Human adipocyte tissue mesenchymal stromal stem cells(AT-MSCs): new therapeutic proposals leading to complete regeneration of injured tissue

• Grant number: 15/25968-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2016
• End date: July 31, 2016
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Ângela Cristina Malheiros Luzo
• Grantee: Guilherme Moura Sales
• Host Institution: Centro de Hematologia e Hemoterapia (HEMOCENTRO). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

Crohn's disease still remains a therapeutic challenge, being an inflammatory disease affect any part of digestive tract. One of the less desirable complications is the involvement with digestive fistula (DC) which lead to significant negative impact on quality of life. For such comorbidity, isn't yet known a really effective treatment procedure to cure or stabilize the evolution of the clinical course. These difficulties might change with the ongoing stem cells therapy research, especially with the use of stromal mesenchymal stem cells (MSCs). Besides their great capacity of proliferation and differentiation to other lineages MSCs have also immunomodulatory properties, being easily obtained from adipose tissue (AT). In a previous study we demonstrated that AT- MSCs could be attached into suture filaments. These filaments were used in the treatment of an animal model with enterocutaneous fistula, similar to those of Crohn's disease and have provided a better and faster fistula healing. These results demonstrated statistical significance (p<0.05). This group was compared with other two groups; Control group (untreated) and Injection group (suspension containing 1.0 x 10E6 AT-MSCs were injected subcutaneously around the fistula). The adhesion method of AT- MSCs in suture filaments was patented and the preclinical results published by the group. The actual study aimed evaluate the healing and immunomodulatory processes related with the faster and better healing obtained when suture filaments with AT- MSCs attached were accomplished. Adipose tissue was obtained from lipoaspirate procedures during esthetical lipoaspiration performed in healthy donors which have previously assigned a written informed consent form. Adipose tissue was submitted to collagenase digestion and the cells were cultured in DMEM low glucose medium, fetal bovine serum added. At the 4th passages, cells were characterized by the criteria requested for the International Society for Cellular Therapy (ISCT "Mesenchymal and Tissue Stem Cell Committee"). The experiments were performed with poliglactine suture filament (4-0 Poly Vicryl / Poliglactine 910). AT-MSCs, 1.0 x 10E6 cells, were fixed in the suture filament by adding fibrin glue (20uL Fibrinogen, 30uL Thrombin e 10uL Calcium Chloride). Samples were analyzed by confocal and scanning electron microscopy. The animal experiments were performed on male Wistar rats divided into 3 groups with 5 animals each. The animals were sacrificed on the 7th (Group 1), 14th (Group 2) and 21th (Group 3) days. All groups were carried out with 4-0 Vicryl with 1x106 AT-MSCs attached in the filaments with fibrin glue. After animals sacrificed, histological sections will be stained with H.E for cytology analyses. AT-MSCs tracking analysis will be performed by confocal immunofluorescence method. Immunohistochemistry analyses due to investigate lymphocyte T CD3 action, angiogenesis, myofibroblast quantification, macrophages and interleukin IL-6 IL-12 IL-1 TNF-± CD209 TLR2 TLR4 detection are being performed in another project of the group. Collagen fibers characterization and quantification will be performed by immunhistochemistry with Anti-collagen I + II + III (I.BB.749 - Abcan) and Anti-collagen X (COL-10) and cytochemistry analyses with sirius red F3AB stain will be performed in this study. (AU)