The basement membrane complex (BMC) is a critical component of the extracellular matrix (ECM) that supports and facilitates the growth of cells. This study investigates four detergents commonly used in the process of tissue decellularization and their effect upon the BMC. The BMC of porcine urinary bladder was subjected to either 3% Triton-X 100, 8 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate, or 1% sodium dodecyl sulfate (SDS) for 24 hours. The BMC structure for each treatment group was assessed by immunolabeling, scanning electron 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 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.

There is a major need for scaffold-based tissue engineered vascular grafts and heart valves with long-term patency and durability to be used in diabetic cardiovascular patients. We hypothesized that diabetes, by virtue of glycoxidation reactions, can directly crosslink implanted scaffolds, drastically altering their properties. In order to investigate the fate of tissue engineered scaffolds in diabetic conditions, we prepared valvular collagen scaffolds and arterial elastin scaffolds by decellularization and implanted them subdermally in diabetic rats. Both types of scaffolds exhibited significant levels of advanced glycation end products (AGEs), chemical crosslinking and stiffening - alterations which are not favorable for cardiovascular tissue engineering. Pre-implantation treatment of collagen and elastin scaffolds with penta-galloyl glucose (PGG), an antioxidant and matrix-binding polyphenol, chemically stabilized the scaffolds, reduced their enzymatic degradation, and protected them from diabetes-related complications by reduction of scaffold-bound AGE levels. PGG-treated scaffolds resisted diabetes-induced crosslinking and stiffening, were protected from calcification, and exhibited controlled remodeling in vivo, thereby supporting future use of diabetes-resistant scaffolds for cardiovascular tissue engineering in patients with diabetes.

Key to most implanted cell free scaffolds for tissue regeneration is the ability to sequester and retain undifferentiated mesenchymal stem cells at the repair site. In this report, syndecan-4, a heparan sulfate containing proteoglycan, was investigated as a unique molecule for use in scaffold functionalization. An electrospun hybrid scaffold comprised of poly (glycerol) sebacate (PGS), silk fibroin and type I collagen (PFC) was used as a model scaffold to develop a procedure and test the hypothesis that functionalization would result in increased scaffold binding of endothelial progenitor cells (EPCs). For these studies both Syndecan-4 and stromal derived factor-1α (SDF-1α) were used in functionalization PFC. Syndecan-4 functionalized PFC bound 4.8 fold more SDF-1α compared to nonfunctionalized PFC. Binding was specific as determined by heparin displacement studies. After culture for 7 days, significantly, more EPCs were detected on PFC scaffolds having both syndecan-4 and SDF-1α compared to scaffolds of PFC with only syndecan-4, or PFC adsorbed with SDF-1α, or PFC alone. Taken together, this study demonstrates that EPCs can be bound to and significantly expanded on PFC material through syndecan-4 mediated growth factor binding. Syndecan-4 with a multiplicity of binding sites has the potential to functionalize and expand stem cells on a variety of scaffold materials for use in tissue regeneration.