Case report: Long-term follow-up of a large full-thickness skin defect treated with a photosynthetic scaffold for dermal regeneration.

It is broadly described that almost every step of the regeneration process requires proper levels of oxygen supply; however, due to the vascular disruption in wounds, oxygen availability is reduced, being detrimental to the regeneration process. Therefore, the development of novel biomaterials combined with improved clinical procedures to promote wound oxygenation is an active field of research in regenerative medicine. This case report derives from a cohort of patients enrolled in a previously published ongoing phase I clinical trial (NCT03960164), to assess safety of photosynthetic scaffolds for the treatment of full skin defects. Here, we present a 56 year old patient, with a scar contracture in the cubital fossa, which impaired the elbow extension significantly affecting her quality of life. As part of the treatment, the scar contracture was removed, and the full-thickness wound generated was surgically covered with a photosynthetic scaffold for dermal regeneration, which was illuminated to promote local oxygen production. Then, in a second procedure, an autograft was implanted on top of the scaffold and the patient's progress was followed for up to 17 months. Successful outcome of the whole procedure was measured as improvement in functionality, clinical appearance, and self-perception of the treated area. This case report underscores the long-term safety and applicability of photosynthetic scaffolds for dermal regeneration and their stable compatibility with other surgical procedures such as autograft application. Moreover, this report also shows the ability to further improve the clinical outcome of this procedure by means of dermal vacuum massage therapy and, more importantly, shows an overall long-term improvement in patient´s quality of life, supporting the translation of photosynthetic therapies into human patients.

Método de aislamiento, cultivo y precondicionamiento con hipoxia de células madre derivadas de tejido adiposo en ratas / Method of isolation, culture and hypoxia preconditioning of adipose tissue stem cells in rats

Introducción y objetivo. Las células madre son candidatas terapéuticas para una amplia gama de enfermedades. Resultan de gran interés en Cirugía Plástica para el tratamiento de heridas crónicas, transferencia de tejido adiposo y colgajos. El objetivo de este estudio es describir el método de aislamiento, cultivo y caracterización de células madre derivadas de tejido adiposo y cómo estas pueden precondicionarse con hipoxia y generar cambios in vitro en su capacidad proliferativa y migratoria. El trabajo es un complemento didáctico a otro publicado por nosotros en esta misma revista utilizando esta metodología en comparación al grupo de retardo de colgajo y grupo control en colgajos cutáneos aleatorizados en ratas. Métodos. Obtuvimos las células madre de tejido graso ínguino-abdominal de ratas adultas: 10 en el grupo de células madre derivadas de tejido adiposo y otras 10 en el grupo de células madre derivadas de tejido adiposo precondicionadas con hipoxia (2% O2 y 5% CO2). Realizamos análisis morfológico directo y con inmunofluorescencia con el marcador vimentina y CD90 y estudio de proliferación y migración celular in vitro. Resultados: Utilizamos en promedio 1.64 +/- 1.13 gr de tejido adiposo en el grupo sin precondicionamiento y 0.93 +/- 0.34 gr en el grupo con precondicionamiento con hipoxia para el aislamiento. Las células madre derivadas de tejido adiposo precondicionadas con hipoxia presentaron un aumento de la capacidad migratoria a las 24 horas de 2.44 +/- 0.85 mm frente a 2.24 +/- 0.82 mm (p ≤ 0.01) y proliferativa 5.42 x105 +/- 1.03 x105 céls/ml frente a 3.26 x105 +/- 8.61 x104 céls/ml) (p ≤ 0.001) de forma significativa en comparación a las sin precondicionamiento. Conclusiones. Describimos en detalle un método de precondicionamiento de células madre mediante hipoxia. Logramos potenciar el efecto de las células madre aumentando en forma significativa su capacidad migratoria y proliferativa de forma precoz

Background and objective. Stem cells are therapeutic candidates for a wide range of diseases. They are of great interest in Plastic Surgery for the management of chronic wounds, adipose tissue transfer and flaps. The objective of this study is to describe the method of isolation, culture and characterization of stem cells derived from adipose tissue and how these can be preconditioned with hypoxia and generate in vitro changes in their proliferative and migratory capacity. This study is a didactic supplement to a paper published by us in this same journal using this methodology in comparison to the group of flap delay and control group in skin flaps randomized in rats. Methods. Stem cells were obtained from inguinal-abdominal fatty tissue of adult rats: 10 in the group of stem cells derived from adipose tissue and another 10 in the group of stem cells derived from adipose tissue preconditioned with hypoxia (2% O2 and 5% CO2). Direct morphological analysis was carried out and with immunofluorescence (vimentin and CD90 marker). Study proliferation and in vitro cell migration was performed. Results. An average of 1.64 +/- 1.13 gr of adipose tissue of the inguinoabdominal area was used in the group of stem cells without preconditioning and 0.93 +/- 0.34 gr. in the group with hypoxic preconditioning for the isolation. Stem cells derived from adipose tissue preconditioned with hypoxia showed an increase in migratory capacity at 24 hours of 2.44 +/- 0.85 mm v/s 2.24 +/- 0.82 mm (p ≤ 0.01) and proliferative of 5.42 x105 +/- 1.03 x105 cells / ml v/s 3.26 x 105 +/- 8.61 x104 cells / ml) (p ≤0.001) significantly compared to those without preconditioning. Conclusions. A method of preconditioning stem cells by hypoxia is described in detail. It is possible to enhance the effect of the stem cells, significantly increasing their early migratory and proliferative capacity

