Development of a full-thickness acellular dermal graft from human skin: Case report of first patient rotator cuff patch augmentation repair.

The processing and initial testing of a new human tissue preparation is described. Full-thickness Acellular Dermal Matrix (ftADM) is the extracellular matrix (ECM) obtained by decellularization of full-thickness human skin from cadaveric donors. The safety, stability and usability of the graft are discussed with respect to the results of the residual cellular content, maintenance of ECM components, and biomechanical properties. Quantitative and qualitative analysis of the ECM demonstrated the absence of cell debris, while the native structure of human dermis was maintained. Biomechanical testing showed stiffness values comparable to other commercial products used for tendon reinforcement, suggesting that our ftADM could be successfully used not only in soft tissue regeneration surgeries, but also in tendon reinforcement. First case of ftADM in rotator cuff augmentation is described. Technical management of the patch during surgery and clinical outcomes are discussed.

Fast protocol for the processing of split-thickness skin into decellularized human dermal matrix.

BACKGROUND: Dermal scaffolds for tissue regeneration are nowadays an effective alternative in not only wound healing surgeries but also breast reconstruction, abdominal wall reconstruction and tendon reinforcement. The present study describes the development of a decellularization protocol applied to human split-thickness skin from cadaveric donors to obtain dermal matrix using an easy and quick procedure. METHODS: Complete split-thickness donor was decellularized through the combination of hypertonic and enzymatic methods. To evaluate the absence of epidermis and dermal cells, and ensure the integrity of the extracellular matrix (ECM) structure, histological analysis was performed. Residual genetic content and ECM biomolecules (collagen, elastin, and glycosaminoglycan) were quantified and tensile strength was tested to measure the effect of the decellularization technique on the mechanical properties of the tissue. RESULTS: Biomolecules quantification, residual genetic content (below 50 ng/mg dry tissue) and histological structure assessment showed the efficacy of the decellularization process and the preservation of the ECM. The biomechanical tests confirmed the preservation of native properties in the acellular tissue. CONCLUSIONS: The acellular dermal matrix obtained from whole split-thickness skin donor with the newly developed decellualrization protocol, maintains the desired biomechanical and structural properties and represents a viable treatment option for patients.

Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol.

Injuries to the peripheral nerves represent a frequent cause of permanent disability in adults. The repair of large nerve lesions involves the use of autografts, but they have several inherent limitations. Overcoming these limitations, the use of decellularized nerve matrix has emerged as a promising treatment in tissue regenerative medicine. Here, we generate longer human decellularized nerve segments with a novel decellularization method, using nonionic, zwitterionic, and enzymatic incubations. Efficiency of decellularization was measured by DNA quantification and cell remnant analysis (myelin, S100, neurofilament). The evaluation of the extracellular matrix (collagen, laminin, and glycosaminoglycans) preservation was carried out by enzyme-linked immunosorbent assay (ELISA) or biochemical methods, along with histological and immunofluorescence analysis. Moreover, biomechanical properties and cytocompatibility were tested. Results showed that the decellularized nerves generated with this protocol have a concentration of DNA below the threshold of 50 ng/mg of dry tissue. Furthermore, myelin, S100, and MHCII proteins were absent, although some neurofilament remnants could be observed. Moreover, extracellular matrix proteins were well maintained, as well as the biomechanical properties, and the decellularized nerve matrix did not generate cytotoxicity. These results show that our method is effective for the generation of decellularized human nerve grafts. The generation of longer decellularized nerve segments would allow the understanding of the regenerative neurobiology after nerve injuries in both clinical assays and bigger animal models. Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol. Combination of zwitterionic, non-ionic detergents, hyperosmotic solution and nuclease enzyme treatment remove cell remnants, maintain collagen, laminin and biomechanics without generating cytotoxic leachables.

