Search

Objective: To observe the effects of kidney-tonifying and blood circulation-promoting recipes and platelet-rich plasma (PRP) on the activity and expression of type I collagen in co-culture system. Methods: The subcutaneous adipose tissue and degenerative nucleus pulposus tissue of patients with nucleus pulposus removal under lumbar intervertebral disc degeneration were isolated and cultured by multiple enzyme digestion methods to establish a co-culture system of ADSCs/NPCs. Patients with lumbar intervertebral disc degeneration were enrolled for 2 weeks.The venous blood was taken 50mL before the first dose and one hour after the last dose. Platelet-rich plasma was prepared by secondary centrifugation and cultured with different plasma. The experiment was divided into five groups: platelet-rich plasma group (PRP), platelet-poor plasma group (PPP), drug-rich platelet-rich plasma group (M-PRP) and drug-poor platelet plasma group (M-PPP), fetal bovine serum group (FBS). The morphological changes of the cells in each group were observed under an inverted microscope. The changes of type I collagen activity were detected by immunofluorescence assay. The expression of CCOL1A1 gene was detected by RT-PCR two weeks after the intervention. Results: In this study, the results of kidney-tonifying and blood circulation-promoting recipes and PRP group intervention were shown. The results of the group intervention showed that the M-PRP group and the PRP group had clearer cell contours and better refractive index than the other groups, and there was no significant difference between the two groups. The results of immunofluorescence staining showed that the number of cells in PRP group and M-PRP group was higher, but the staining intensity of single cells was significantly lower than that in FBS group. RT-PCR results showed that the expression of COL1A1 mRNA in PPP group, PRP group, M-PRP group and M-PPP group was significantly down-regulated (P

Different stimulants might induce different extracellular matrix profiles. It is essential to gain an understanding and quantification of these changes to allow for focused anti-fibrotic drug development. This study investigated the expression of extracellular matrix by dermal fibroblast mimicking fibrotic skin diseases as SSc using clinically validated biomarkers. Primary healthy human dermal fibroblasts were grown in media containing FICOLL. The cells were stimulated with PDGF-AB, TGF-β1, or IL-6. Anti-fibrotic compounds (iALK-5, Nintedanib) were added together with growth factors. Biomarkers of collagen formation and degradation together with fibronectin were evaluated by ELISAs in the collected supernatant. Immunohistochemical staining was performed to visualize fibroblasts and proteins, while selected gene expression levels were examined through qPCR. TGF-β and PDGF, and to a lesser extent IL-6, increased the metabolic activity of the fibroblasts. TGF-β primarily increased type I collagen and fibronectin protein and gene expression together with αSMA. PDGF stimulation resulted in increased type III and VI collagen formation and gene expression. IL-6 decreased fibronectin levels. iALK5 could inhibit TGF-β induced fibrosis while nintedanib could halt fibrosis induced by TGF-β or PDGF. Tocilizumab could not inhibit fibrosis induced in this model. The extent and nature of fibrosis are dependent on the stimulant. The model has potential as a pre-clinical model as the fibroblasts fibrotic phenotype could be reversed by an ALK5 inhibitor and Nintedanib.

Metastasis or the progression of malignancy poses a major challenge in cancer therapy and is the principal reason for increased mortality. The epithelial-mesenchymal transition (EMT) of the basement membrane (BM) allows cells of epithelial phenotype to transform into a mesenchymal-like (quasi-mesenchymal) phenotype and metastasize via the lymphovascular system through a metastatic cascade by intravasation and extravasation. This helps in the progression of carcinoma from the primary site to distant organs. Collagen, laminin, and integrin are the prime components of BM and help in tumor cell metastasis, which makes them ideal cancer drug targets. Further, recent studies have shown that collagen, laminin, and integrin can be used as a biomarker for metastatic cells. In this review, we have summarized the current knowledge of such therapeutics, which are either currently in preclinical or clinical stages and could be promising cancer therapeutics.Data availability: Not applicable Scientific Research at Majmaah University [R-2022-117] Dr. Niraj Kumar Jha is thankful to Sharda University for the infra-structure and facility. The author would like to thank Deanship of Sci-entific Research at Majmaah University for supporting this work under project number No. R-2022-117. The authors would like to acknowledge the support from their respective institutes throughout the review writing process.

