The Interplay of ECM-Based Graft Materials and Mechanisms of Tissue Remodeling

Wound healing is a complex natural process that involves the recruitment of cells, the renewal of tissue composition, and the reinforcement of structural tissue architecture. Following ischemic injury or chronic disease, wound healing is delayed, and can often result in chronic inflammation or permanent morbidity. Tissue engineering strategies to harness the wound healing process include the use of naturally derived extracellular matrix (ECM) scaffolds with inherent bioactivity to both passively facilitate and actively direct healing toward a successful resolution. As the body heals, the properly designed ECM scaffold is gradually remodeled and integrated into the body, leaving behind organized tissue that provides long-term strength. Herein we explain the interplay of the ECM (i.e., its complex composition and bioactivity) with the cells of the body throughout the process of tissue remodeling, thus explaining how even a tissue-engineered xenograft material can direct the body to restore itself

Two Group A Streptococcal Peptide Pheromones Act through Opposing Rgg Regulators to Control Biofilm Development

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum

The mechanical properties of porcine small-intestinal submucosa as a vascular graft biomaterial

Each year, hundreds of thousands of small-diameter, arterial graft procedures are performed in the United States. While the autogenous saphenous vein is presently the only acceptable material for these procedures, many patients do not have suitable saphenous veins to transplant. Therefore, an acute need exists for a new biomaterial to aid these patients. Small-intestinal submucosa (SIS) is able to withstand the stresses of arterial blood pressure while stimulating the host to fashion a new vascular conduit in its place. The studies contained herein describe the mechanical properties of SIS--specifically, porosity, circumferential stress-strain, and changes which occur in these properties after implantation. Porosity of a graft material is thought to be very important in the host incorporation process. Specimens of SIS were porosity tested with a typical result of 0.52 ml/min/cm\sp2, which is much higher than polytetrafluoroethylene grafts and much lower than polyester grafts. The circumferential stress-strain curve for SIS is useful in predicting its performance in specific applications and was determined using pressurized air or water while measuring the outer diameter. This curve is a nonlinear, sigmoid shape which ends with burst at an approximate stress of 5.25 MPa, but the ultimate stress is source dependent with larger pigs producing stronger SIS. The sterilization methods of glutaraldehyde, peracetic acid, and gamma irradiation all decreased the strength of SIS, but the last study described herein proves that peracetic acid sterilized SIS is sufficiently strong to serve as an infrarenal aortic graft in the dog. Sterilized SIS grafts placed in this aortic position in dogs for 1 or 2 months transformed into new vascular conduits. The wall thickness of the grafts increased to over 1 mm from an original thickness of only 0.1 mm, and the overall strength of the 2-month graft explants was greater than that of the natural aorta. The overall stiffness of the graft explants and immunocyto-chemical staining show that a portion of the original SIS remains in the grafts at this time. Porcine SIS is a promising new biomaterial for vascular graft applications, and these studies provide new information concerning this material. SIS has a porosity that is suitable for rapid host incorporation, and a strength after peracetic acid sterilization that is sufficient for use in the infrarenal aortic position. Because of its remodelling characteristics, SIS is not only a potential alternative to autogenous saphenous vein but perhaps a general biomaterial for many soft tissue replacement applications

Suppression and activation of the malignant phenotype by extracellular matrix in xenograft models of bladder cancer: a model for tumor cell "dormancy".

