Resumen de Un nuevo modelo para el estudio de úlceras por presión y sus aplicaciones: hormona de crecimiento
- español
La úlcera por presión es un problema de elevada importancia por la repercusión sociosanitaria y económica que conlleva. La investigación actual está limitada por la escasez de modelos animales apropiados. Presentamos el primer modelo animal de úlcera por presión sobre piel humana, y la aplicación de hormona de crecimiento para el estudio del proceso de cicatrización. Dicha hormona tiene un papel en la homeostasis de la piel y cicatrización de heridas cutáneas. No obstante, su uso local en úlceras por presión apenas está documentado en la literatura. Nos planteamos si es posible mejorar la cicatrización de las úlceras por presión aplicando la hormona de crecimiento de manera local, utilizando para ello el modelo de piel humana.
Ratones NOD/Scid (n=40) fueron injertados con piel humana tras la creación de defectos cutáneos de 4x3cm en su dorso. Después de 60 días con injertos estables, los ratones fueron sometidos a 3 ciclos de isquemia-reperfusión para la creación de una úlcera sobre la piel humana, utilizando una pinza de presión. El estudio de la piel humana se llevó a cabo mediante análisis visual, histológico, inmunohistoquímico y citogenético. Se determinó el prendimiento, viabilidad de la piel, contracción, mantenimiento de estructura a largo plazo, elasticidad y proteínas de matriz extracelular. El estudio de la úlcera por presión se realizó mediante análisis visual e histológico hasta su cierre completo. Se comparó mediante inmunohistoquímica la expresión de las principales proteínas fibrilares de matriz extracelular en la piel humana injertada antes y después de aplicar presión mecánica. En la última fase del estudio se dividió a los ratones con úlcera en dos grupos: tratamiento con hormona de crecimiento recombinante humana (grupo rhGH, n=4) y grupo control (n=4). Se administró la hormona de manera local mediante cuatro inyecciones intradérmicas de 0,15 mg en los bordes de la úlcera, una vez por semana durante 4 semanas. Se realizó evaluación de la cicatrización en todos los casos mediante análisis macroscópico y cálculo de las velocidades de cicatrización. Se realizó análisis histológico de las muestras de piel obtenidas al finalizar el proceso de cicatrización en ambos grupos.
Los resultados obtenidos mostraron piel humana viable y estable, que conservó sus características macroscópicas y microscópicas durante 220 días. La úlcera por presión en piel humana se caracterizó por necrosis de todas las capas epidérmicas y dérmicas tras la aplicación de los ciclos de isquemia-reperfusión, revelando una úlcera por presión estadio III en todos los casos. El estudio de las proteínas de matriz extracelular reveló un incremento de las mismas tras la úlcera. Con respecto a la aplicación de rhGH, la velocidad de cicatrización fue dos veces superior en el grupo rhGH (1,25 ± 0,33 mm2/día) comparado con el control (0,61 ± 0,27 mm2/día), siendo esta diferencia estadísticamente significativa (p=0,03). Histológicamente, el grupo tratado con hormona presentó mayor celularidad de tipo inflamatorio en dermis, mayor depósito de colágeno y mayor grosor de piel.
Podemos concluir que se ha desarrollado el primer modelo animal de úlcera por presión sobre piel humana. Este modelo posee relevancia clínica por permitir testar diferentes estrategias terapéuticas directamente sobre piel humana en el contexto de un organismo vivo, sin las consideraciones éticas que conlleva la investigación en humanos. Una de sus aplicaciones ha sido la administración local de rhGH. En nuestro modelo, la aplicación de la hormona duplica de forma estadísticamente significativa la velocidad de cicatrización de la úlcera por presión. Su uso e implicaciones en humanos deberán ser estudiados con mayor profundidad.
- English
The incidence and prevalence of pressure ulcers (PU) in today's society is high, despite the implementation of prevention strategies. Because of population aging and medical advances, this disease has become a health problem with a significant socio-economic impact. Minimal improvements in treatment would have a major impact not only economically but also psychologically on families and caregivers, as well as on patients’ quality of life. Understanding the pathophysiology is essential for PU prevention and management. Although many factors are involved, external pressure is considered to be the main factor. In addition, ischemia-reperfusion injury has been shown to contribute to its pathophysiology significantly. However, many aspects are still not fully understood.
The management of these patients calls for a comprehensive, multidisciplinary team approach. Among the many therapeutic options, tissue engineering is growing and providing new tools for the development of more efficient strategies to fight against ulcers, but it is still in its infancy regarding PU. Research on the pathogenesis and mechanisms involved in wound healing is crucial. Because of practical, ethical, and safety considerations, research into PU is limited to studies involving patients with pre-existing ulcers. Therefore, animal models are required for this purpose.
There are several experimental animal models for PU, but not on human skin. In this context, this doctoral thesis hypothesizes the development of a novel model of human skin pressure ulcer, as well as its applicability.
