Abnormal collagen fibrils in cartilage of matrilin-1/matrilin-3-deficient mice

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.

Absorbable collagen membranes for periodontal regeneration: a systematic review

Guided tissue regeneration (GTR) with bioabsorbable collagen membranes (CM) is commonly used for the treatment of periodontal defects. The objective of this systematic review of randomized clinical trials was to assess the clinical efficacy of GTR procedures with CM, with or without bone substitutes, in periodontal infrabony defects compared with that of open flap debridement (OFD) alone. Primary outcomes were tooth loss and gain in clinical attachment level (CAL). Screening of records, data extraction, and risk-of-bias assessments were performed by two reviewers. Weighted mean differences were estimated by random effects meta-analysis. We included 21 reports on 17 trials. Risk of bias was generally high. No data were available for the primary outcome tooth loss. The summary treatment effect for change in CAL for GTR with CM compared with OFD was 1.58 mm (95% CI, 1.27 to 1.88). Despite large between-trial heterogeneity (I2 = 75%, p < .001), all trials favored GTR over OFD. No differences in treatment effects were detected between trials of GTR with CM alone and trials of GTR with CM in combination with bone substitutes (p for interaction, .31). GTR with CM, with or without substitutes, may result in improved clinical outcomes compared with those achieved with OFD alone. Our findings support GTR with CM for the treatment of infrabony periodontal defects.

Treatment of multiple adjacent Miller class I and II gingival recessions with a Modified Coronally Advanced Tunnel (MCAT) technique and a collagen matrix or palatal connective tissue graft: a randomized, controlled clinical trial

BACKGROUND A newly developed collagen matrix (CM) of porcine origin has been shown to represent a potential alternative to palatal connective tissue grafts (CTG) for the treatment of single Miller Class I and II gingival recessions when used in conjunction with a coronally advanced flap (CAF). However, at present it remains unknown to what extent CM may represent a valuable alternative to CTG in the treatment of Miller Class I and II multiple adjacent gingival recessions (MAGR). The aim of this study was to compare the clinical outcomes following treatment of Miller Class I and II MAGR using the modified coronally advanced tunnel technique (MCAT) in conjunction with either CM or CTG. METHODS Twenty-two patients with a total of 156 Miller Class I and II gingival recessions were included in this study. Recessions were randomly treated according to a split-mouth design by means of MCAT + CM (test) or MCAT + CTG (control). The following measurements were recorded at baseline (i.e. prior to surgery) and at 12 months: Gingival Recession Depth (GRD), Probing Pocket Depth (PD), Clinical Attachment Level (CAL), Keratinized Tissue Width (KTW), Gingival Recession Width (GRW) and Gingival Thickness (GT). GT was measured 3-mm apical to the gingival margin. Patient acceptance was recorded using a Visual Analogue Scale (VAS). The primary outcome variable was Complete Root Coverage (CRC), secondary outcomes were Mean Root Coverage (MRC), change in KTW, GT, patient acceptance and duration of surgery. RESULTS Healing was uneventful in both groups. No adverse reactions at any of the sites were observed. At 12 months, both treatments resulted in statistically significant improvements of CRC, MRC, KTW and GT compared with baseline (p < 0.05). CRC was found at 42% of test sites and at 85% of control sites respectively (p < 0.05). MRC measured 71 ± 21% mm at test sites versus 90 ± 18% mm at control sites (p < 0.05). Mean KTW measured 2.4 ± 0.7 mm at test sites versus 2.7 ± 0.8 mm at control sites (p > 0.05). At test sites, GT values changed from 0.8 ± 0.2 to 1.0 ± 0.3 mm, and at control sites from 0.8 ± 0.3 to 1.3 ± 0.4 mm (p < 0.05). Duration of surgery and patient morbidity was statistically significantly lower in the test compared with the control group respectively (p < 0.05). CONCLUSIONS The present findings indicate that the use of CM may represent an alternative to CTG by reducing surgical time and patient morbidity, but yielded lower CRC than CTG in the treatment of Miller Class I and II MAGR when used in conjunction with MCAT.

