The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1.

Bone morphogenic protein-1 (BMP-1) was originally identified as one of several BMPs that induced new bone formation when implanted into ectopic sites in rodents. BMP-1, however, differed from other BMPs in that it its structure was not similar to transforming growth factor beta. Instead, it had a large domain homologous to a metalloendopeptidase isolated from crayfish, an epidermal growth-factor-like domain, and three regions of internal sequence homology referred to as CUB domains. Therefore, BMP-1 was a member of the "astacin families" of zinc-requiring endopeptidases. Many astacins have been shown to play critical roles in embryonic hatching, dorsal/ventral patterning, and early developmental decisions. Here, we have obtained amino acid sequences and isolated cDNA clones for procollagen C-proteinase (EC 3.4.24.19), an enzyme that is essential for the processing of procollagens to fibrillar collagens. The results demonstrate that procollagen C-proteinase is identical to BMP-1.

Ca2+ channel blockers modulate metabolism of collagens within the extracellular matrix.

The extracellular matrix (ECM) is an intricate network composed of an array of macromolecules capable of regulating the functional responsiveness of cells. Its composition greatly varies among different types of tissue, and dysregulation of its metabolism may contribute to vascular remodeling during the pathogenesis of various diseases, including atherosclerosis. In view of their antiatherosclerotic effects, the role of Ca2+ channel blockers in the metabolism of ECM was examined. Nanomolar concentrations of the five Ca2+ channel blockers amlodipine, felodipine, manidipine, verapamil, or diltiazem significantly decreased both the constitutive and platelet-derived growth factor BB-dependent collagen deposition in the ECM formed by human vascular smooth muscle cells and fibroblasts. The drugs inhibited the expression of fibrillar collagens type I and III and of basement membrane type IV collagen. Furthermore, Ca2+ channel blockers specifically increased the proteolytic activity of the 72-kDa type IV collagenase as shown by gelatin zymography and inhibited the transcription of tissue inhibitor of metalloproteinases-2.

A nutritional intervention study with hydrolyzed collagen in pre-pubertal Spanish children: influence on bone modeling biomarkers

Aim: The aim of the study was to investigate the influence of dietary intake of commercial hydrolyzed collagen (Gelatine Royal ®) on bone remodeling in pre-pubertal children. Methods: A randomized double-blind study was carried out in 60 children (9.42 ± 1.31 years) divided into three groups according to the amount of partially hydrolyzed collagen taken daily for 4 months: placebo (G-I, n = 18), collagen (G-II, n = 20) and collagen + calcium (G-III, n = 22) groups. Analyses of the following biochemical markers were carried out: total and bone alkaline phosphatase (tALP and bALP), osteocalcin, tartrate-resistant acid phosphatase (TRAP), type I collagen carboxy terminal telopeptide, lipids, calcium, 25-hydroxyvitamin D, insulin-like growth factor 1 (IGF-1), thyroid-stimulating hormone, free thyroxin and intact parathormone. Results: There was a significantly greater increase in serum IGF-1 in G-III than in G II (p < 0.01) or G-I (p < 0.05) during the study period, and a significantly greater increase in plasma tALP in G-III than in G-I (p < 0.05). Serum bALP behavior significantly (p < 0.05) differed between G-II (increase) and G-I (decrease). Plasma TRAP behavior significantly differed between G-II and G-I (p < 0.01) and between G-III and G-II (p < 0.05). Conclusion: Daily dietary intake of hydrolyzed collagen seems to have a potential role in enhancing bone remodeling at key stages of growth and development.

Characterization of Adult Stem/Progenitor Cell Populations From Bone Marrow in a Three-Dimensional Collagen Gel Culture System.

Stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a useful strategy. Although MSCs are commonly isolated by exploiting their plastic adherence, several studies have suggested that there are other populations of stem and/or osteoprogenitor cells which are removed from primary culture during media replacement. Therefore, we developed a three-dimensional (3D) culture system in which adherent and non-adherent stem cells are selected and expanded. Here, we described the characterization of 3D culture-derived cell populations in vitro and the capacity of these cells to differentiate into bone and/or cartilage tissue when placed inside of demineralized bone matrix (DBM) cylinders, implanted subcutaneously into the backs of rat for 2, 4 and 8 weeks. Our results demonstrates that 3D culture cells were a heterogeneous population of uncommitted cells that express pluripotent, hematopoietic, mesenchymal and endothelial specific markers in vitro and can undergo osteogenic differentiation in vivo.

Osteoarticular Expression of Musashi-1 in an Experimental Model of Arthritis.

