In vitro assessment of decellularized porcine dermis as a matrix for urinary tract reconstruction

Abstract

The contribution by the units in York and Leeds to tissue‐engineering research is considerable, and they describe here their work to assess a natural matrix for use in the urinary tract, and to develop an in vitro regimen for assessing the biocompatibility of potential biomaterials. They describe how they have developed a simple, reproducible and rigorous regimen which will help to identify the causes of potential bio‐incompatibility. Buccal mucosa has become the tissue of choice for urethroplasty. Authors from Edinburgh report on the oral complications after its harvesting; they found that most patients were satisfied with its use, but that it had long‐term complications, about which patients should be informed. OBJECTIVES To assess the potential of PermacolTM (Tissue Science Laboratories, Swillington, UK), a natural matrix derived from decellularized porcine dermis, as a matrix for urological tissue engineering, and thus to develop an in vitro regimen for assessing the biocompatibility of potential biomaterials before experimentation in animal models. MATERIALS AND METHODS Urinary tract‐derived normal human urothelial (NHU) and smooth muscle (SM) cells were grown in monoculture as autologous cell lines. Permacol was assessed for its ability to support colonization by NHU and SM cells. The failure of the Permacol matrix to be infiltrated by SM cells was further investigated using the highly invasive EJ bladder cancer cell line. RESULTS NHU cells readily attached and grew as a monolayer on the surface of Permacol. Cells stratified when the culture medium was supplemented with 2 mmol/L calcium. EJ cells initially grew on the surface and subsequently invaded the matrix, while SM cells only colonized the surface of Permacol when cocultured with NHU cells. Cytoxicity, evaluated by contact inhibition and conditioned‐medium assays, excluded the presence of soluble toxins in the biomaterial. CONCLUSIONS We developed a simple, reproducible and rigorous regimen for assessing potential biomaterials in vitro. Applying this system might reduce the use of animals and help to identify causes of potential bio‐incompatibility. The inability of SM cells to penetrate the Permacol matrix suggests that required matrix‐bound signalling factors are absent, possibly as a result of the procedures used for processing Permacol. Identifying the key regulatory factors that regulate SM cell growth and orchestrate regenerative processes in the urinary tract will be important for developing suitable biomaterials for the bladder.

Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering

“…Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Paisley, UK) supplemented with 10% (v/v) fetal bovine serum (FBS) (Harlan, Loughborough, UK) and 1% (v/v) L-glutamine (Sigma, Poole, UK) at 37 1C in a humidified atmosphere of 10% CO 2 in air. Morphological examination and immunocytochemical labelling for SMA (detailed above) was used to confirm cell strain identity and homogeneity of the cultures, as described [15]. Porcine smooth muscle (PSM) cell cultures were subcultured at confluence and maintained as finite cell lines through at least 15 passages with no change in phenotype, or were cryopreserved for long-term storage.…”

“…Pieces (1 cm 2 ) of bladder tissue were incubated in stripping medium (Ca 2+ /Mg 2+ -free HBSS containing 10 mM HEPES, pH 7.6, 20 KIU/ml Trasylol and 0.1%, w/v EDTA) at 37 1C for 4 h to detach urothelium from the stroma [41]. The stroma was finely minced, partially digested by incubation for 2 h in 100 U/ml collagenase type IV (Sigma, Poole, UK) and smooth muscle cells isolated as previously described for human smooth muscle cells [15]. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Paisley, UK) supplemented with 10% (v/v) fetal bovine serum (FBS) (Harlan, Loughborough, UK) and 1% (v/v) L-glutamine (Sigma, Poole, UK) at 37 1C in a humidified atmosphere of 10% CO 2 in air.…”

“…Some clinical success has been reported from implanting tissue constructs prepared by seeding bladderderived cells into composite matrices of collagen and synthetic polyglycolic acid into patients with end-stage bladder disease [3]. There has also been considerable interest in developing natural decellularised matrices from a variety of tissue derivations, including small intestine submucosa (SIS) [8][9][10][11][12][13], porcine dermis (Permacol s ) [14,15] and the urinary bladder itself [16][17][18][19][20].…”

Biocompatibility evaluation of vessel extracellular matrix as a matrix for urethral reconstruction

“…Indirect cytocompatibility study was done b y e x p o s i n g R B S M C s t o V E C M e x t r a c t s , a s recommended by current published standards [7,8] . This stage is particularly important in the urinary tract because in clinical applications biomaterials may be exposed to urine, which acts as an extraction vehicle.…”

Development and characterization of an ex vivo arterial long-term proliferation model for restenosis research

“…A 1 cm 2 sheet of decellularized specimen was put into the well of a 24-well culture plate. The assay was performed as previously described (Kimuli et al, 2004). Briefly, smooth muscle cells and endothelial cells (passage 4, at a density of 1 x 10 4 cells/10 µl) were added to the luminal surface of the specimen.…”