Extracellular Matrices Proteins Research Articles

Photoactivatable substrates show diverse phenotypes of leader cells in collective migration when moving along different extracellular matrix proteins.

In cancer metastasis, collectively migrating clusters are discriminated into leader and follower cells that move through extracellular matrices (ECMs) with different characteristics. The impact of changes in ECM protein types on leader cells and migrating clusters is unknown. To address this, we investigated the response of leader cells and migrating clusters upon moving from one ECM protein to another using a photoactivatable substrate bearing photocleavable PEG (PCP), whose surface changes from protein-repellent to protein-adhesive in response to light. We chose laminin and collagen I for our study since they are abundant in two distinct regions in living tissues, namely basement membrane and connective tissue. Using the photoactivatable substrates, the precise deposition of the first ECM protein in the irradiated areas was achieved, followed by creating well-defined cellular confinements. Secondary irradiation enabled the deposition of the second ECM protein in the new irradiated regions, resulting in region-selective heterogeneous and homogenous ECM protein-coated surfaces. Different tendencies in leader cell formation from laminin into laminin compared to those migrating from laminin into collagen were observed. The formation of focal adhesion and actin structures for cells within the same cluster in the ECM proteins responded according to the underlying ECM protein type. Finally, integrin β1 was crucial for the appearance of leader cells for clusters migrating from laminin into collagen. However, when it came to laminin into laminin, integrin β1 was not responsible. This highlights the correlation between leader cells in collective migration and the biochemical signals that arise from underlying extracellular matrix proteins.

Mesenchymal Stem Cells in Burn Wound Management

Mesenchymal stem cells have a known regenerative potential and are used in many indications. They secrete many growth factors, including for fibroblasts (FGF), endothelium (VEGF), as well as 14 anti-inflammatory cytokines, and they stimulate tissue regeneration, promoting the secretion of proteins and glycosaminoglycans of extracellular matrices, such as collagen I, II, III, and V, elastin, and also metalloproteinases. They secrete exosomes that contain proteins, nucleic acids, lipids, and enzymes. In addition, they show the activity of inactivating free radicals. The aim of this study was an attempt to collect the existing literature on the use of stem cells in the treatment of a burn wound. There were 81 studies included in the analysis. The studies differed in terms of the design, burn wound model, source of stem cells, and methods of cellular therapy application. No major side effects were reported, and cellular therapy reduced the healing time of the burn wound. Few case reports on human models did not report any serious adverse events. However, due to the heterogeneity of the evidence, cellular therapy in burn wound treatment remains an experimental method.

Extracellular matrix protein gelatin provides higher expansion, reduces size heterogeneity, and maintains cell stiffness in a long-term culture of mesenchymal stem cells

Extracellular matrices (ECM) present in our tissues play a significant role in maintaining tissue homeostasis through various physical and chemical cues such as topology, stiffness, and secretion of biochemicals. They are known to influence the behavior of resident stem cells. It is also known that ECM type and coating density on cell culture plates strongly influence in vitro cellular behavior. However, the influence of ECM protein coating on long-term mesenchymal stem cell expansion has not been studied yet. To address this gap, we cultured bone-marrow derived hMSCs for multiple passages on the tissue culture plastic plates coated with 25 μg/ml of various ECM proteins. We found that cells on plates coated with ECM proteins had much higher proliferation compared to the regular tissue culture plates. Further, gelatin-coated plates helped the cells to grow faster compared to collagen, fibronectin, and laminin coated plates. Additionally, the use of gelatin showed less size heterogeneity among the cells when expanded from passages 3 to 9 (P3 to P9). Gelatin also helped in maintaining cellular stiffness which was not observed across other ECM proteins. In summary, in this research, we have shown that gelatin which is the least expensive compared to other ECM proteins, provides a better platform for mesenchymal stem cell expansion.