The majority of patients with osteogenesis imperfecta (OI) have mutations in the COL1A1 or COL1A2 gene, which has consequences for the composition of the bone matrix and bone architecture. The mutations result in overmodified collagen molecules, thinner collagen fibres and hypermineralization of bone tissue at a bone matrix level. Trabecular bone in OI is characterized by a lower trabecular number and connectivity as well as a lower trabecular thickness and volumetric bone mass. Cortical bone shows a decreased cortical thickness with less mechanical anisotropy and an increased pore percentage as a result of increased osteocyte lacunae and vascular porosity.Most OI patients have mutations at different locations in the COL1 gene. Disease severity in OI is probably partly determined by the nature of the primary collagen defect and its location with respect to the C-terminus of the collagen protein. The overall bone biomechanics result in a relatively weak and brittle structure. Since this is a result of all of the above-mentioned factors as well as their interactions, there is - considerable variation between patients, and accurate prediction on bone strength in the individual patient with OI is difficult.Current treatment of OI focuses on adequate vitamin-D levels and interventions in the bone turnover cycle with bisphosphonates. Bisphosphonates increase bone mineral density, but the evidence on improvement of clinical status remains limited. Effects of newer drugs such as antibodies against RANKL and sclerostin are currently under investigation.This paper was written under the guidance of the Study Group Genetics and Metabolic Diseases of the European Paediatric Orthopaedic Society.

Biomimetic scaffolds have been extensively studied for guiding osteogenesis through structural cues. Inspired by the natural bone growth process, we have employed a hierarchical outer-inner dual reinforcing strategy, which relies on the interfacial ionic bond interaction between amine/calcium and carboxyl groups, to build a nanofiber/particle dual strengthened hierarchical silk fibroin scaffold. This scaffold can provide an applicable form of osteogenic structural cue and mimic the natural bone forming process. Owing to the active interaction between compositions located in the outer pore space and the inner pore wall, the scaffold has over 4 times improvement in the mechanical properties, followed by a significant alteration of the cell-scaffold interaction pattern, demonstrated by over 2 times elevation in the spreading area and enhanced osteogenic activity potentially involving the activities of integrin, vinculin and Yes-associated protein (YAP). The in vivo performance of the scaffold identified the inherent osteogenic effect of the structural cue, which promotes rapid and uniform regeneration. Overall, the hierarchical scaffold is promising in promoting uniform bone regeneration through its specific structural cue endowed by its micro-nano construction.

The extracellular matrix can trigger cellular responses through its composition and structure. Major extracellular matrix components are the proteoglycans, which are composed of a core protein associated with glycosaminoglycans, among which the small leucine-rich proteoglycans (SLRPs) are the largest family. This review highlights how the codon usage pattern can be used to modulate cellular response and discusses the biological impact of post-translational events on SLRPs, including the substitution of glycosaminoglycan moieties, glycosylation, and degradation. These modifications are listed, and their impacts on the biological activities and structural properties of SLRPs are described. We narrowed the topic to skeletal tissues undergoing dynamic remodeling. ispartof: BIOMOLECULES vol:10 issue:1 ispartof: location:Switzerland status: published