Clasificación y grado de necrosis en colgajo cutáneo aleatorio en ratas tras tratamiento con células madre precondicionadas con hipoxia y retardo del colgajo / Classification and score of necrosis in random skin flap in rats after treatment with transplantation of hypoxia preconditioned adipose-derived stem cells and flap delay

Introducción y Objetivo: Realizar una clasificación de necrosis en colgajos cutáneos aleatorios (predispuesto a necrosis) según los hallazgos macroscópicos e histopatológicos, con o sin tratamiento para aumentar la supervivencia del colgajo, podría ser útil para analizar de forma global el colgajo y evaluar distintas modalidades para disminuir la necrosis. Nuestro objetivo es evaluar si existe más de un patrón de necrosis cutánea del colgajo cutáneo aleatorio al séptimo día de postoperatorio realizando una clasificación de necrosis y comparar la efectividad de los métodos de retardo del colgajo y células madres derivadas de tejido adiposo (ASCs) con y sin precondicionamiento para aumentar la supervivencia del colgajo. Material y Método: Utilizamos 40 ratas macho adultas sometidas a colgajo cutáneo aleatorio en el área dorsal de 2 x 8 cm agrupadas en control, RTC (retardo del colgajo), ASCs y ASCs-H (células madre derivadas de tejido adiposo precondicionadas con hipoxia). Realizamos cálculo del área, puntuación de necrosis y estudio histológico de los colgajos al séptimo día con hematoxilina-eosina, inmunohistoquímica con VEGF (factor de crecimiento endotelial vascular) y cuantificación del colágeno tipo I y III. Resultados: Según la clasificación, el tipo 0 fue considerado como piel normal; tipo 0+ con cambio adaptativo reversible; tipo 1 necrosis menor con inflamación superficial; tipo 2 necrosis menor con inflamación profunda; y finalmente el tipo 3 necrosis en su grado máximo. La piel sana mantiene la coloración y textura elástica y blanda. A medida que progresa la necrosis cambia hacia un tono más oscuro, y la textura y consistencia van aumentando hasta tornarse acartonada y delgada. En los tipos 0+ y 1 observamos focos de reepitelización temprana al séptimo día, mientras que en estadios más avanzados la necrosis fue completa. El grupo de RTC presenta una menor puntuación y por lo tanto necrosis de menor grado respecto al resto de los grupos tratados. Por otra parte, el porcentaje global de necrosis fue menor en los grupos tratados con ASCs y ASCs-H respecto al grupo control (p ≤ 0.05). Conclusiones: La clasificación y puntuación de necrosis parece ser un método adecuado para comprender la evolución de la necrosis del colgajo cutáneo aleatorio y como herramienta para investigación. La necrosis de bajo grado permitiría un afrontamiento conservador y expectante respecto a la de mayor grado. El grupo RTC presentó un puntuación menor al séptimo día con mayor capacidad de reepitelización, mientras que los tratamientos con ASCs y ASCs-H lograron una menor área de necrosis de forma global al séptimo día

Background and Objective: Perform a characterization and classification of necrosis in random skin flaps (predisposed to necrosis) according to the macroscopic and histopathological findings, with or without treatment to increase the survival of the flap, it could be useful to analyze in a global way the flap and evaluate different modalities for decrease necrosis. Our aim is to evaluate if there is more than one skin cutaneous necrosis pattern of the random cutaneous flap on the seventh postoperative day, performing a necrosis classification and to compare the effectiveness of the flap delay and adipose tissue derived stem cells (ASCs) with and without preconditioning methods to increase the survival of the flap. Methods: Forty adult male rats subjected to random skin flap in the dorsal area of 2 x 8 cm grouped into control group, RTC (flap delay), ASCs (adipose-derived stem cells) and ASCs-H (hypoxic preconditioning of adipose-derived stem cells), were used. Area calculation, necrosis score and histological study of flaps on the seventh day with hematoxylin-eosin, immunohistochemistry with VEGF (vascular endothelial growth factor) and quantification of type I and III collagen were performed. Results: According to the classification of necrosis, type 0 was considered normal skin; type 0+ with reversible adaptive change; type 1 minor necrosis with superficial inflammation; type 2 minor necrosis with deep inflammation; and finally type 3 necrosis in its maximum degree. Healthy skin maintains coloring, elastic and soft texture. As necrosis progresses shifts towards a darker tone, texture and consistency increases until it becomes stiff and thin. In types 0+ and 1, foci of early reepithelialization was observed on the seventh day, while more advanced in the classification stages, necrosis is complete. The group of RTC presents a lower score and therefore lesser degree of necrosis with respect to the rest of the treated groups. Moreover the overall percentage of necrosis was lower in the groups treated with ASCs and ASCs-H in the control group (p ≤ 0.05). Conclusions: The classification and the score of necrosis seems to be a suitable method for understanding the evolution of skin flap necrosis and as a tool for research. Low-grade necrosis allows conservative and expectant confrontation with respect to higher grades. The RTC group presented a lower necrosis score on the seventh day with greater reepithelialization capacity, whereas treatment with ASCs and ASCs-H achieved a lower area of necrosis overall on the seventh day