Expression of cadherins and some connective tissue components in cow uterus and placenta during pregnancy

The implantation and placental development processes are regulated with cell adhesion molecules and remodeling of the maternal endometrium's extracellular matrices (ECM) and fetal chorion. This study aimed to investigate the distribution and localization of some classical cadherins (E-, N-, and P-cadherins) and extracellular matrix components collagen type 5α1, fibronectin, and laminin in the cow placentomes during pregnancy using immunohistochemical and Western blotting analyses. The study results confirmed the expression of E- and P-cadherins, collagen type Vα1 (COLVα1), fibronectin, and laminin in the cow placentomes, but not N-cadherin. Throughout the pregnancy, E- and P- cadherins, COLVα1, and laminin were localized in the luminal and glandular epithelium of the inter-caruncular endometrium, caruncular epithelium, and the uninucleate (UNCs) and binucleate trophoblast giant cells (BNCs/TGCs). E- cadherin immunoreactivity in the first pregnancy period was strong in the UNCs while moderate in the BNCs/TGCs. However, it was weak in both trophoblast in the second and third pregnancy periods. In the fetal trophoblasts, P- cadherin and laminin immunostainings were more intense in the BNCs/TGCs than UNCs. The fetal and maternal stromal cells were also positive for P- cadherin, COLVα1, fibronectin, and laminin. The immunostaining intensity of COLVα1 and fibronectin in the stromal extracellular matrix of the placentomes decreased as the pregnancy progressed. The endothelia of fetal and maternal vessels were positive for all proteins. The presence and distinct localization of cadherins and ECM proteins in the cow placentome components support the role of these molecules in regulating placental cell growth, migration, and matrix production during pregnancy.

Multi-level consistent changes of the ECM pathway identified in a typical keratoconus twin\u2019s family by multi-omics analysis

BackgroundKeratoconus (KC) is a common, degenerative disorder of the cornea, and genetic factors play a key role in its development. However, the genetic etiology of KC is still unclear. This study used the family of twins as material, using, for the first time, multi-omics analysis, to systematically display the changes in KC candidate factors in patients at the DNA, RNA, and protein levels. These can evaluate candidate pathogenic factors in depth and lock onto pathogenic targets.ResultsThe twins in this study presented classic phenotypes, clear diagnoses, complete case data, and clinical samples, which are excellent materials for genetically studying KC. Whole-exome sequencing was conducted on both the twins and their parents. Transcriptome sequencing was conducted on proband’s and health individual’s primary human corneal fibroblast cells. Quantitative Real-time PCR and western blot were used to validate the differential gene expressions between the proband and controls. By integrating genomics, transcriptome, and protein level data, multiple consecutive events of KC were systematically analyzed to help better understand the molecular mechanism and genetic basis of KC. The results showed that the accumulation of rare, micro-effect risk variants was the pathogenic factor in this Chinese KC family. Consistent changes in extracellular matrices (ECMs) at the DNA and RNA levels suggested that ECM related changes play a key role in KC pathogenesis. The major gene variants (WNT16, CD248, COL6A2, COL4A3 and ADAMTS3) may affect the expression of related collagens or ECM proteins, thus reducing the amount of ECM in corneas and resulting in KC.ConclusionsThis study, the first to explore the genetic etiology of KC via multi-omics analysis under the polygenetic model, has provided new insights into the genetic mechanisms underlying KC and an effective strategy for studying KC pathogenesis in the future.

Engineering Stem Cell-Derived Extracellular Matrices: Decellularization, Characterization, and Biological Function.

Stem cells, including mesenchymal stem cells and pluripotent stem cells, have attracted considerable attention in tissue engineering and regenerative medicine primarily because of their unique ability in self-renewal and multilineage differentiation. However, stem cells also have important secretory functions that form a specialized in vivo microenvironment and direct tissue development and regeneration. Extracellular matrices (ECMs) derived from stem cells retain the functional properties of their native environment and exhibit unique signaling that mediates stem cell self-renewal and lineage commitment. Stem cell-derived ECMs (scECMs) also have tunable properties corresponding to their developmental stages, suggesting that their lineage- and developmental specificity can be engineered for a wide range of applications. Hence, there is a growing interest in reconstructing stem cell microenvironment through decellularization and obtaining decellularized matrices that exhibit unique biological properties. This article summarizes recent advances in the use and understanding of scECMs. Moreover, future directions to extend the spectrum of applications of stem-derived ECMs in tissue engineering by comprehensively elucidating and engineering their regulatory function is highlighted. Impact statement Stem cells bear unique potency for multilineage differentiation as well as the capacity to secrete a vast amount of regulatory molecules. At different developmental stages, the extracellular matrices (ECMs) secreted by stem cells regulate their microenvironment and direct tissue development. The decellularization of stem cells effectively preserves ECM functional properties and can provide suitable templates to regulate stem cell fate decision, which can hardly be reproduced using single ECM proteins or synthetic scaffolds. This review highlights the unique regulatory functions of stem cell-derived ECMs, which can serve as novel sources of highly bioactive materials for tissue engineering and cell therapy.

