Résumé
Objective To evaluate the effect of living skin equivalents (LSE) constructed of mixed autologous and allogeneic skin cells and human amnion which served as a matrix on repairing scar contracture of the hand in a patient with recessive dystrophic epidermolysis bullosa (RDEB).Methods Skin tissues were obtained from a patient with RDEB and her mother,and epidermal keratinocytes and dermal fibroblasts were isolated from these tissues and cultured in vitro separately.Human amnion was obtained from the placenta of an unrelated healthy parturient undergoing cesarean delivery,and served as a matrix of the LSE.The autologous and allogeneic fibroblasts were mixed and seeded on the stromal side of the amnion,and then the autologous and allogeneic keratinocytes were mixed and seeded on the epithelial side of the amnion,so as to construct the human amnion-LSE (AM-LSE).Histological examination was performed to observe the structure of the skin tissues obtained from the patient and her mother,and immunofluorescence staining was conducted to detect the expression of type Ⅶ collagen in the skin tissues of the patient and her mother and in the AM-LSE.The AM-LSE was grafted on the skin defects of the patient after release of scar contracture of the hand.After grafting,the survival status of the AM-LSE graft and repairing effect on the wounds were evaluated.Results The constructed AM-LSE consisted of dermis,multilayered and fully differentiated epidermis and well-developed basement membrane.Immunofluorescence examination revealed that type Ⅶ collagen was linearly distributed along the basement membrane.Half a year after grafting,the AM-LSE survived well,and no obvious rejection reaction was observed.No blisters or ulcers occurred at the recipient sites,and the scar contracture was mild.The grafted area showed normal skin color with soft texture.The patient could grab and hold things,which had met self-care requirements of daily living.Conclusions The AM-LSE constructed of mixed autologous and allogeneic skin cells have good histological structures,and can be grafted on the wounds after resection of the scars in a RDEB patient.After grafting,no obvious rejection reaction was observed,and the skin was not liable to develop blisters,ulcers or scar contracture due to friction.
Résumé
Cell therapy has shown encouraging perspectives for human and veterinary medicine. Experimentally, genetic manipulation allows to mark and locate allogeneic cells. However, this makes their genotype/phenotype different from non-marked cells used clinically. Alternatively, the presence of the Y-chromosome enables male donor cells detection in female organisms. However, the concentration of engrafted cells may be minimal in tissues, due to systemic distribution. In this study, a nested-PCR multiplex test was developed, aiming to increase the sensitivity of the presence/absence diagnosis of male mice adipose-derived (ADSC-Y) and bone marrow mononuclear (BMNC-Y) cells in samples of blood and lungs from females, after endovenous transplantation. Four females received placebos; four females received ADSC-Y from two males; and four females received BMNC-Y from two males. The PCR first-step included two primer sets (multiplex): one for amplification of a Y-chromosome fragment (SRYout; 300bp); the other for amplification of an X-chromosome (DXNds3 gene) fragment. In the PCR second-step, one primer set (SRYinn) was used for amplification of a 110bp fragment, restrained in the SRYout amplification product. The PCR internal control (DXNds3 gene) was detected in all DNA samples, whereas the SRY gene external fragment (300bp) was detected exclusively in ADSC-Y and BMNC-Y pure DNA samples. The SRY gene internal fragment (110bp) was detected in 100% of the blood and lung samples from the ADSC-Y and BMNC-Y female recipients. The nested-PCR technique increased sensitivity and reliability for molecular diagnostic of presence or absence of male mice cells in body fluids and tissues of female recipients after endovenous transplantation.
A terapia celular traz perspectivas encorajadoras à medicina humana e veterinária. Experimentalmente, a manipulação genética permite a marcação e a localização de células alogênicas. Porém, isso torna seu genótipo/fenótipo diferente daquelas usadas clinicamente, sem marcação. Alternativamente, a presença do cromossomo Y possibilita detectar células de doadores machos no organismo de fêmeas. Todavia, a concentração de células transplantadas pode ser mínima em certos tecidos, pela distribuição sistêmica. Neste estudo, foi desenvolvida uma nested-PCR multiplex, visando a aumentar a sensibilidade do diagnóstico de presença/ausência de células derivadas do tecido adiposo (CDTA-Y) e derivadas da fração mononuclear da medula óssea (CFMO-Y) de camundongos machos, em amostras de sangue e de pulmões de camundongos fêmeas, após transplante endovenoso. Quatro fêmeas receberam placebo; quatro fêmeas receberam CDTA-Y de dois machos; e quatro fêmeas receberam CFMO-Y de dois machos. A primeira fase da PCR teve dois pares de primers (multiplex): um para amplificação de fragmento do cromossomo Y (SRYout; 300pb); outro para amplificação de fragmento do cromossomo X (gene DXNds3). Na segunda fase da PCR, foi usado um par de primers para amplificação de fragmento de 110pb (SRYinn) interno ao produto amplificado pelo SRYout. O controle interno da reação (gene DXNds3) foi detectado em todas as amostras de DNA testadas, enquanto que o fragmento externo do gene SRY (300pb) foi detectado apenas nas amostras puras de DNA de CDTA-Y e CFMO-Y. O fragmento interno do gene SRY (110pb) foi detectado no sangue e nos pulmões de 100% das receptoras de CDTA-Y e CFMO-Y. A técnica de nested-PCR aumentou a sensibilidade e a segurança do diagnóstico molecular de presença ou ausência de células de camundongos machos em fluidos e tecidos de receptoras fêmeas após transplante endovenoso.
Résumé
La piel es un tejido complejo vulnerable a procesos que alteran su estructura, integridad y funcionalidad como, por ejemplo, quemaduras, heridas crónicas y diversas enfermedades congénitas. Los avances tecnológicos en la fabricación de biomateriales y en el cultivo de células han permitido la producción de sustitutos cutáneos que han sido una alternativa terapéutica para algunas de estas complicaciones. Esta revisión pretende actualizar los aspectos generales, composición, perspectivas futuras y de aplicación de los principales sustitutos cutáneos que se ofrecen actualmente en el mercado internacional. Igualmente, presentará algunas experiencias del Grupo de Ingeniería de Tejidos y Terapias Celulares (GITTC) de la Facultad de Medicina de la Universidad de Antioquia.
The skin is a complex tissue vulnerable to different processes that may alter its structure, integrity and functionality, among them: burns, chronic wounds and various congenital diseases. Technological advances in biomaterials manufacture and cell culture have allowed the production of skin substitutes thus providing an alternative therapy for some of these complications. This review aims to update the general aspects, composition, future prospects and implementation of the most common skin substitutes currently available in the international market. Some experiences of the Tissue Engineering and Cellular Therapy Group (GITTC), at the University of Antioquia, Faculty of Medicine, in Medellín, Colombia, are also presented.