Treatment of multiple adjacent Miller Class I and II gingival recessions with collagen matrix and the modified coronally advanced tunnel technique

OBJECTIVE To clinically evaluate the treatment of Miller Class I and II multiple adjacent gingival recessions using the modified coronally advanced tunnel technique combined with a newly developed bioresorbable collagen matrix of porcine origin. METHOD AND MATERIALS Eight healthy patients exhibiting at least three multiple Miller Class I and II multiple adjacent gingival recessions (a total of 42 recessions) were consecutively treated by means of the modified coronally advanced tunnel technique and collagen matrix. The following clinical parameters were assessed at baseline and 12 months postoperatively: full mouth plaque score (FMPS), full mouth bleeding score (FMBS), probing depth (PD), recession depth (RD), recession width (RW), keratinized tissue thickness (KTT), and keratinized tissue width (KTW). The primary outcome variable was complete root coverage. RESULTS Neither allergic reactions nor soft tissue irritations or matrix exfoliations occurred. Postoperative pain and discomfort were reported to be low, and patient acceptance was generally high. At 12 months, complete root coverage was obtained in 2 out of the 8 patients and 30 of the 42 recessions (71%). CONCLUSION Within their limits, the present results indicate that treatment of Miller Class I and II multiple adjacent gingival recessions by means of the modified coronally advanced tunnel technique and collagen matrix may result in statistically and clinically significant complete root coverage. Further studies are warranted to evaluate the performance of collagen matrix compared with connective tissue grafts and other soft tissue grafts.

Computational and experimental methodology for site-matched investigations of the influence of mineral mass fraction and collagen orientation on the axial indentation modulus of lamellar bone

Relationships between mineralization, collagen orientation and indentation modulus were investigated in bone structural units from the mid-shaft of human femora using a site-matched design. Mineral mass fraction, collagen fibril angle and indentation moduli were measured in registered anatomical sites using backscattered electron imaging, polarized light microscopy and nano-indentation, respectively. Theoretical indentation moduli were calculated with a homogenization model from the quantified mineral densities and mean collagen fibril orientations. The average indentation moduli predicted based on local mineralization and collagen fibers arrangement were not significantly different from the average measured experimentally with nanoindentation (p=0.9). Surprisingly, no substantial correlation of the measured indentation moduli with tissue mineralization and/or collagen fiber arrangement was found. Nano-porosity, micro-damage, collagen cross-links, non-collagenous proteins or other parameters affect the indentation measurements. Additional testing/simulation methods need to be considered to properly understand the variability of indentation moduli, beyond the mineralization and collagen arrangement in bone structural units.

Importance of the collagen adhesin ace in pathogenesis and protection against Enterococcus faecalis experimental endocarditis.

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFDeltaace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFDeltaace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

Role played by serum, a biological cue, in the adherence of Enterococcus faecalis to extracellular matrix proteins, collagen, fibrinogen, and fibronectin.

BACKGROUND: Most previous studies have found that Enterococcus faecalis isolates do not show significant adherence to fibronectin and fibrinogen. METHODS: The influence of various conditions on E. faecalis adherence to extracellular matrix (ECM) proteins was evaluated using a radiolabeled-cell adherence assay. RESULTS: Among the conditions studied, growth in 40% horse serum (a biological cue with potential clinical relevance) elicited adherence of all 46 E. faecalis strains tested to fibronectin and fibrinogen but not to elastin; adherence levels were independent of strain source, and adherence was eliminated by treating cells with trypsin. As previously reported, serum also elicited adherence to collagen. Although prolonged exposure to serum during growth was needed for enhancement of adherence to fibrinogen, brief exposure (<5 >min) to serum had an immediate, although partial, enhancing effect on adherence to fibronectin and, to a lesser extent, collagen; pretreatment of bacteria with chloramphenicol did not decrease this enhanced adherence to fibronectin and collagen, indicating that protein synthesis is not required for the latter effect. CONCLUSION: Taken together, these data suggest that serum components may serve (1) as host environmental stimuli to induce the production of ECM protein-binding adhesin(s), as previously seen with collagen adherence, and also (2) as activators of adherence, perhaps by forming bridges between ECM proteins and adhesins.

Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen.

Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and early-onset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the timing of growth plate closure when other extracellular proteins are absent.

Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis.

Enterococcus faecium is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating E. faecium virulence determinants. Our previous studies showed that some clinical E. faecium isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic acm deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Deltaacm::cat is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P < 0.0001) and for valve colonization 1 and 3 hours after infection (P or=50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that acm transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in E. faecium collagen adherence by affinity-purified anti-Acm antibodies from E. faecium endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.

A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen.

Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P