Background. Collagen-induced arthritis (CIA), a murine experimental disease model induced by immunization with type II collagen (CII), is used to evaluate novel therapeutic strategies for rheumatoid arthritis. Adult stem cell marker Musashi-1 (Msi1) plays an important role in regulating the maintenance and differentiation of stem/precursor cells. The objectives of this investigation were to perform a morphological study of the experimental CIA model, evaluate the effect of TNFα-blocker (etanercept) treatment, and determine the immunohistochemical expression of Msi1 protein. Methods. CIA was induced in 50 male DBA1/J mice for analyses of tissue and serum cytokine; clinical and morphological lesions in limbs; and immunohistochemical expression of Msi1. Results. Clinically, TNFα-blocker treatment attenuated CIA on day 32 after immunization (P < 0.001). Msi1 protein expression was significantly higher in joints damaged by CIA than in those with no lesions (P < 0.0001) and was related to the severity of the lesions (Spearman's rho = 0.775, P = 0.0001). Conclusions. Treatment with etanercept attenuates osteoarticular lesions in the murine CIA model. Osteoarticular expression of Msi1 protein is increased in joints with CIA-induced lesion and absent in nonlesioned joints, suggesting that this protein is expressed when the lesion is produced in order to favor tissue repair.

Distribution of collagen types I, III, and IV in gastric tissue of marmosets (Callithrix spp., Callitrichidae: Primates)

Extracellular matrix (ECM) components such as fibrillar collagens play a fundamental role in wound repair and have also been studied in association with the gastric ulcer healing process in gastroenterology. Nevertheless, there have been no studies in the literature to date regarding the description and characterization of ECM components, neither in normal nor in injured gastric tissue of primate species. The objective of this study was to investigate the expression of gastric collagen types I, III, and IV in marmosets (Callithrix sp.). Histological specimens from the stomach of 6 Callithrix jacchus, 12 C. kuhli, and 12 C. geoffroyi were evaluated. The specimens were immunostained with anti-types I and III collagen polyclonal antibodies and anti-type IV collagen monoclonal antibody. Collagen types I and III were detected in the submucosa and lamina propria between the mucosal glands while collagen type IV was detected in the muscularis mucosae, muscular layers, blood vessels, and gastric mucosa between the mucosal glands. It is hoped that these findings can contribute to future studies on the gastric extracellular matrix components in primates and to comparative studies in the area of gastroenterology.

Collagenase specificity in chondrosarcoma metastasis

The treatment of some mesenchymal malignancies has made significant gains over the past few decades with the development of effective systemic therapies. In contrast, the treatment of chondrosarcoma has been limited to surgical resection, with the most significant prognostic indicators being surgical margins and histologic grade. We have reported that MMP-1/TIMP-1 gene expression serves to prognosticate for tumor recurrence in this group of patients. This led to the hypothesis that collagenase activity facilitates cell egression from the cartilaginous matrix. In the current study we examine the specificity of collagenase gene expression in archival human chondrosarcoma samples using semi-quantitative PCR. Messenger RNA was affinity extracted and subject to reverse transcription. The subsequent cDNA was amplified using novel primers and quantitated by densitometry. Ratios of gene expression were constructed and compared to disease-free survival. The data demonstrate that the significance of the MMP-1/TIMP-1 ratio as a predictor of recurrence is confirmed with a larger number of patients. Neutrophil collagenase or MMP-8 was observed in only 5 of 29 samples. Collagenase-3 or MMP-13 was observed in all samples but the level did not correlate with disease-free survival. Since the collagenases have similar activity for fibrillar collagens and cleave the peptide in the same location, post-transcriptional regulatory mechanisms may account for the observed specificity. The determination of the MMP-1/TIMP-1 gene expression ratio not only serves to identify those patients at risk for recurrence but may also serve as a novel therapeutic avenue as an adjunct to surgical resection.

Tensile bond strength of self-etching versus total-etching adhesive systems under different dentinal substrate conditions

The use of acid etchants to produce surface demineralization and collagen network exposure, allowing adhesive monomers interdiffusion and consequently the formation of a hybrid layer, has been considered the most efficient mechanism of dentin bonding. The aim of this study was to compare the tensile bond strength to dentin of three adhesive systems, two self-etching ones (Clearfil SE Bond - CSEB and One Up Bond F - OUBF) and one total-etching one (Single Bond - SB), under three dentinal substrate conditions (wet, dry and re-wet). Ninety human, freshly extracted third molars were sectioned at the occlusal surface to remove enamel and to form a flat dentin wall. The specimens were restored with composite resin (Filtek Z250) and submitted to tensile bond strength testing (TBS) in an MTS 810. The data were submitted to two-way ANOVA and Tukey's test (p = 0.05). Wet dentin presented the highest TBS values for SB and CSEB. Dry dentin and re-wet produced significantly lower TBS values when using SB. OUBF was not affected by the different conditions of the dentin substrate, producing similar TBS values regardless of the surface pretreatments.