Background Lipofilling is a treatment modality to restore tissue volume, but it may also rejuvenate the aging skin. Platelet-rich plasma has been reported to augment the efficacy of lipofilling, both on graft take and rejuvenation, by altering the adipose-derived stem cells. The authors hypothesized that addition of platelet-rich plasma would increase the rejuvenating effect and shorten recovery time. Methods The study conducted was a single-center, double-blind, placebo-controlled, randomized trial (2012 to 2015). In total, a well-defined cohort of 32 healthy female patients enrolled in the study, with 25 completing the follow-up. All patients underwent aesthetic facial lipofilling with either saline or platelet-rich plasma added. Outcome was determined by changes in skin elasticity, volumetric changes of the nasolabial fold, recovery time, and patient satisfaction during follow-up (1 year). Results Platelet-rich plasma did not improve the outcome of facial lipofilling when looking at skin elasticity improvement, graft volume maintenance in the nasolabial fold. Reversal of the correlation between age and elasticity, however, might suggest a small effect size, and thus might not be significant with our small study population. Conclusions This randomized, double-blind, placebo-controlled study clearly has shown that platelet-rich plasma significantly reduces postoperative recovery time but does not improve patient outcome when looking at skin elasticity, improvement of the nasolabial fold, or patient satisfaction. The reversal of the correlation between age and elasticity might indicate some effect on skin but requires more power in future studies. Clinical question/level of evidence Therapeutic, II.

Background : Osteogenesis imperfecta (OI) is a heterogeneous hereditary connective tissue disorder clinically hallmarked by increased susceptibility to bone fractures. Methods : We analyzed a cohort of 77 diagnosed OI patients from 49 unrelated Palestinian families. Next-generation sequencing technology was used to screen a panel of known OI genes. Results : In 41 probands, we identified 28 different disease-causing variants of 9 different known OI genes. Eleven of the variants are novel. Ten of the 28 variants are located in COL1A1, five in COL1A2, three in BMP1, three in FKBP10, two in TMEM38B, two in P3H1, and one each in CRTAP, SERPINF1, and SERPINH1. The absence of disease-causing variants in the remaining eight probands suggests further genetic heterogeneity in OI. In general, most OI patients (90%) harbor mainly variants in type I collagen resulting in an autosomal dominant inheritance pattern. However, in our cohort almost 61% (25/41) were affected with autosomal recessive OI. Moreover, we document a 21-kb genomic deletion in the TMEM38B gene identified in 29% (12/41) of the tested probands, making it the most frequent OI-causing variant in the Palestinian population. Conclusion : This is the first genetic screening of an OI cohort from the Palestinian population. Our data are important for genetic counseling of OI patients and families in highly consanguineous populations.

The majority of patients with osteogenesis imperfecta (OI) have mutations in the COL1A1 or COL1A2 gene, which has consequences for the composition of the bone matrix and bone architecture. The mutations result in overmodified collagen molecules, thinner collagen fibres and hypermineralization of bone tissue at a bone matrix level. Trabecular bone in OI is characterized by a lower trabecular number and connectivity as well as a lower trabecular thickness and volumetric bone mass. Cortical bone shows a decreased cortical thickness with less mechanical anisotropy and an increased pore percentage as a result of increased osteocyte lacunae and vascular porosity. Most OI patients have mutations at different locations in the COL1 gene. Disease severity in OI is probably partly determined by the nature of the primary collagen defect and its location with respect to the C-terminus of the collagen protein. The overall bone biomechanics result in a relatively weak and brittle structure. Since this is a result of all of the above-mentioned factors as well as their interactions, there is considerable variation between patients, and accurate prediction on bone strength in the individual patient with OI is difficult. Current treatment of OI focuses on adequate vitamin-D levels and interventions in the bone turnover cycle with bisphosphonates. Bisphosphonates increase bone mineral density, but the evidence on improvement of clinical status remains limited. Effects of newer drugs such as antibodies against RANKL and sclerostin are currently under investigation. This paper was written under the guidance of the Study Group Genetics and Metabolic Diseases of the European Paediatric Orthopaedic Society. Cite this article: Nijhuis WH, Eastwood DM, Allgrove J, Hvid I, Weinans HH, Bank RA, Sakkers RJ. Current concepts in osteogenesis imperfecta: bone structure, biomechanics and medical management.

