver nanoparticles (AgNPs); which have proved to be effective against non-multidrug-resistant and multidrug-resistant bacteria. In addition, the use of mesenchymal stem cells (MSC) is an excellent option to improve wound healing due to their capability for differentiation and release of relevant growth factors. Finally, radiosterilized pig skin (RPS) is a biomatrix successfully used as wound dressing to avoid massive water loss, which represents an excellent carrier to deliver MSC into wound beds. Together, AgNPs, RPS and MSC represent a potential dressing to control massive water loss, prevent bacterial infection and enhance skin regeneration; three essential processes for appropriate wound healing with minimum scaring. We synthesized stable 10 nm-diameter spherical AgNPs that showed 21- and 16-fold increase in bacteria growth inhibition (in comparison to antibiotics) against clinical strains Staphylococcus aureus and Stenotrophomonas maltophilia, respectively. RPS samples were impregnated with different AgNPs suspensions to develop RPS-AgNPs nanocomposites with different AgNPs concentrations. Nanocomposites showed inhibition zones, in Kirby–Bauer assay, against both clinical bacteria tested. Nanocomposites also displayed antibiofilm properties against S. aureus and S. maltophilia from RPS samples impregnated with 250 and 1000 ppm AgNPs suspensions, respectively. MSC were isolated from adipose tissue and seeded on nanocomposites; cells survived on nanocomposites impregnated with up to 250 ppm AgNPs suspensions, showing 35% reduction in cell viability, in comparison to cells on RPS. Cells on nanocomposites proliferated with culture days, although the number of MSC on nanocomposites at 24 h of culture was lower than that on RPS. AgNPs with better bactericide activity than antibiotics were synthesized. RPS-AgNPs nanocomposites impregnated with 125 and 250 ppm AgNPs suspensions decreased bacterial growth, decreased biofilm formation and were permissive for survival and proliferation of MSC; constituting promising multi-functional dressings for successful treatment of skin wounds.

Human adipose-derived mesenchymal stem cells (hADMSCs) are believed to be potential key factors for starting the regenerative process after tissue injury. However, an efficient method of delivering these regenerative cells to an external wound site is still lacking. Human amnion and pig skin have long been used as skin wound dressings for the treatment of burns and other skin lesions. Herein, we present the generation of two constructs using these two biomaterials as effective scaffolds for the culture of hADMSCs. It was found that hADMSCs seeded onto radiosterilized human amnion and pig skin are viable and proliferate. These cells are able to migrate over these scaffolds as demonstrated by using time-lapse microscopy. In addition, the scaffolds induce hADMSCs to secrete interleukin-10, an important negative regulator of inflammation, and interleukin-1β, a proinflammatory protein. The interplay between these two proteins has been proven to be vital for a balanced restoration of all necessary tissues. Thus, radiosterilized human amnion and pig skin are likely suitable scaffolds for delivery of hADMSCs transplants that could promote tissue regeneration in skin injuries like patients with burn injuries.

Extensive burns represent a significant challenge in biomedicine due to the multiple systemic and localized complications resulting from the major skin barrier loss. The functionalization of xenografts with nanostructured antibacterial agents proposes a fast and accessible application to restore barrier function and prevent localized bacterial contamination. Based on this, the objective of this work was to functionalize a xenograft by electrospray deposition with silver nanoparticles (AgNPs) and to evaluate its antibiofilm and cytotoxic effects on human fibroblasts. Initially, AgNPs were synthesized by a green microwave route with sizes of 2.1, 6.8, and 12.2 nm and concentrations of 0.055, 0.167, and 0.500 M, respectively. The AgNPs showed a size relationship directly proportional to the concentration of AgNO₃, with a spherical and homogeneous distribution determined by high-resolution transmission electron microscopy. The surface functionalization of radiosterilized porcine skin (RPS) via electrospray deposition with the three AgNP concentrations (0.055, 0.167, and 0.500 M) in the epidermis and the dermis showed a uniform distribution on both surfaces by energy-dispersive X-ray spectroscopy. The antibiofilm assays of clinical multidrug-resistant Pseudomonas aeruginosa showed significant effects at the concentrations of 0.167 and 0.500 M, with a log reduction of 1.3 and 2.6, respectively. Additionally, viability experiments with human dermal fibroblasts (HDF) exposed to AgNPs released from functionalized porcine skin showed favorable tolerance, with retention of viability more significant than 90% for concentrations of 0.05 and 0.167 M after 24 h exposure. Antibacterial activity combined with excellent biocompatibility makes this biomaterial a candidate for antibacterial protection by inhibiting bacterial biofilms in deep burns during early stages of development.

Radiosterilized pig skin (RPS) has been used as a dressing for burns since the 1980s. Its similarity to human skin in terms of the extracellular matrix (ECM) allows the attachment of mesenchymal stem cells, making it ideal as a scaffold to create cellularized constructs. The use of silver nanoparticles (AgNPs) has been proven to be an appropriate alternative to the use of antibiotics and a potential solution against multidrug-resistant bacteria. RPS can be impregnated with AgNPs to develop nanomaterials capable of preventing wound infections. The main goal of this study was to assess the use of RPS as a scaffold for autologous fibroblasts (Fb), keratinocytes (Kc), and mesenchymal stem cells (MSC) in the treatment of second-degree burns (SDB). Additionally, independent RPS samples were impregnated with AgNPs to enhance their properties and further develop an antibacterial dressing that was initially tested using a burn mouse model. This protocol was approved by the Research and Ethics Committee of the INRLGII (INR 20/19 AC). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis of the synthesized AgNPs showed an average size of 10 nm and rounded morphology. Minimum inhibitory concentrations (MIC) and Kirby–Bauer assays indicated that AgNPs (in solution at a concentration of 125 ppm) exhibit antimicrobial activity against the planktonic form of S. aureus isolated from burned patients; moreover, a log reduction of 1.74 ± 0.24 was achieved against biofilm formation. The nanomaterial developed with RPS impregnated with AgNPs solution at 125 ppm (RPS-AgNPs125) facilitated wound healing in a burn mouse model and enhanced extracellular matrix (ECM) deposition, as analyzed by Masson’s staining in histological samples. No silver was detected by energy-dispersive X-ray spectroscopy (EDS) in the skin, and neither by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in different organs of the mouse burn model. Calcein/ethidium homodimer (EthD-1), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and scanning electron microscopy (SEM) analysis demonstrated that Fb, Kc, and MSC could attach to RPS with over 95% cell viability. Kc were capable of releasing FGF at 0.5 pg above control levels, as analyzed by ELISA assays. An autologous RPS-Fb-Kc construct was implanted in a patient with SDB and compared to an autologous skin graft. The patient recovery was assessed seven days post-implantation, and the patient was followed up at one, two, and three months after the implantation, exhibiting favorable recovery compared to the gold standard, as measured by the cutometer. In conclusion, RPS effectively can be used as a scaffold for the culture of Fb, Kc, and MSC, facilitating the development of a cellularized construct that enhances wound healing in burn patients.