In vivo preservation of the hybrid layer by chlorhexidine

Host-derived proteases have been reported to degrade the collagen matrix of incompletely-resin-infiltrated dentin. This study tested the hypothesis that interfacial degradation of resin-dentin bonds may be prevented or delayed by the application of chlorhexidine (CHX), a matrix metalloproteinase inhibitor, to dentin after phosphoric acid-etching. Contralateral pairs of resin-bonded Class I restorations in non-carious third molars were kept under intra-oral function for 14 months. Preservation of resin-dentin bonds was assessed by microtensile bond strength tests and TEM examination. In vivo bond strength remained stable in the CHX-treated specimens, while bond strength decreased significantly in control teeth. Resin-infiltrated dentin in CHX-treated specimens exhibited normal structural integrity of the collagen network. Conversely, progressive disintegration of the fibrillar network was identified in control specimens. Auto-degradation of collagen matrices can occur in resin-infiltrated dentin, but may be prevented by the application of a synthetic protease inhibitor, such as chlorhexidine.

Smear layer removal and collagen fiber exposure using tetracycline hydrochloride conditioning

Aim: Smear layer removal and collagen fiber exposure may improve periodontal treatment and regeneration. This in vitro study assessed smear layer removal and collagen fiber exposure after tetracycline hydrochloride (TTC) application on root surfaces using scanning electron microscopy (SEM). Methods and Materials: Root cementum was removed with diamond burs followed by scaling and root planning. Four hundred fifty samples were divided into ten groups: a control (saline application) and nine different TTC concentrations were applied at doses of 10, 25, 50, 75, 100, 125, 150, 200, and 250 mg/ml. The TTC application was performed in all groups in three different ways (passive, brushing, and burnishing) and at three different periods of conditioning (1, 2, and 3 minutes). A previously trained, calibrated, and blind examiner evaluated photomicrographs of the samples using Sampaio's index (2005). Statistical analysis was performed using the Kruskal-Wallis' and Dunn's tests. Results: The concentrations of 50 mg/mL and 75 mg/mL applied by burnishing were the most effective in smear layer removal and collagen fiber exposure. Both the passive mode of application (p=0.0001) and 1 minute period of application (p=0.002) were the least effective. Conclusions: The concentrations of 50 mg/mL and 75 mg/mL applied by burnishing during 2 or 3 minutes were the most effective. Clinical Significance: These parameters may be applied in periodontal procedures involving TTC root conditioning to optimize results.

Collagen XII interacts with avian tenascin-X through its NC3 domain

Large oligomeric proteins often contain several binding sites for different molecules and can therefore induce formation of larger protein complexes. Collagen XII, a multidomain protein with a small collagenous region, interacts with fibrillar collagens through its C-terminal region. However, no interactions to other extracellular proteins have been identified involving the non-collagenous N-terminal NC3 domain. To further elucidate the components of protein complexes present close to collagen fibrils, different extracellular matrix proteins were tested for interaction in a solid phase assay. Binding to the NC3 domain of collagen XII was found for the avian homologue of tenascin-X that in humans is linked to Ehlers-Danlos disease. The binding was further characterized by surface plasmon resonance spectroscopy and supported by immunohistochemical co-localization in chick and mouse tissue. On the ultrastructural level, detection of collagen XII and tenascin-X by immunogold labeling confirmed this finding.

How best to preserve and reveal the structural intricacies of cartilaginous tissue.

No single processing technique is capable of optimally preserving each and all of the structural entities of cartilaginous tissue. Hence, the choice of methodology must necessarily be governed by the nature of the component that is targeted for analysis, for example, fibrillar collagens or proteoglycans within the extracellular matrix, or the chondrocytes themselves. This article affords an insight into the pitfalls that are to be encountered when implementing the available techniques and how best to circumvent them. Adult articular cartilage is taken as a representative pars pro toto of the different bodily types. In mammals, this layer of tissue is a component of the synovial joints, wherein it fulfills crucial and diverse biomechanical functions. The biomechanical functions of articular cartilage have their structural and molecular correlates. During the natural course of postnatal development and after the onset of pathological disease processes, such as osteoarthritis, the tissue undergoes structural changes which are intimately reflected in biomechanical modulations. The fine structural intricacies that subserve the changes in tissue function can be accurately assessed only if they are faithfully preserved at the molecular level. For this reason, a careful consideration of the tissue-processing technique is indispensable. Since, as aforementioned, no single methodological tool is capable of optimally preserving all constituents, the approach must be pre-selected with a targeted structure in view. Guidance in this choice is offered.