Collagen-bound nucleation inhibitor ameliorates mineralization via caching of prenucleation clusters., Contemporary models of intrafibrillar mineralization mechanisms are established using collagen fibrils as templates without considering the contribution from collagen-bound apatite nucleation inhibitors. However, collagen matrices destined for mineralization in vertebrates contain bound matrix proteins for intrafibrillar mineralization. Negatively charged, high–molecular weight polycarboxylic acid is cross-linked to reconstituted collagen to create a model for examining the contribution of collagen-ligand interaction to intrafibrillar mineralization. Cryogenic electron microscopy and molecular dynamics simulation show that, after cross-linking to collagen, the bound polyelectrolyte caches prenucleation cluster singlets into chain-like aggregates along the fibrillar surface to increase the pool of mineralization precursors available for intrafibrillar mineralization. Higher-quality mineralized scaffolds with better biomechanical properties are achieved compared with mineralization of unmodified scaffolds in polyelectrolyte-stabilized mineralization solution. Collagen-ligand interaction provides insights on the genesis of heterogeneously mineralized tissues and the potential causes of ectopic calcification in nonmineralized body tissues.

International audience; In this review, recent progress in understanding the direct effects of radiation on the structure and stability of collagen, the most abundant protein in the human body, and other proteins is surveyed. Special emphasis is placed on the triple‐helical structure of collagen, as studied by means of collagen mimetic peptides. The emerging patterns are the dose dependence of radiation processes and their abundance, the crucial role of radicals in covalent‐bond formation (crosslinking) or cleavage, and the influence of the radiation energy and nature. Future research should allow fundamental questions, such as charge transfer and fragmentation dynamics triggered by ionization, to be answered, as well as developing applications such as protein‐based biomaterials, notably with properties controlled by irradiation.

Procollagen C-proteinase enhancer-1 (PCPE-1) is a secreted protein that specifically accelerates proteolytic release of the C-propeptides from fibrillar procollagens, a crucial step in fibril assembly. As such, it is a potential therapeutic target to improve tissue repair and prevent fibrosis, a major cause of mortality worldwide. Here we present the crystal structure of the active CUB1CUB2 fragment of PCPE-1 bound to the C-propeptide trimer of procollagen III (CPIII). This shows that the two CUB domains bind to two different chains of CPIII and that the N-terminal region of one CPIII chain, close to the proteolytic cleavage site, lies in the cleft between CUB1 and CUB2. This suggests that enhancing activity involves unraveling of this chain from the rest of the trimer, thus facilitating the action of the proteinase involved. Support for this hypothesis comes from site-directed mutagenesis, enzyme assays, binding studies, and molecular modeling.

Osteogenesis imperfecta (OI) is a skeletal dysplasia characterized by brittle bones and extraskeletal manifestations. The disease phenotype varies greatly. Most commonly, OI arises from monoallelic mutations in one of the two genes encoding type I collagen, COL1A1 and COL1A2 and is inherited as an autosomal dominant trait. Here, we describe a consanguineous family with autosomal recessive OI caused by a novel homozygous glycine substitution in COL1A2, NM_000089.3: c.604G > A, p.(Gly202Ser), detected by whole-genome sequencing. The index patient is a 31-year-old Greek woman with severe skeletal fragility. She had mild short stature, low bone mineral density of the lumbar spine and blue sclerae. She had sustained multiple long bone and vertebral fractures since childhood and had been treated with bisphosphonates for several years. She also had an affected sister with similar clinical manifestations. Interestingly, the parents and one sister, all carriers of the COL1A2 glycine mutation, did not have manifestations of OI. In summary, we report on autosomal recessive OI caused by a homozygous glycine-to-serine substitution in COL1A2, leading to severe skeletal fragility. The mutation carriers lacked OI manifestations. This family further expands the complex genetic spectrum of OI and underscores the importance of genetic evaluation for correct genetic counselling.

