Radiosterilized Pig Skin, Silver Nanoparticles and Skin Cells as an Integral Dressing Treatment for Burns: Development, Pre-Clinical and Clinical Pilot Study.

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.

Development of Radiosterilized Porcine Skin Electrosprayed with Silver Nanoparticles Prevents Infections in Deep Burns.

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.

Allogeneic Limbal Epithelial Transplantation Modified With Solid Platelet-Rich Plasma for Bilateral Limbal Stem Cell Deficiency.

PURPOSE: To present successful management of bilateral limbal stem cell deficiency (LSCD) by using an allogeneic limbal epithelial stem cell transplantation together with solid activated platelet-rich plasma (PRP). METHODS: A 59-year-old man with a history of bilateral LSCD due to penicillin-induced Stevens-Johnson Syndrome suffered from a lime corneal burn in his right eye, leading to a total LSCD with severely reduced visual acuity. After stabilizing the ocular surface, we performed an allogeneic limbal epithelial transplantation from a cadaveric donor using an autologous clot of PRP to cover the limbal grafts to nourish the ocular surface microenvironment. RESULTS: At the first week after the procedure, the corneal epithelium had fully reepithelized. At month 3, visual acuity improved from hand motion to 20/70. CONCLUSIONS: In this case, this new modified procedure was a promising, easy-to-perform, apparently safe, and effective treatment option to enhanced epithelial wound healing in ocular surface diseases. To our knowledge, this is the first report describing the incorporation of solid PRP in limbal transplantation procedures.

Overview on radiation and tissue banking in Latin America.

The International Atomic Energy Agency (IAEA) played an important role in the establishment of new tissue banks and the improvement of already existing ones in Latin America. The Agency strongly supported, through regional, interregional and national technical cooperation projects, providing equipment, expert missions and training for the production and application of human tissues for transplantation. From 1999 to 2005 five regional courses were given in Buenos Aires under the modality of 1-year distance learning training courses and 1-week face to face courses. The courses were organized by the IAEA, through the National Atomic Energy Commission (CNEA) and the Faculty of Medicine of Buenos Aires University as Post Graduate Specialization Course. In 2005 the Latin American countries joined with Spain and Portugal, and created the Ibero American Network Council of Donation and Transplant (Red Consejo Iberoamericano de Donación y Trasplantes-RCIDT). The objective of this network is to cooperate among twenty-one Ibero American countries in organizational and legislative aspects, training of professionals, and ethical and social issues related to the donation and transplantation of organs, tissues and cells. The members of this Network work actively to harmonize the regulations and the control of donation and transplantation of human organs, tissues and cells. At present, in Latin America, more than 220 facilities of tissues banks are operating and tissue allografts are being produced by single and multi-tissue banks. The efforts made by the governments and professionals from the region allow the tissue banks to operate under quality systems and introduce new technologies.

Generation of Two Biological Wound Dressings as a Potential Delivery System of Human Adipose-Derived Mesenchymal Stem Cells.

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-1ß, 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.

Use of irradiated human amnion as a matrix for limbal stem cell culture.

Several ocular diseases affect the corneal surface; the development of effective technologies for the treatment of corneal lesions has brought about an improvement in the quality of life of affected patients. The aim of this study is to culture and characterize limbal stem cells cultured on gamma ((60)Co) radiosterilized human amnion (RHA). Limbal stem cells were isolated from ten preserved samples of corneal transplant. The cells were cultured since primary culture until expanded cells on RHA and stained with monoclonal antibodies to establish their immunophenotype, after which cytokeratin 12 and Vimentin were positive by immunohistochemistry. The immunophenotype remained constant since primary culture until expanded cells in RHA. The RHA and cells construct were structurally integrated. Immunohistochemistry was cytokeratin 12, Vimentin positive, and cytokeratin 19 negative. In vitro limbal cells maintain a constant epithelial transition immunophenotype in culture up to primary culture until expanded cells on RHA.

A review of the International Atomic Energy Agency (IAEA) international standards for tissue banks.

The IAEA International Standards for Tissue Banks published in 2003 were based on the Standards then currently in use in the USA and the European Union, among others, and reflect the best practices associated with the operation of a tissue bank. They cover legal, ethical and regulatory controls as well as requirements and procedures from donor selection and tissue retrieval to processing and distribution of finished tissue for clinical use. The application of these standards allows tissue banks to operate with the current good tissue practice, thereby providing grafts of high quality that satisfy the national and international demand for safe and biologically useful grafts. The objective of this article is to review the IAEA Standards and recommend new topics that could improve the current version.