A major problem in cancer research is the lack of a tractable model for delayed metastasis. Herein we show that cancer cells suppressed by SISgel, a gel-forming normal ECM material derived from Small Intestine Submucosa (SIS), in flank xenografts show properties of suppression and re-activation that are very similar to normal delayed metastasis and suggest these suppressed cells can serve as a novel model for developing therapeutics to target micrometastases or suppressed cancer cells. Co-injection with SISgel suppressed the malignant phenotype of highly invasive J82 bladder cancer cells and highly metastatic JB-V bladder cancer cells in nude mouse flank xenografts. Cells could remain viable up to 120 days without forming tumors and appeared much more highly differentiated and less atypical than tumors from cells co-injected with Matrigel. In 40% of SISgel xenografts, growth resumed in the malignant phenotype after a period of suppression or dormancy for at least 30 days and was more likely with implantation of 3 million or more cells. Ordinary Type I collagen did not suppress malignant growth, and tumors developed about as well with collagen as with Matrigel. A clear signal in gene expression over different cell lines was not seen by transcriptome microarray analysis, but in contrast, Reverse Phase Protein Analysis of 250 proteins across 4 cell lines identified Integrin Linked Kinase (ILK) signaling that was functionally confirmed by an ILK inhibitor. We suggest that cancer cells suppressed on SISgel could serve as a model for dormancy and re-awakening to allow for the identification of therapeutic targets for treating micrometastases

Functional role of ILK signaling in SISgel suppressed cells in culture.

<p>J82 cells grown on Matrigel for five days with no ILK inhibitor (top) or with 10 µM QLT0267 ILK inhibitor (bottom panel). Note suppression of invasion. All magnifications are 200X. Images are representative of 3 experiments.</p

Comparison of the phenotypes of J82 bladder cancer cell lines grown on (A) Matrigel, (B) collagen or (C) SISgel in 3-D culture.

<p>Note the loss of invasion and of the more orderly appearance of the cells grown on SISgel. All magnifications are 200X. Images are representative of a minimum of 4 experiments.</p

Comparison of Ki67 (proliferation) and vimentin in suppressed cell tissue and actively growing tumors.

<p>JB-V cells co-injected with Matrigel and labeled for Ki67 (A) and vimentin (C). JB-V cells co-injected with SISgel and in the suppressed phenotype labeled for Ki67 (B) and vimentin (D). Controls omitting the primary antibody (not shown) were entirely negative for both markers. Images at 200X.</p

Signature of proteins that are statistically significantly differentially expressed comparing cells from 4 lines grown on plastic, SISgel and Matrigel.

<p>The symbol (V) indicates the antibody was validated to show a single band by Western blot. A “p” indicates that the antibody is against a phosphorylated form of the protein, with the amino acid position and identity provided. The two mTOR entries represent duplicates. A complete list of the antibodies used at the time of the analysis is provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064181#pone.0064181.s001" target="_blank">Table S1</a>.</p

Tumor growth at 28 days from 3 million cells as a function of ECM preparation on which J82 bladder cancer cells were grown and ECM preparation co-injected with J82 bladder cancer cells.

<p>Tumor growth at 28 days from 3 million cells as a function of ECM preparation on which J82 bladder cancer cells were grown and ECM preparation co-injected with J82 bladder cancer cells.</p

Histopathology of cells from flank xenografts.

<p>Features identified histopathologically are shown as follows. Areas of malignant cells are outlined in black, and areas of coagulative necrosis are outlined in yellow. Black arrows illustrate apoptotic bodies. Yellow arrows identify mitotic figures. At the zoom level shown here these are difficult to distinguish but were identified under high power. (A) Cells co-injected with Matrigel that immediately presented a malignant growth pattern. The pathologic description noted large areas of coagulative necrosis with acute inflammation (the small, dense cells are neutrophils) with an area of highly atypical cells revealing marked nuclear pleomorphism, all indicative of high grade neoplasia. (B) Cells co-injected with SISgel that remained in the suppressed or dormant phenotype. This slide was read as cells with moderate cellular atypia and pleomorphism with minimal evidence of coagulative necrosis, mitosis and apoptosis. (C) Cells co-injected with SISgel that initially presented a suppressed phenotype for 8 weeks but then resumed growth in the malignant phenotype. This was read as containing an area of coagulative necrosis with acute inflammation and foci of markedly atypical cells along with prominent mitosis and apoptosis. Some fields contained multinucleated tumor giant cells usually indicative of high-grade neoplasia (D) Cells co-injected with Collagen I demonstrating a malignant growth pattern similar to those illustrated in panels A and C. All images are at 400X.</p