INTRODUCTION The components of bone assemble hierarchically to provide stiffness and toughness. Deciphering the specific organization and relationship between bone’s principal components—mineral and collagen—requires answers to three main questions: whether the association of the mineral phase with collagen follows an intrafibrillar or extrafibrillar pattern, whether the morphology of the mineral building blocks is needle- or platelet-shaped, and how the mineral phase maintains continuity across an extensive network of cross-linked collagen fibrils. To address these questions, a nanoscale level of three-dimensional (3D) structural characterization is essential and has now been performed. RATIONALE Because bone has multiple levels of 3D structural hierarchy, 2D imaging methods that do not detail the structural context of a sample are prone to interpretation bias. Site-specific focused ion beam preparation of lamellar bone with known orientation of the analyzed sample regions allowed us to obtain imaging data by 2D high-resolution transmission electron microscopy (HRTEM) and to identify individual crystal orientations. We studied higher-level bone mineral organization within the extracellular matrix by means of scanning TEM (STEM) tomography imaging and 3D reconstruction, as well as electron diffraction to determine crystal morphology and orientation patterns. Tomographic data allowed 3D visualization of the mineral phase as individual crystallites and/or aggregates that were correlated with atomic-resolution TEM images and corresponding diffraction patterns. Integration of STEM tomography with HRTEM and crystallographic data resulted in a model of 3D mineral morphology and its association with the organic matrix. RESULTS To visualize and characterize the crystallites within the extracellular matrix, we recorded imaging data of the bone mineral in two orthogonal projections with respect to the arrays of mineralized collagen fibrils. Three motifs of mineral organization were observed: “filamentous” (longitudinal or in-plane) and “lacy” (out-of-plane) motifs, which have been reported previously, and a third “rosette” motif comprising hexagonal crystals. Tomographic reconstructions showed that these three motifs were projections of the same 3D assembly. Our data revealed that needle-shaped, curved nanocrystals merge laterally to form platelets, which further organize into stacks of roughly parallel platelets separated by gaps of approximately 2 nanometers. These stacks of platelets, single platelets, and single acicular crystals coalesce into larger polycrystalline aggregates exceeding the lateral dimensions of the collagen fibrils, and the aggregates span adjacent fibrils as continuous, cross-fibrillar mineralization. CONCLUSION Our findings can be described by a model of mineral and collagen assembly in which the mineral organization is hierarchical at the nanoscale. First, the data reveal that mineral particles are neither exclusively needle- nor platelet-shaped, but indeed are a combination of both, because curved acicular elements merge laterally to form slightly twisted plates. This can only be detected when the organic extracellular matrix is preserved in the sample. Second, the mineral particles are neither exclusively intrafibrillar nor extrafibrillar, but rather form a continuous cross-fibrillar phase where curved and merging crystals splay beyond the typical dimensions of a single collagen fibril. Third, in the organization of the mineral phase of bone, a helical pattern can be identified. This 3D observation, integrated with previous studies of bone hierarchy and structure, illustrates that bone (as a material, as a tissue, and as an organ) follows a fractal-like organization that is self-affine. The assembly of bone components into nested, helix-like patterns helps to explain the paradoxical combination of enhanced stiffness and toughness of bone and results in an expansion of the previously known hierarchical structure of bone to at least 12 levels.

Adherence to oral bisphosphonates is low. A screening strategy is proposed based on the response of biochemical markers of bone turnover after 3 months of therapy. If no change is observed, the clinician should reassess the adherence to the treatment and also other potential issues with the drug.INTRODUCTION: Low adherence to oral bisphosphonates is a common problem that jeopardizes the efficacy of treatment of osteoporosis. No clear screening strategy for the assessment of compliance is widely accepted in these patients.METHODS: The International Osteoporosis Foundation and the European Calcified Tissue Society have convened a working group to propose a screening strategy to detect a lack of adherence to these drugs. The question to answer was whether the bone turnover markers (BTMs) PINP and CTX can be used to identify low adherence in patients with postmenopausal osteoporosis initiating oral bisphosphonates for osteoporosis. The findings of the TRIO study specifically address this question and were used as the basis for testing the hypothesis.RESULTS: Based on the findings of the TRIO study, specifically addressing this question, the working group recommends measuring PINP and CTX at baseline and 3 months after starting therapy to check for a decrease above the least significant change (decrease of more than 38% for PINP and 56% for CTX). Detection rate for the measurement of PINP is 84%, for CTX 87% and, if variation in at least one is considered when measuring both, the level of detection is 94.5%.CONCLUSIONS: If a significant decrease is observed, the treatment can continue, but if no decrease occurs, the clinician should reassess to identify problems with the treatment, mainly low adherence.