Hyaluronan‐based hydrogels as versatile tumor‐like models: Tunable ECM and stiffness with genipin‐crosslinking

Three-dimensional (3D) biomimetic cell culture platforms offer more realistic microenvironments that cells naturally experience in vivo. We developed a tunable hyaluronan-based hydrogels that could easily be modified to mimic healthy or malignant extracellular matrices (ECMs). For that, we pre-functionalized our hydrogels with an adhesive polypeptide (poly-l-lysine, PLL) or ECM proteins (type III and type IV collagens), naturally present in tumorous tissues, and next, we tuned their stiffness by crosslinking with gradual concentrations of genipin (GnP). Then, we thoroughly characterized our substrates before testing them with glioblastoma and breast cancer cells, and thereafter with endothelial cells. Overall, our hydrogels exhibited (a) increasing stiffness with GnP concentration for every pre-functionalization and (b) efficient enzyme resistance with PLL treatment, and also with type IV collagen but to a lesser extent. While PLL-treated hydrogels were not favorable to the culture of any glioblastoma cell lines, they enhanced the proliferation of breast cancer cells in a stiffness-dependent manner. Contrary to type III collagen, type IV collagen pre-treated hydrogels supported the proliferation of glioblastoma cells. The as-desired HA-based 3D tumor-like models we developed may provide a useful platform for the study of various cancer cells by simply tuning their biochemical composition and their mechanical properties.

Strategy to replace animal-derived ECM by a modular and highly defined matrix_suppl2

Many extracellular matrices (ECM) used for modern cell culture are derived from animals. An alternative approach is the recombinant production of individual matrix protein components. A further development of this strategy uses a constant core protein polymer that is modifiable with functional domains of various ECM proteins. This way, a single, highlydefined ECM system could be used for a large variety of cell types. Self-assembling protein domains from human muscle sarcomeres, termed ZT material (ZT), have been shown to be suitable for this modular approach of generating ECMs. We explored in a proof-of-concept study whether ZT modified with the fibronectin 10 domain (ZTFn10) is able to substitute bovine serum-derived fibronectin as a coating for neural crest cell (NCC)-based toxicity testing. Human NCCs were generated from pluripotent stem cells and used in the automated version of an NCC migration assay (cMINC). ZTFn10, but not the unmodified core material (ZT), allowed high migration activity. The classical cMINC setup, with bovine fibronectin coating, was used as positive control, and detailed analysis of NCC migration by time-lapse recording indicated that the novel ECM fully matched the bioactivity of the traditional ECM. A final set of experiments showed that various positive controls of the cMINC assay (PCB180, LiCl, cytochalasin D) showed nearly identical inhibition curves on the traditional and the novel ECM. Thus, the cMINC, and possibly other bioassays, can be performed with a ZT-based ECM instead of traditional animal-derived protein coatings.

Strategy to replace animal-derived ECM by a modular and highly defined matrix_suppl5

