Mario Alberto Pérez-Díaz

Cross-linked polymer blends from natural compounds, namely gelatin (Gel), chitosan (CS), and synthetic poly (vinyl alcohol) (PVA), have received increasing scrutiny because of their versatility, biocompatibility, and ease of use for tissue engineering. Previously, Gel/CS/PVA [1:1:1] hydrogel produced via the freeze-drying process presented enhanced mechanical properties. This study aimed to investigate the biocompatibility and chondrogenic potential of a steam-sterilized Gel/CS/PVA hydrogel using differentiation of human adipose-derived mesenchymal stromal cells (AD-hMSC) and cartilage marker expression. AD-hMSC displayed fibroblast-like morphology, 90% viability, and 69% proliferative potential. Mesenchymal profiles CD73 (98.3%), CD90 (98.6%), CD105 (97.0%), CD34 (1.11%), CD45 (0.27%), HLA-DR (0.24%); as well as multilineage potential, were confirmed. Chondrogenic differentiation of AD-hMSC in monolayer revealed the formation of cartilaginous nodules composed of glycosaminoglycans after 21 days. Compared to nonstimulated cells, hMSC-derived chondrocytes shifted the expression of CD49a from 2.82% to 40.6%, CD49e from 51.4% to 92.2%, CD54 from 9.66 to 37.2%, and CD151 from 45.1% to 75.8%. When cultured onto Gel/CS/PVA hydrogel during chondrogenic stimulation, AD-hMSC changed to polygonal morphology, and chondrogenic nodules increased by day 15, six days earlier than monolayer-differentiated cells. SEM analysis showed that hMSC-derived chondrocytes adhered to the surface with extended filopodia and abundant ECM formation. Chondrogenic nodules were positive for aggrecan and type II collagen, two of the most abundant components in cartilage. This study supports the biocompatibility of AD-hMSC onto steam-sterilized GE/CS/PVA hydrogels and its improved potential for chondrocyte differentiation. Hydrogel properties were not altered after steam sterilization, which is relevant for biosafety and biomedical purposes.

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.

Apical periodontitis is an inflammation leading to the injury and destruction of periradicular tissues. It is a sequence of events that starts from root canal infection, endodontic treatment, caries, or other dental interventions. Enterococcus faecalis is a ubiquitous oral pathogen that is challenging to eradicate because of biofilm formation during tooth infection. This study evaluated a hydrolase (CEL) from the fungus Trichoderma reesei combined with amoxicillin/clavulanic acid as a treatment against a clinical E. faecalis strain. Electron microscopy was used to visualize the structure modification of the extracellular polymeric substances. Biofilms were developed on human dental apices using standardized bioreactors to evaluate the antibiofilm activity of the treatment. Calcein and ethidium homodimer assays were used to evaluate the cytotoxic activity in human fibroblasts. In contrast, the human-derived monocytic cell line (THP-1) was used to evaluate the immunological response of CEL. In addition, the secretion of the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine IL-10 were measured by ELISA. The results demonstrated that CEL did not induce the secretion of IL-6 and TNF-α when compared with lipopolysaccharide used as a positive control. Furthermore, the treatment combining CEL with amoxicillin/clavulanic acid showed excellent antibiofilm activity, with a 91.4% reduction in CFU on apical biofilms and a 97.6% reduction in the microcolonies. The results of this study could be used to develop a treatment to help eradicate persistent E. faecalis in apical periodontitis.

Psoriasis and atopic dermatitis (AD) are characterized by enhanced skin inflammation, which results in hyperproliferation and the recruitment of immune cells into the skin. For that reason, it is needed a chemical capable to reduce cell proliferation and the recruitment of cells. The search for new molecules for therapeutic skin treatment mainly focuses on the antioxidant and anti-inflammatory properties, highlighting the rheological properties of polymeric polypeptides. We studied L-arginine (L-Arg) grafted (-g-) to enzymatic poly(gallic acid) (PGAL). The latter is a multiradical antioxidant with greater properties and thermal stability. The derivative was enzymatically polymerized in an innocuous procedure. The poly(gallic acid)-g-L-Arg molecule (PGAL-g-L-Arg) inhibits bacterial strains which also have been involved in the progression of psoriasis and AD. However, it is important to analyze their biological effect on skin cells. The cell viability was analyzed by calcein/ethidium homodimer assays and crystal violet. The proliferation and cell attachment were determined by a curve of time and quantitation of the optical density of crystal violet. To analyze the cell migration a wound-healing assay was performed. This synthesis demonstrates that it is not cytotoxic at high concentrations (250 μg/mL). We observed a decrease in the proliferation, migration, and adhesion of dermal fibroblasts in vitro but the compound could not avoid the increase of reactive oxygen species in the cell. Based on our findings, PGAL-g-L-Arg is a promising candidate for treating skin diseases such as psoriasis and AD where decreasing the proliferation and cell migration could help to avoid inflammation.

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.

Hyaluronic acid (HA) is one of the most attractive natural polymers employed in biomaterials with biological applications. This polysaccharide is found in different tissues of the body because it is a natural component of the extracellular matrix; furthermore, it has crucial functions in cell growth, migration, and differentiation. Since its biological characteristics, HA has been utilized for the new biomaterial’s development for tissue engineering, such as hydrogels. These hydrophilic macromolecular networks have gained significant attention due to their unique properties, making them potential candidates to be applied in biomedical fields. Different mechanisms to obtain hydrogels have been described. However, the research of new non-toxic methods has been growing in recent years. In this study, we prepared a new hydrogel of HA and polyvinyl alcohol by the cost-effective technique of cross-linking by gamma irradiation. The hydrogel was elaborated for the first time and was characterized by several methods such as Fourier Transform Infrared Spectroscopy, Differential Scanning Calorimetry, Thermogravimetric Analysis, and Scanning Electron Microscopy. Likewise, we evaluated the cytotoxicity of the biomaterial and its influence on cell migration in human fibroblasts. Furthermore, we provide preliminary evidence of the wound closure effect in a cellular wound model. The novel hydrogel offers an increase of HA stability with the potential to expand the useful life of HA in its different medical applications.

The purpose of our study was to obtain new wound dressings in the form of hydrogels that promote wound healing taking advantage of the broad activities of elastin (ELT) in physiological processes. The hydrogel of ELT and polyvinylpyrrolidone (PVP; ELT–PVP) was obtained by cross-linking induced by gamma irradiation at a dose of 25 kGy. The physicochemical changes attributed to cross-linking were analyzed through scanning electron microscopy (SEM), infrared spectroscopy analysis with Fourier transform (FTIR), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Furthermore, we performed a rheological study to determine the possible changes in the fluidic macroscopic properties produced by the cross-linking method. Finally, we accomplished viability and proliferation analyses of human dermal fibroblasts in the presence of the hydrogel to evaluate its biological characteristics. The hydrogel exhibited a porous morphology, showing interconnected porous with an average pore size of 16 ± 8.42 µm. The analysis of FTIR, DSC, and TGA revealed changes in the chemical structure of the ELT–PVP hydrogel after the irradiation process. Also, the hydrogel exhibited a rheological behavior of a pseudoplastic and thixotropic fluid. The hydrogel was biocompatible, demonstrating high cell viability, whereas ELT presented low biocompatibility at high concentrations. In summary, the hydrogel obtained by gamma irradiation revealed the appropriate morphology to be applied as a wound dressing. Interestingly, the hydrogel exhibited a higher percentage of cell viability compared with ELT, suggesting that the cross-linking of ELT with PVP is a suitable strategy for biological applications of ELT without generating cellular damage.

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.