1. APPLICATION OF ALLOGENIC BONE MARROW STROMAL CELLS ON THE UROLOGY .
NM Yudintceva1, YA Nashchekina1, MI Blinova1, NV Orlova2, MG Shejhov2, TI Vinogradova2, AN Muravjev2
1Cell Culture Department, Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russia Federation
2Saint-Petersburg Scientific-Research Institute of Phthisiopulmonology, Russian Ministry of Health Care, Russian Federation
INTRODUCTION: Urinary bladder pathology is actual problem nowadays. Bowel reconstruction of non-functioning bladder remains the gold standard despite various complications. Recently were published results of successful attempts of bladder replacement with in vitro generated neo-organ containing autologous cells. But that method is unsuitable for patients with total fibrous transformation of the bladder. The purpose of this study is creation of graft containing allogenic cells to restore injured bladder tissue.
MATERIAL AND METHODS:
CELL CULTURE
The bone marrow stromal cells (BMSCs) and the dermal fibroblasts (DFs) were isolated from variety of rabbit’s tissues. Both types of cells were cultivated following standard methods.
PREPARATION 3D SCAFFOLDS
Scaffolds were prepared by particle-leaching method. The poly-L-lactic acid was dissolved in chloroform with 2% concentration (w/v) and with room temperature. NaCl particles (100–150 mm) were stirred with silk fibroin. The mixture of salt/silk fibroin was put into the wells (diameter: 18 mm; height: 3 mm). The polymer solution was added to salt/silk fibroin mixture. To remove some remaining solvent was executed 48 h air dried and
24 h subsequently vacuum-dried. The resulting polymer/salt/silk fibroin composites were immersed 24 h in distilled water for to leach out the salt. Thickness of sponge-like foams was about 2 mm. The scaffolds were sterilized by ozonizing. a b
Fig.1. Cell cultures: a – BMSCs; b – DFs.
The scaffolds were filled by the mixture of solution of collagen Type I with different cells, then this mixture was polymerized into the gel. This grafts were incubated in CO2 - incubator and transplanted to rabbits after bladder resection. a b c d
Fig.2. The poly-L-lactide/silk fibroin scaffold: a - outward appearence; b - SEM of transverse section of scaffold without cells; c, d - SEM of scaffolds surface with growing BMSCs and DFs. a b c d
Fig.4. Resection model of rabbit’s bladder. Photographs of various surgical stages of grafts implantation: a, b - abdominal incision and extrusion of rabbit bladder; c - partial resection of the bladder;
d - scaffold tailored to area of the defect site was stitched into this zone.
Fig.3. Bladder tissue engineering strategies.
RESULTS: After 2 months post-surgery of observation was noticed the transplantation of DFs-containing scaffold caused inflammatory reaction and rejection of implant. As in case of transplantation BMSCs-containing scaffold there were no evidence of graft rejection and signs of vascularization of the transplanted graft. Histological examination revealed initial stages of tissue reparation. Graft demonstrated safeness of application, caused mild inflammation in implantation area and efficiently initiated the process of reconstruction of injured tissue. a c a b b
Fig.5. Post-surgery view of bladder after using the BMSCs-containing scaffold: a - external surface; b - internal surface; c - transverse section;
Fig.6. Post-surgery view of bladder after using the DFs-containing scaffold: a - external surface; b - internal surface. d c a b
Fig.7. Histological examination of implantation area after BMSCs-containing scaffold transplantation: a – the native epithelium; b – the epithelium of zone implantation;
c – inflammation infiltrate; d – fatty tissue with edema; Stained by hematoxylin and eosin. 40x
DISCUSSION: This experiment was shown the positive result of application of BMSCs-containing scaffold for regeneration of injured bladder tissue. There is a huge interest in further research of ability of BMSCs to differentiate into various types of bladder cells especially to urothelial cells.
This work was supported by a grant from the Russian Science Foundation (№ )
and with financial support from the Federal Agency of Scientific Organizations (Russia) and RFBR № ofi-m.