Many extracellular matrices (ECM) used for modern cell culture are derived from animals. An alternative approach is the recombinant production of individual matrix protein components. A further development of this strategy uses a constant core protein polymer that is modifiable with functional domains of various ECM proteins. This way, a single, highlydefined ECM system could be used for a large variety of cell types. Self-assembling protein domains from human muscle sarcomeres, termed ZT material (ZT), have been shown to be suitable for this modular approach of generating ECMs. We explored in a proof-of-concept study whether ZT modified with the fibronectin 10 domain (ZTFn10) is able to substitute bovine serum-derived fibronectin as a coating for neural crest cell (NCC)-based toxicity testing. Human NCCs were generated from pluripotent stem cells and used in the automated version of an NCC migration assay (cMINC). ZTFn10, but not the unmodified core material (ZT), allowed high migration activity. The classical cMINC setup, with bovine fibronectin coating, was used as positive control, and detailed analysis of NCC migration by time-lapse recording indicated that the novel ECM fully matched the bioactivity of the traditional ECM. A final set of experiments showed that various positive controls of the cMINC assay (PCB180, LiCl, cytochalasin D) showed nearly identical inhibition curves on the traditional and the novel ECM. Thus, the cMINC, and possibly other bioassays, can be performed with a ZT-based ECM instead of traditional animal-derived protein coatings.

Strategy to replace animal-derived ECM by a modular and highly defined matrix.

Many extracellular matrices (ECM) used for modern cell culture are derived from animals. An alternative approach is the recombinant production of individual matrix protein components. A further development of this strategy uses a constant core protein polymer that is modifiable with functional domains of various ECM proteins. This way, a single, highly defined ECM system could be used for a large variety of cell types. Self-assembling protein domains from human muscle sarcomeres, termed here ZT material (ZT), have been shown to be suitable for this modular approach of generating ECMs. We explored in a proof-of-concept study, whether ZT, modified with the fibronectin 10 domain (ZTFn10) is able to substitute bovine serum-derived fibronectin as coating for neural crest cell (NCC)-based toxicity testing. Human NCC were generated from pluripotent stem cells and used in the automated version of a NCC migration assay (cMINC). ZTFn10, but not the unmodified core material (ZT), allowed for a high migration activity. The classical cMINC setup, with bovine fibronectin coating, was used as positive control, and detailed analysis of NCC migration by time-lapse recording indicated that the novel ECM fully matched the bioactivity of the traditional ECM. A final set of experiments showed that various positive controls of the cMINC assay (PCB180, LiCl, cytochalasin D) showed nearly identical inhibition curves on the traditional and the novel ECM. Thus, the cMINC, and possibly other bioassays, can be performed with a ZT-based ECM instead of traditional animal-derived protein coatings.

Acoustic modification of collagen hydrogels facilitates cellular remodeling.

Developing tunable biomaterials that have the capacity to recreate the physical and biochemical characteristics of native extracellular matrices (ECMs) with spatial fidelity is important for a variety of biomedical, biological, and clinical applications. Several factors have made the ECM protein, collagen I, an attractive biomaterial, including its ease of isolation, low antigenicity and toxicity, and biodegradability. However, current collagen gel formulations fail to recapitulate the range of collagen structures observed in native tissues, presenting a significant challenge in achieving the full potential of collagen-based biomaterials. Collagen fiber structure can be manipulated in vitro through mechanical forces, environmental factors, or thermal mechanisms. Here, we describe a new ultrasound-based fabrication technology that exploits the ability of ultrasound to generate localized mechanical forces to control the collagen fiber microstructure non-invasively. The results indicate that exposing soluble collagen to ultrasound (7.8 or 8.8 ​MHz; 3.2–10 ​W/cm2) during hydrogel formation leads to local variations in collagen fiber structure and organization that support increased levels of cell migration. Furthermore, multiphoton imaging revealed increased cell-mediated collagen remodeling of ultrasound-exposed but not sham-exposed hydrogels, including formation of multicellular aggregates, collagen fiber bundle contraction, and increased binding of collagen hybridizing peptides. Skin explant ​cultures obtained from diabetic mice showed similar enhancement of cell-mediated remodeling of ultrasound-exposed but not sham-exposed collagen hydrogels. Using the mechanical forces associated with ultrasound to induce local changes in collagen fibril structure and organization ​to functionalize native biomaterials is a promising non-invasive and non-toxic technology for tissue engineering and regenerative medicine.