Immunohistochemical evaluation of fibrillar components of the extracellular matrix of transversalis fascia and anterior abdominal rectus sheath in men with inguinal hernia

OBJECTIVE: to evaluate the role of fibrillar extracellular matrix components in the pathogenesis of inguinal hernias. METHODS: samples of the transverse fascia and of the anterior sheath of the rectus abdominis muscle were collected from 40 men aged between 20 and 60 years with type II and IIIA Nyhus inguinal hernia and from 10 fresh male cadavers (controls) without hernia in the same age range. The staining technique was immunohistochemistry for collagen I, collagen III and elastic fibers; quantification of fibrillar components was performed with an image analysis processing software. RESULTS: no statistically significant differences were found in the amount of elastic fibers, collagen I and collagen III, and the ratio of collagen I / III among patients with inguinal hernia when compared with subjects without hernia. CONCLUSION: the amount of fibrillar extracellular matrix components did not change in patients with and without inguinal hernia.

Collagens and proteoglycans of the corneal extracellular matrix

The cornea is a curved and transparent structure that provides the initial focusing of a light image into the eye. It consists of a central stroma that constitutes 90% of the corneal depth, covered anteriorly with epithelium and posteriorly with endothelium. Its transparency is the result of the regular spacing of collagen fibers with remarkably uniform diameter and interfibrillar space. Corneal collagen is composed of heterotypic fibrils consisting of type I and type V collagen molecules. The cornea also contains unusually high amounts of type VI collagen, which form microfibrillar structures, FACIT collagens (XII and XIV), and other nonfibrillar collagens (XIII and XVIII). FACIT collagens and other molecules, such as leucine-rich repeat proteoglycans, play important roles in modifying the structure and function of collagen fibrils.Proteoglycans are macromolecules composed of a protein core with covalently linked glycosaminoglycan side chains. Four leucine-rich repeat proteoglycans are present in the extracellular matrix of corneal stroma: decorin, lumican, mimecan and keratocan. The first is a dermatan sulfate proteoglycan, and the other three are keratan sulfate proteoglycans. Experimental evidence indicates that the keratan sulfate proteoglycans are involved in the regulation of collagen fibril diameter, and dermatan sulfate proteoglycan participates in the control of interfibrillar spacing and in the lamellar adhesion properties of corneal collagens. Heparan sulfate proteoglycans are minor components of the cornea, and are synthesized mainly by epithelial cells. The effect of injuries on proteoglycan synthesis is discussed.

The role of protecting groups in the formation of organogels through a nano-fibrillar network formed by self-assembling terminally protected tripeptides

A series of eight synthetic self-assembling terminally blocked tripeptides have been studied for gelation. Some of them form gels in various aromatic solvents including benzene, toluene, xylene, and chlorobenzene. It has been found that the protecting groups play an important role in the formation of organogels. It has been observed that, if the C-terminal has been changed from methyl ester to ethyl ester the gelation property does not change significantly (keeping the N-terminal protecting group same), while the change of the protecting group from ethyl ester to isopropyl ester completely abolishes the gelation property. Similarly, keeping the identical C-terminal protecting group (methyl ester) the results of the gelation study indicate that the substitution of N-terminal protection Boc-(tert-butyloxycarbonyl) to Cbz-(benzyloxycarbonyl) does change the gelation property insignificantly, while the change from Boc- to pivaloyl (Piv-) or acetyl (Ac-) group completely eliminates the gelation property. Morphological studies of the dried gels of two of the peptides indicate the presence of an entangled nano-fibrillar network that might be responsible for gelation. FTIR studies of the gels demonstrate that an intermolecular hydrogen bonding network is formed during gelation. Results of X-ray powder diffraction studies for these gelator peptides in different states (dried gels, gel, and bulk solids) reflected that the structure in the wet gel is distinctly different from the dried gel and solid state structures. Single crystal X-ray diffraction studies of a non-gelator peptide, which is structurally similar to the gelator molecules reveal that the peptide forms an antiparallel beta-sheet structure in crystals. (c) 2007 Elsevier Ltd. All rights reserved.