Terapia fotodinámica antimicrobiana in vitro aplicada sobre Trichophyton mentagrophytes con nuevo azul de metileno como fotosensibilizador / In vitro antimicrobial photodynamic therapy against Trichophyton mentagrophytes using new methylene blue as the photosensitizer

INTRODUCCIÓN Y OBJETIVOS: La terapia fotodinámica antimicrobiana combina el uso de un fármaco fotosensibilizante, la luz y el oxígeno para erradicar microorganismos patógenos. Trichophyton mentagrophytes es un hongo dermatofito capaz de invadir la piel y tejidos queratinizados. El objetivo de este trabajo es aplicar la terapia fotodinámica antimicrobiana para la inactivación in vitro de T. mentagrophytes utilizando el nuevo azul de metileno como agente fotosensibilizador. MATERIAL Y MÉTODOS: Se aplica un diseño factorial completo para optimizar los parámetros que permiten la fotoinactivación del dermatofito. Se tiene en cuenta la concentración del nuevo azul de metileno, el tiempo de contacto entre el fotosensibilizador y el hongo antes del tratamiento con luz y la fluencia de luz roja aplicada entre 620 y 645 nm. RESULTADOS: La mínima concentración de nuevo azul de metileno que produce una mortalidad de todas las células de T. mentagrophytes de la suspensión inicial (concentración∼106 ufc/ml) es 50 M para una fluencia de 81 J/cm-2 y un tiempo previo de contacto hongo-fotosensibilizador de 10 min. Si se aumenta la concentración a 100 M la fluencia que se necesita disminuye a 9 J/cm-2. CONCLUSIONES: La comparación de nuestros datos con otros publicados muestra que la susceptibilidad de T. mentagrophytes a la terapia fotodinámica antimicrobiana con nuevo azul de metileno es cepa-dependiente. El nuevo azul de metileno es un fotosensibilizador a tener en cuenta para el tratamiento de las micosis cutáneas causadas por este dermatofito

INTRODUCTION AND OBJECTIVES: Antimicrobial photodynamic therapy combines the use of a photosensitizing drug with light and oxygen to eradicate pathogens. Trichophyton mentagrophytes is a dermatophytic fungus able to invade the skin and keratinized tissues. We have investigated the use of new methylene blue as the photosensitizing agent for antimicrobial photodynamic therapy to produce the in vitro inactivation of T mentagrophytes. MATERIAL AND METHODS: A full factorial design was employed to optimize the parameters for photoinactivation of the dermatophyte. The parameters studied were new methylene blue concentration, contact time between the photosensitizing agent and the fungus prior to light treatment, and the fluence of red light (wavelength, 620---645 nm) applied. RESULTS: The minimum concentration of new methylene blue necessary to induce the death of all T. mentagrophytes cells in the initial suspension (approximate concentration, 106 colony forming units per milliliter) was 50 M for a fluence of 81 J/cm2 after a contact time of 10 minutes with the photosensitizing-agent. Increasing the concentration to 100 M allowed the fluence to be decreased to 9 J/cm2. CONCLUSIONS: Comparison of our data with other published data shows that the susceptibility of T. mentagrophytes to antimicrobial photodynamic therapy with new methylene blue is straindependent. New methylene blue is a photosensitizing agent that should be considered for the treatment of fungal skin infections caused by this dermatophyte

In Vitro Antimicrobial Photodynamic Therapy Against Trichophyton mentagrophytes Using New Methylene Blue as the Photosensitizer. / Terapia fotodinámica antimicrobiana in vitro aplicada sobre Trichophyton mentagrophytes con nuevo azul de metileno como fotosensibilizador.

INTRODUCTION AND OBJECTIVES: Antimicrobial photodynamic therapy combines the use of a photosensitizing drug with light and oxygen to eradicate pathogens. Trichophyton mentagrophytes is a dermatophytic fungus able to invade the skin and keratinized tissues. We have investigated the use of new methylene blue as the photosensitizing agent for antimicrobial photodynamic therapy to produce the in vitro inactivation of T mentagrophytes. MATERIAL AND METHODS: A full factorial design was employed to optimize the parameters for photoinactivation of the dermatophyte. The parameters studied were new methylene blue concentration, contact time between the photosensitizing agent and the fungus prior to light treatment, and the fluence of red light (wavelength, 620-645nm) applied. RESULTS: The minimum concentration of new methylene blue necessary to induce the death of all T. mentagrophytes cells in the initial suspension (approximate concentration, 106 colony forming units per milliliter) was 50µM for a fluence of 81J/cm2 after a contact time of 10minutes with the photosensitizing-agent. Increasing the concentration to 100µM allowed the fluence to be decreased to 9J/cm2. CONCLUSIONS: Comparison of our data with other published data shows that the susceptibility of T. mentagrophytes to antimicrobial photodynamic therapy with new methylene blue is strain-dependent. New methylene blue is a photosensitizing agent that should be considered for the treatment of fungal skin infections caused by this dermatophyte.