The basement membrane complex (BMC) is a critical component of the
The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. microscopy (SEM) and second harmonic generation (SHG) imaging of the fiber network. The composition was assessed by quantification of dsDNA glycosaminoglycans (GAGs) and collagen content. Results showed that collagen fibers within samples treated with 1% SDS and 8 mM CHAPS were denatured and the ECM contained less GAGs compared to samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMECs) were seeded onto each BMC FK-506 and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin β-1 SEM imaging and semi-quantitative assessment of cellular infiltration phenotype and confluence. HMECs cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared to HMECs cultured on a BMC treated with 4% sodium deoxycholate 8 mM CHAPS and 1% SDS. Both 8 mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMECs were able to infiltrate the damaged sub-basement membrane tissue showed decreased confluence and an atypical phenotype. The choice of detergents used for tissue decellularization can have a marked effect upon the integrity of the BMC of the resultant bioscaffold. Keywords: Re-endothelization Organ engineering Extracellular matrix Biologic scaffold Regenerative medicine Decellularization 1 Introduction The decellularization of tissues for the purpose of utilizing the extracellular matrix (ECM) as a bioscaffold for reconstructive surgical procedures or whole organ engineering involves the use of various enzymes detergents and mechanical/physical methods[1-3]. During the process of decellularization parenchymal cells within the source tissues and organs such as the dermis small intestine urinary bladder liver and lung are destroyed and/or removed[1 2 4 However the less abundant but equally important non-parenchymal cells are also removed in the process. Such cells include the endothelial cells of the resident vascular network structures and any site appropriate epithelial cell populations. The remaining vascular network devoid of endothelial cells has been FK-506 proposed as a potential guide and substrate for revascularization[8-11]. Therefore the effects BHR1 of decellularization methods upon the structure and composition of the basement membrane complex (BMC) are critical for subsequent in-vitro or in-vivo recellularization. FK-506 There have been several published methods for decellularizing tissues and generating biologic scaffolds composed of ECM each of which describes a unique and specific recipe of enzymes and detergents. Commonly used detergents include Triton X-100[11 12 3 (CHAPS) sodium deoxycholate and sodium dodecyl sulfate (SDS)[8 14 Detergents are able to solubilize cell membranes and dissociate DNA from proteins making such agents attractive for the decellularization process. Studies have shown that ionic detergents can be more effective for cellular removal than non-ionic and zwitterionic detergents. However subjecting tissue to harsh detergents such as SDS can disrupt the ECM structure eliminate growth factors and/or denature essential proteins. The present study compared the effects of four commonly used decellularization agents upon the BMC and its ability to support endothelial cells in FK-506 vitro. The findings have relevance for decellularization strategies used in the production of ECM derived biologic scaffolds and whole organ engineering. 2 Materials and Methods 2.1 Scaffold Preparation and Decellularization Porcine urinary bladders were obtained from animals (~120 kg) at a local abattoir (Thoma’s Meat Market Saxonburg PA). Bladders were frozen (>16 h at ?80 °C) and thawed completely before use. The BMC and underlying lamina propria were isolated and harvested from the bladders as previously described [7 22 23 The tissue was then placed in FK-506 0.02% Trypsin/0.05% EGTA solution for two hours at 37°C with physical agitation to detach cells from the FK-506 extracellular matrix. Tissue samples were then subjected to either 3 Triton-X 100 (Sigma-Aldrich) 8 mM CHAPS (Sigma-Aldrich) 4 sodium deoxycholate (Sigma-Aldrich) 1 SDS (Bio-Rad) or Type I water (non-detergent control) for 24 hours with physical agitation (300 rpm on an orbital shaker). Scaffolds were next rinsed.