Photoactivatable substrates for systematic study of the impact of an extracellular matrix ligand on appearance of leader cells in collective cell migration

Epithelial cells migrate as multicellular units. The directionality and speed of these units are determined by actively moving leader cells. It is important to understand how external cues affect the appearance of these leader cells in physiological and pathological processes. However, the impact of extracellular matrices (ECMs) is still controversial, because physically-adsorbed ECM proteins are amenable to protein remodeling, and uncontrolled cluster geometry can vary migration phenotypes. Here, we demonstrate a photoactivatable substrate, which we used to study the impact of a cyclic Arg-Gly-Asp (cRGD) ligand on leader cell formation in MDCK cells. This robust platform allowed us to investigate the effect of cRGD density on leader cell formation, in any given cluster geometry, with minimized ECM remodeling. Our results show a biphasic response of leader cell appearance upon reducing the surface cRGD density. The increase, in leader cell appearance, within the higher density range, is not only associated with the weakening of circumferential actomyosin belts, but also reduction of cellular mechanical tension and intercellular junctional E-cadherin. These results indicate that cRGD-mediated cell-ECM interactions positively regulate mechanical and biochemical coupling within cell clusters; both are critical for the coordination of cell collectives and eventual reduction in the appearance of leader cells.

Elastin-PLGA hybrid electrospun nanofiber scaffolds for salivary epithelial cell self-organization and polarization.

Regenerating the salivary glands by mimicking the extracellular matrix (ECM) is a promising approach for long term treatment of salivary gland damage. Despite their topographic similarity to the ECM, electrospun fibers of synthetic polymers lack the biochemical complexity, elasticity and hydrophilicity of the ECM. Elastin is an ECM protein abundant in the salivary glands and responsible for tissue elasticity. Although it's widely used for tissue regeneration of other organs, little is known about its utility in regenerating the salivary tissue. This study describes the use of elastin to improve the elasticity, hydrophilicity and biochemical complexity of synthetic nanofibers and its potential in directing in vivo-like organization of epithelial salivary cells which helps the design of efficient salivary gland regeneration scaffolds.

Design of bFGF-tethered self-assembling extracellular matrix proteins via coiled-coil triple-helix formation

Self-assembling peptides are attractive materials for tissue engineering applications because of their functionality including high biocompatibility and biodegradability. Modification of self-assembling peptides with functional motifs, such as the cell-adhesive tripeptide sequence RGD leads to functional artificial extracellular matrices (ECMs). In this study, we developed an artificial self-assembling ECM protein tethered with a growth factor via heterotrimer triple-helix (helix A/B/C) formation. The helix A and helix C peptides, which are capable of forming a heterodimer coiled-coil structure, were fused to both ends of a matrix protein composed of the elastin-derived structural unit (APGVGV)12 with an RGD motif. The helix B peptide, which constituents the third helix of the triple-helix structure, was fused with basic fibroblast growth factor (bFGF) for tethering to the artificial ECM proteins. Each recombinant protein exhibited cell adhesion and cell proliferation activities similar to the original, while the designed bFGF-tethered ECM protein exhibited superior cell proliferation activity. These results demonstrate that the approach of creating growth factor-tethered self-assembling proteins via triple-helix formation can be applied to develop functional ECMs for tissue engineering applications.

Induced Remodeling of Porcine Tendons to Human Anterior Cruciate Ligaments by α-GAL Epitope Removal and Partial Cross-Linking.