Male, non-obese diabetic (NOD) / severe combined immunodeficiency (Scid) (NOD.CB17- Prkdscid/NCrHsd) mice (n=40) were engrafted under general anesthesia with human skin. Fullthickness human skin graft was placed onto a 4x3 cm wound created on the dorsum of the animals. After 60 days, mice were classified into two groups: one for studying the engraftment process and long-term features of the human skin (n=10), and another one for creating a pressure ulcer (n=22). A compression device that delivered a pressure of 150 mmHg, was applied to the human skin graft. A total of three cycles of ischemia-reperfusion were performed, each consisting of 8 hours of clamping followed by 16 hours of release. Evaluations of the human skin were conducted with visual, histological, immunohistochemical analyses and cytogenetic testing. Skin graft take, viability, retraction, structural stability, elasticity, and extracellular matrix protein profiles and distributions were determined. For PU assessment, visual and histological analyses were conducted through to its complete closure. Dermal extracellular fibrillar proteins before and after applying mechanical pressure were compared by immunohistochemical staining.
The results revealed viable and stable human skin, which retained its macroscopic and microscopic features for 220 days: keratinized stratified squamous epithelium on a dermis with well-defined papillae and blood vessels. Neither specific accumulations of lymphocytic cells nor areas of edema were observed. Fluorescent in situ hybridization (FISH) enabled us to confirm the human origin of the skin. The human skin graft was clearly distinguished from receptor mouse skin with a gradual mouse-human skin transition. The graft showed shrinkage over time, but the achieved human skin dimensions are superior to those published by other authors, and they provide enough tissue to develop skin diseases and to test different therapies directly on human skin.
As a further step, the first PU model has been developed directly on human skin. As macroscopically and histologically observed, placing the compression device for three cycles of ischemia-reperfusion induced a PU, reproducible in all cases. It was characterized by full-thickness skin loss, involving deep dermal and subcutaneous tissue, as a stage III pressure sore. The repair capacity of the human skin could be assessed by removing pressure. A homogeneous and uniform section of human skin, showing the characteristics expected for a neodermis, was observed. Protein expression such as collagen I, collagen III, LOX and fibrillin-1 showed increased activity with significant differences between the human skin after PU and the human skin before the mechanical injury. These results indicate that the human skin graft maintained its ability to react against an insult, and was able to synthesize and organize the necessary proteins as the native human skin.
Having established the model described above, we focused on potential applications.
Previously published PU models have been developed using animal skin, which imposes limitations when studying the wound healing process and the extrapolation of results and the effect of treatments on humans. Tissue engineering applied to wound healing, and specifically to PU, is an expanding field, and its challenge is prompt tissue recovery. Following this line, the growth hormone, the anabolic hormone par excellence, plays a role in skin homeostasis and cutaneous wound healing, as shown by several animal and burn patient studies. In most of them its use has been systemic, and there are only a few case reports about PU and the growth hormone in the literature. We hypothesize whether it is possible to improve PU healing by locally applying the recombinant human growth hormone (rhGH) in our model. We designed a protocol for local administration of the hormone. Four mice with PU were treated with four local intradermal injections, each of 0.15 mg (0.5IU), applied to the PU edges always on human skin, once per week for four weeks. Four mice with PU received the same care but without the local rhGH injections (control group). Evaluation of the wound healing was conducted with photographic and visual assessments and the healing rate was calculated. Histological analysis was performed at the end of the process, and the results were compared between both groups (rhGH vs control).
The results showed a healing rate twice as fast in mice subjected to treatment with the growth hormone - rhGH group (1.25 ± 0.33 mm2 per day) compared with those which did not receive it - control group (0.61 ± 0.27 mm2 per day), with a statistically significant difference (pvalue = 0.03). Kinetics was compared between groups, with faster healing for the first 30 days in rhGH group (time in which the hormone was administered once per week). From day 30 there was no significant differences between the two groups (healing rate of rhGH group = 1.3 mm2/day versus healing rate of control group = 1.25 mm2/day). Histological differences in skin thickness, cellularity and quantity and distribution of collagen between the two groups were visualized. The group treated with the hormone had thicker skin, increased cellularity of the dermis due to inflammatory cell infiltrate, and increased collagen deposition in the dermis, showing no histological signs of malignancy.
The conclusions drawn from this study are as follows:
- NOD/Scid mouse allows transplantation of human skin, characterized by its viability and long-term stability. Although the graft showed shrinkage over time, it provides enough tissue to develop skin diseases and to test different therapies directly on human skin.
- The first animal model of PU directly on human skin has been developed. The healing process has been described and standardized and our skin model has retained its ability to react to mechanical insult.
- Local administration of the growth hormone (rhGH) accelerates pressure ulcer healing in our model. The rhGH may have a clinical use in pressure ulcer treatment. However, further studies remain to be performed.
- The pressure ulcer model has clinical relevance in terms of allowing the testing of different therapeutic strategies directly on human skin in the context of a living organism, without the ethical considerations involved in human research.
