Mechanical strength vs. degradation of a biologically-derived surgical mesh over time in a rodent full thickness abdominal wall defect.

The use of synthetic surgical mesh materials has been shown to decrease the incidence of hernia recurrence, but can be associated with undesirable effects such as infection, chronic discomfort, and adhesion to viscera. Surgical meshes composed of extracellular matrix (i.e., biologically-derived mesh) are an alternative to synthetic meshes and can reduce some of these undesirable effects but are less frequently used due to greater cost and perceived inadequate strength as the mesh material degrades and is replaced by host tissue. The present study assessed the temporal association between mechanical properties and degradation of biologic mesh composed of urinary bladder matrix (UBM) in a rodent model of full thickness abdominal wall defect. Mesh degradation was evaluated for non-chemically crosslinked scaffolds with the use of (14)C-radiolabeled UBM. UBM biologic mesh was 50% degraded by 26 days and was completely degraded by 90 days. The mechanical properties of the UBM biologic mesh showed a rapid initial decrease in strength and modulus that was not proportionately associated with its degradation as measured by (14)C. The loss of strength and modulus was followed by a gradual increase in these values that was associated with the deposition of new, host derived connective tissue. The strength and modulus values were comparable to or greater than those of the native abdominal wall at all time points.

The effect of detergents on the basement membrane complex of a biologic scaffold material.

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 3% Triton-X 100, 8mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 4% sodium deoxycholate or 1% sodium dodecyl sulfate (SDS) for 24h. 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 (GAG) and collagen content. The results showed that collagen fibers within samples treated with 1% SDS and 8mM CHAPS were denatured, and the ECM contained fewer GAG compared with samples treated with 3% Triton X-100 or 4% sodium deoxycholate. Human microvascular endothelial cells (HMEC) were seeded onto each BMC and cultured for 7 days. Cell-ECM interactions were investigated by immunolabeling for integrin ß-1, SEM imaging and semi-quantitative assessment of cellular infiltration, phenotype and confluence. HMEC cultured on a BMC treated with 3% Triton X-100 were more confluent and had a normal phenotype compared with HMEC cultured on a BMC treated with 4% sodium deoxycholate, 8mM CHAPS and 1% SDS. Both 8mM CHAPS and 1% SDS damaged the BMC to the extent that seeded HMEC 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.

Fractionation of an ECM hydrogel into structural and soluble components reveals distinctive roles in regulating macrophage behavior.

Extracellular matrix (ECM) derived from mammalian tissues has been utilized to repair damaged or missing tissue and improve healing outcomes. More recently, processing of ECM into hydrogels has expanded the use of these materials to include platforms for 3-dimensional cell culture as well as injectable therapeutics that can be delivered by minimally invasive techniques and fill irregularly shaped cavities. At the cellular level, ECM hydrogels initiate a multifaceted host response that includes recruitment of endogenous stem/progenitor cells, regional angiogenesis, and modulation of the innate immune response. Unfortunately, little is known about the components of the hydrogel that drive these responses. We hypothesized that different components of ECM hydrogels could play distinctive roles in stem cell and macrophage behavior. Utilizing a well-characterized ECM hydrogel derived from urinary bladder matrix (UBM), we separated the soluble and structural components of UBM hydrogel and characterized their biological activity. Perivascular stem cells migrated toward and reduced their proliferation in response to both structural and soluble components of UBM hydrogel. Both components also altered macrophage behavior but with different fingerprints. Soluble components increased phagocytosis with an IL-1RA(high), TNFα(low), IL-1ß(low), uPA(low) secretion profile. Structural components decreased phagocytosis with a PGE2(high), PGF2α(high), TNFα(low), IL-1ß(low), uPA(low), MMP2(low), MMP9(low), secretion profile. The biologic activity of the soluble components was mediated by Notch and PI3K/Akt signaling, while the biologic activity of the structural components was mediated by integrins and MEK/ERK signaling. Collectively, these findings demonstrate that soluble and structural components of ECM hydrogels contribute to the host response but through different mechanisms.

Characterization of the 5' end of the mouse Ent gene encoding the basement membrane protein, entactin.

Entactin (Ent) is a 150-kDa basement membrane glycoprotein that forms a complex with laminin and also binds to type-IV collagen. For the initial characterization of the 5' end of the mouse Ent gene, we isolated genomic clones that encompass the first three exons of the gene. A 3.5-kb EcoRI fragment at the 5' end of the gene was sequenced and shown to contain 1226 bp of the 5'-flanking DNA, the 260-bp first exon, and 2060 bp of the first intron. The second exon is separated from the first exon by an intron that spans greater than 18 kb. The major transcription start point (tsp) was mapped to 35 nucleotides upstream from the translation start codon. The 5'-flanking DNA contains a putative TATA box, two CAAT boxes and two GC boxes. Nine potential AP-2- and two potential AP-1-binding sites were found in the upstream region and the first intron. The first 248 bp of the promoter region are G+C rich, and they are 65% identical to the promoter of human Ent. Fragments of the 5'-flanking DNA linked to a reporter gene were shown to have promoter activity in transient transfection assays.

Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells.

The extracellular matrix of rat pheochromocytoma PC12 cells was shown by indirect immunofluorescence to consist of a network of fibronectin. The matrix did not contain laminin. The cells synthesized messenger RNA for fibronectin, laminin B2, and s-laminin but not for entactin or the B1 and A chains of laminin. Laminin B2 but not laminin B1 was detectable in the culture medium and in cell lysates. A full-length cDNA clone for the B1 chain of laminin was constructed in the plasmid p-444, which contains the neomycin-resistance marker and human beta-actin promoter. PC12 cells were transfected with this recombinant plasmid, and stable neomycin-resistant clones were isolated and characterized. Clones that synthesized laminin B1 messenger RNA were found to deposit a laminin-containing matrix. In many of these clones the deposition of the fibronectin matrix was greatly diminished. The laminin matrix was predominantly localized in the intercellular spaces forming a honeycomb pattern. The morphology of the laminin-synthesizing transfected cells was markedly different from the parental cells. The cells grew in tight clusters that were resistant to dissociating agents. It is concluded that the B1 chain of laminin contains information that is required for the formation of a stable laminin-containing extracellular matrix network either by interaction with cell surface receptors or other extracellular matrix components. Furthermore, expression of the laminin B1 gene may be a central regulatory point in determining extracellular matrix composition during embryogenesis.

Entactin forms a complex with fibronectin and co-localizes in the extracellular matrix of the embryonal carcinoma-derived 4CQ cell line.

A novel extracellular matrix that consists of a complex of fibronectin and entactin was synthesized by the embryonal carcinoma-derived cell line 4CQ. The matrix was devoid of laminin. High steady state levels of the messenger RNAs for fibronectin, entactin, and the B2 chain of laminin were detected in these cells. Laminin B1 message was several fold lower while laminin A chain message was undetectable. In contrast, in the sister embryonal carcinoma-derived cell M1536-B3 there were high levels of message for all three chains of laminin and for entactin but very little for fibronectin. The data suggest that the synthesis and deposition of laminin and fibronectin are inversely related. The direct binding of entactin and fibronectin was also demonstrated by affinity column chromatography and solid phase assay.

Human spreading factor: synthesis and response by HepG2 hepatoma cells in culture.

Human serum spreading factor (SF) is a cell adhesion and spreading-promoting glycoprotein purified from serum or plasma that mediates effects in a wide variety of animal cell culture systems. HepG2 human hepatoma cells were found to synthesize and secrete SF into culture medium. Quantitative immunoassay of the protein indicated a concentration of about 1 microgram/ml in 48 hr-conditioned medium from confluent cultures. Although fibronectin also was synthesized and secreted into the culture medium, HepG2 cell spreading was observed in response to human serum SF, but not in response to human plasma fibronectin. Immunoprecipitation of SF from culture medium of cells metabolically-labeled with leucine, fucose or glucosamine identified a single form of the molecule of approximately 70,000 daltons. Treatment of cultures with tunicamycin inhibited incorporation of fucose and glucosamine into immunoprecipitated SF, but did not prevent synthesis and secretion of the protein. Electrophoretic analysis and cell spreading assays showed that SF secreted by tunicamycin-treated HepG2 cells was of molecular weight (mw) approximately 60,000, and was biologically active.

Heparin-binding properties of human serum spreading factor.

Human serum spreading factor (SF) is a blood glycoprotein that promotes attachment and spreading and influences growth, migration, and differentiation of a variety of animal cells in culture. SF purified from human plasma or serum by chromatographic methods reported previously (Barnes, D. W., and Silnutzer, J. (1983) J. Biol. Chem. 258, 12548-12552) does not bind to heparin-Sepharose under conditions of physiological ionic strength and pH. In a further examination of the heparin-binding properties of human serum SF, we found that exposure of purified SF to 8 M urea altered several properties of the protein, including heparin affinity, and these alterations remained after removal of the urea from SF solutions. Urea-treated SF bound to heparin under physiological conditions, and salt concentrations of 0.4 M or higher were required for elution of urea-treated SF from heparin-Sepharose at pH 7.0. The alteration of heparin-binding properties of SF also was observed upon exposure of the protein to heat or acid. Treatment of SF with urea, heat, or acid resulted additionally in greatly decreased cell spreading-promoting activity of the molecule. The decreased biological activity was associated with a reduced ability of the treated SF to bind to the cell culture substratum, a prerequisite for the attachment-promoting activity of the molecule. Experiments examining the heparin-binding properties of native SF in unfractionated human plasma indicated that the major portion of SF in blood did not bind to heparin under conditions of physiological ionic strength and pH.