This review describes a novel method developed for processing porcine tendon and other ligament implants that enables in situ remodeling into autologous ligaments in humans. The method differs from methods using extracellular matrices (ECMs) that provide postoperative orthobiological support (i.e., augmentation grafts) for healing of injured ligaments, in that the porcine bone-patellar-tendon-bone itself serves as the graft replacing ruptured anterior cruciate ligament (ACL). The method allows for gradual remodeling of porcine tendon into autologous human ACL while maintaining the biomechanical integrity. The method was first evaluated in a preclinical model of monkeys and subsequently in patients. The method overcomes detrimental effects of the natural anti-Gal antibody and harnesses anti-non-gal antibodies for the remodeling process in two steps: Step 1. Elimination of α-gal epitopes—this epitope that is abundant in pigs (as in other nonprimate mammals) binds the natural anti-Gal antibody, which is the most abundant natural antibody in humans. This interaction, which can induce fast resorption of the porcine implant, is avoided by enzymatic elimination of α-gal epitopes from the implant with recombinant α-galactosidase. Step 2. Partial cross-linking of porcine tendon with glutaraldehyde—this cross-linking generates covalent bonds in the ECM, which slow infiltration of macrophages into the implant. Anti-non-gal antibodies are produced in recipients against the multiple porcine antigenic proteins and proteoglycans because of sequence differences between human and porcine homologous proteins. Anti-non-gal antibodies bind to the implant ECM, recruit macrophages, and induce the implant destruction by directing proteolytic activity of macrophages. Partial cross-linking of the tendon ECM decreases the extent of macrophage infiltration and degradation of the implant and enables concomitant infiltration of fibroblasts that follow the infiltrating macrophages. These fibroblasts align with the implant collagen fibers and secrete their own collagen and other ECM proteins, which gradually remodel the porcine tendon into human ACL. This ligamentization process lasts ∼2 years and the biomechanical integrity of the graft is maintained throughout the whole period. These studies are the first, and so far the only, to demonstrate remodeling of porcine tendon implants into permanently functional autologous ACL in humans.

Microproteome of dentoalveolar tissues

Proteomic analysis of extracellular matrices (ECM) of dentoalveolar tissues can provide insights into developmental, pathological, and reparative processes. However, targeted dissection of mineralized tissues, dental cementum (DC), alveolar bone (AB), and dentin (DE), presents technical difficulties. We demonstrate an approach combining EDTA decalcification and laser capture microdissection (LCM), followed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), to analyze proteome profiles of these tissues. Using the LCM-LC-MS/MS approach, a total of 243 proteins was identified from all tissues, 193 proteins in DC, 147 in AB, and 135 proteins DE. Ninety proteins (37% of total) were common to all tissues, whereas 52 proteins (21%) were overlapping in only two. Also, 101 (42%) proteins were exclusively detected in DC (60), AB (15), or DE (26). Identification in all tissues of expected ECM proteins including collagen alpha-1(I) chain (COL1A1), collagen alpha-1(XII) chain (COL12A1), biglycan (BGN), asporin (ASPN), lumican (LUM), and fibromodulin (FMOD), served to validate the approach. Principal component analysis (PCA) and hierarchical clustering identified a high degree of similarity in DC and AB proteomes, whereas DE presented a distinct dataset. Exclusively and differentially identified proteins were detected from all three tissues. The protein-protein interaction network (interactome) of DC was notable for its inclusion of several indicators of metabolic function (e.g. mitochondrial proteins, protein synthesis, and calcium transport), possibly reflecting cementocyte activity. The DE proteome included known and novel mineralization regulators, including matrix metalloproteinase 20 (MMP-20), 5′ nucleotidase (NT5E), and secreted phosphoprotein 24 (SPP-24 or SPP-2). Application of the LCM-LC-MS/MS approach to dentoalveolar tissues would be of value in many experimental designs, including developmental studies of transgenic animals, investigation of treatment effects, and identification of novel regenerative factors.

The apical ECM preserves embryonic integrity and distributes mechanical stress during morphogenesis.

Epithelia are bound by both basal and apical extracellular matrices (ECM). Although the composition and function of the former have been intensively investigated, less is known about the latter. The embryonic sheath, the ECM apical to the Caenorhabditis elegans embryonic epidermis, has been suggested to promote elongation of the embryo. In an RNAi screen for the components of the sheath, we identified the zona pellucida domain proteins NOAH-1 and NOAH-2. We found that these proteins act in the same pathway, and in parallel to three other putative sheath proteins, the leucine-rich repeat proteins SYM-1, LET-4 and FBN-1/Fibrillin, to ensure embryonic integrity and promote elongation. Laser nano-ablation experiments to map the stress field show that NOAH-1 and NOAH-2, together with PAK-1/p21-activated kinase, maintain and relay the actomyosin-dependent stress generated within the lateral epidermis before muscles become active. Subsequently, loss-of-function experiments show that apical ECM proteins are essential for muscle anchoring and for relaying the mechanical input from muscle contractions, which are essential for elongation. Hence, the apical ECM contributes to morphogenesis by maintaining embryonic integrity and relaying mechanical stress.