Coding and traceability used by tissue banks in Mexico.

This short communication describes how some Mexican tissue banks have established their own system for coding and traceability of tissues.

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Mexico.

Tissue banking started in Mexico in 1948-1949, when two bone banks were established, one at the Infantile Hospital of Mexico and other at the Central Military Hospital. Mexico has benefited for the implementation of the IAEA program since through it has been able to settle down and to consolidate the Tissue Bank at the Instituto Nacional de Investigaciones Nucleares ININ (National Institute for Nuclear Research). This is the only bank in Latin America that has a Quality Management System in force, certified under ISO 9001:2000 since August 1, 2003. The first tissue processed was amnion. The main products of the BTR are amnion and pig skin. Both are biological tissues which their main use is as a wound dressing in patients with burns, scars, diabetic ulcers, epidermolysis bullosa, damaged ocular surface, etc. The General Health Law, published in 1984 and reformed in June 19, 2007, describes the procedure for the disposal of organs, tissues and human cadavers in its fourteenth title and in the Regulation for Sanitary Control. During the period 2001-2005, the ININ Tissue Bank produced 292 sterilised tissues (amnion, 86,668 cm(2), and frozen pig skin, 164,220 cm(2), at an estimated cost of 1,012,668 Mexican pesos. Until 2006, one hundred eighty five (185) patients have been treated with the use of sterilised tissues produced by the ININ Tissue Bank. The radiation source used for sterilisation of tissues is an industrial Cobalt-60 irradiator model JS-6500 AECL, which belongs to ININ. This equipment is located in other building, close to the BTR, in the Centro Nuclear de México "Dr. Nabor Carrillo Flores" (Nuclear Center of Mexico). Until 2006, six hospitals use in a routine way the sterilised tissues produced by the ININ Tissue Bank, for the treatment of burns originated by diverse agents like flame, electricity, liquids in boil, chemical reagents, as well as for the reconstruction of the ocular surface. Two of these hospitals treat patients of very low economic incomes, mainly needy individuals, who cannot afford to pay this type of treatments in other hospitals due to their high cost. The results obtained up to now are highly promising.

The tissue bank at the Instituto Nacional de Investigaciones Nucleares: ISO 9001:2000 certification of its quality management system.

UNLABELLED: Tissue banking is a complex operation concerned with the organisation and coordination of all the steps, that is, from donor selection up to storage and distribution of the final products for therapeutic, diagnostic, instruction and research purposes. An appropriate quality framework should be established in order to cover all the specific methodology as well as the general aspects of quality management, such as research and development, design, instruction and training, specific documentation, traceability, corrective action, client satisfaction, and the like. Such a framework can be obtained by developing a quality management system (QMS) in accordance with a suitable international standard: ISO 9001:2000. This paper presents the implementation process of the tissue bank QMS at the Instituto Nacional de Investigaciones Nucleares in Mexico. OBJECTIVE: The objective of the paper is to share the experience gained by the tissue bank personnel [radiosterilised tissue bank (BTR)] at the Instituto Nacional de Investigaciones Nucleares (ININ, National Institute of Nuclear Research), during implementation of the ISO 9001:2000 certification process. At present, the quality management system (QMS) of ININ also complies with the Mexican standard NMX-CC-9001:2000. The scope of this QMS is Research, Development and Processing of Biological Tissues Sterilised by Gamma Radiation, among others.

Application of biological dressings from radiosterilized amnios with cobalt 60 and serologic studies on the handling of burns in pediatric patients.

We present the use of biological dressing (amnios) as an alternative for skin replacement due to burn accidents. With the use of serologic tests, it is guaranteed to be free of the possibility of transmitting infectious diseases. The amnios is sterilized with 60Co gamma radiation. In this way, amnios is free of bacteria and fungi. In addition, with the use of serologic tests at the moment of the childbirth and 6 months later, we can be sure that it won't transmit the syphilis, AIDS, hepatitis b and c viruses. This treatment was applied to 12 children with burns of 1 degree and 2 degrees degree (7 girls and 5 boys) that required hospitalization. The application is very simple. It does not require of anesthesic procedure, or the use of surgical room. The pain decreased notoriously, and the procedure can be repeated as frequently as necessary. Once the dressing is applied, it does not require any type of surgical cleaning. The scaring process begins at the borders and under the dressing, where there is a new tissue. It decreases: 1) the possibility of infection, and therefore reduces the consumption of antibiotic; 2) the use of analgesics; 3) the time of scaring and 4) the number of days of hospitalization.