Comparative profiling of extractable proteins in extracellular matrices of porcine cholecyst and jejunum intended for preparation of tissue engineering scaffolds.

Scaffolds prepared from cholecyst and jejunum have differential immunological potential, despite similar biocompatibility, when used as subcutaneous grafts. The reason for differential immunogenicity is probably due to differences in the nature of protein composition and biomolecules in the extracellular matrices (ECMs) of source organs that are used for preparation of the scaffolds. Against this background, the present study aims to identify the extractable proteins of ECMs derived from porcine cholecyst and jejunum. The proteins were extracted and identified through a conventional database search following sodium dodecyl sulfate-polyacrylamide gel-electrophoresis separation and mass spectroscopy. The resultant protein profile was analyzed and at least 154 proteins in cholecyst-derived extracellular matrix (CDE) and 186 proteins in jejunum-derived extracellular matrix (JDE) were identified. Both the matrices contained several extracelluar proteins including fibronectin, nidogen, decorin, and lumican that are known to participate in wound healing responses. However, the CDE had fewer cellular proteins than JDE, especially the latter contained class-I and class-II histocompatibility antigens which are incriminated as potent immunogens responsible for graft rejection. The results of the study suggested that the ECMs used for the scaffold preparation need not be "acellular" and differences in the protein composition of the ECMs might have caused the differential wound healing responses. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 489-496, 2017.

A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability.

Biofilm-forming bacteria embedded in polymeric extracellular matrices (ECMs) that consist of polysaccharides, proteins and/or extracellular DNAs (eDNAs) acquire high resistance to antimicrobial agents and host immune systems. To understand molecular mechanisms of biofilm formation and maintenance and to develop therapeutic countermeasures against chronic biofilm-associated infections, reliable methods to isolate ECMs are inevitable. In this study, we refined the ECM extraction method recently reported and evaluated its applicability. Using three Staphylococcus aureus biofilms in which proteins, polysaccharides or eDNAs are major contributors to their integrity, ECMs were extracted using salts and detergents. We found that extraction with 1.5 M sodium chloride (NaCl) could be optimum for not only ECM proteins but also polysaccharides and eDNAs. In addition, long-time incubation was not necessary for efficient ECM isolation. Lithium chloride (LiCl) was comparative to NaCl but is more expensive. In contrast to SDS, NaCl hardly caused leakage of intracellular proteins and did not affect viability of bacterial cells within biofilms. Furthermore, this method is applicable to other bacteria such as Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli and Pseudomonas aeruginosa. Thus, this refined method is very simple, rapid, low cost and non-invasive and could be used for a broad range of applications.

Cnm is a major virulence factor of invasive Streptococcus mutans and part of a conserved three‐gene locus

SummaryCnm, a collagen‐ and laminin‐binding protein present in a subset of Streptococcus mutans strains, mediates binding to extracellular matrices (ECM), intracellular invasion and virulence in the Galleria mellonella model. Antibodies raised against Cnm were used to confirm expression and the cell surface localization of Cnm in the highly invasive OMZ175 strain. Sequence analysis identified two additional genes (cnaB and cbpA) encoding putative surface proteins immediately upstream of cnm. Inactivation of cnaB and cbpA in OMZ175, individually or in combination, did not decrease the ability of this highly invasive and virulent strain to bind to different ECM proteins, invade human coronary artery endothelial cells (HCAEC), or kill G. mellonella. Similarly, expression of cnaB and cbpA in the cnm− strain UA159 revealed that these genes did not enhance Cnm‐related phenotypes. However, integration of cnm in the chromosome of UA159 significantly increased its ability to bind to collagen and laminin, invade HCAEC, and kill G. mellonella. Moreover, the presence of antibodies against Cnm nearly abolished the ability of OMZ175 to bind to collagen and laminin and invade HCAEC, and significantly protected G. mellonella against OMZ175 infection. We concluded that neither CnaB nor CbpA is necessary for the expression of Cnm‐related traits. We also provided definitive evidence that Cnm is an important virulence factor and a suitable target for the development of novel preventive and therapeutic strategies to combat invasive S. mutans strains.