Collagen forms an organized network in articular cartilage to give tensile stiffness to the tissue. Due to its long half-life, collagen is susceptible to cross-links caused by advanced glycation end-products. The current standard method for determination of cross-link concentrations in tissues is the destructive high-performance liquid chromatography (HPLC). The aim of this study was to analyze the cross-link concentrations nondestructively from standard unstained histological articular cartilage sections by using Fourier transform infrared (FTIR) microspectroscopy. Half of the bovine articular cartilage samples ( n = 27 ) were treated with threose to increase the collagen cross-linking while the other half ( n = 27 ) served as a control group. Partial least squares (PLS) regression with variable selection algorithms was used to predict the cross-link concentrations from the measured average FTIR spectra of the samples, and HPLC was used as the reference method for cross-link concentrations. The correlation coefficients between the PLS regression models and the biochemical reference values were r = 0.84 ( p < 0.001 ), r = 0.87 ( p < 0.001 ) and r = 0.92 ( p < 0.001 ) for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), and pentosidine (Pent) cross-links, respectively. The study demonstrated that FTIR microspectroscopy is a feasible method for investigating cross-link concentrations in articular cartilage, published version, peerReviewed

Collagens are subjected to extensive posttranslational modifications, such as lysine hydroxylation. Bruck syndrome (BS) is a connective tissue disorder characterized at the molecular level by a loss of telopeptide lysine hydroxylation, resulting in reduced collagen pyridinoline cross-linking. BS results from mutations in the genes coding for lysyl hydroxylase (LH) 2 or peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65. Given that the immunophilin FKBP65 does not exhibit LH activity, it is likely that LH2 activity is somehow dependent on FKPB65. In this report, we provide insights regarding the interplay between LH2 and FKBP65. We found that FKBP65 forms complexes with LH2 splice variants LH2A and LH2B but not with LH1 and LH3. Ablating the catalytic activity of FKBP65 or LH2 did not affect complex formation. Both depletion of FKBP65 and inhibition of FKBP65 PPIase activity reduced the dimeric (active) form of LH2 but did not affect the binding of monomeric (inactive) LH2 to procollagen I alpha 1. Furthermore, we show that LH2A and LH2B cannot form heterodimers with each other but are able to form heterodimers with LH1 and LH3. Collectively, our results indicate that FKBP65 is linked to pyridinoline cross-linking by specifically mediating the dimerization of LH2. Moreover, FKBP65 does not interact with LH1 and LH3, explaining why in BS triple-helical hydroxylysines are not affected. Our results provide a mechanistic link between FKBP65 and the loss of pyridinolines and may hold the key to future treatments for diseases related to collagen cross-linking anomalies, such as fibrosis and cancer.

Several studies have demonstrated the presence of B-cell follicles and autoantibodies in chronic obstructive pulmonary disease (COPD). It is unclear against which antigens this B-cell response is directed and whether it contributes to development or worsening of disease.We assessed different B-cell subsets in blood and lung tissue from COPD patients and controls, and compared differences in B-cell responsiveness to stimulation with lung-specific antigens.Active smoking induced an adaptive immune response with relatively high levels of (class-switched) memory B-cells in blood and immunoglobulin (Ig)G memory B-cells in the lung. COPD smokers showed more switching to IgG, whereas healthy smokers switched more to IgA. COPD patients had higher levels of memory B-cells in the lung and stimulation with lung-specific antigens induced higher numbers of anti-decorin antibody-producing cells in COPD patients compared with healthy controls.Differential switching to IgG and IgA indicates that the adaptive immune response to smoke differs between COPD patients and healthy controls. A higher level of memory B-cells in the lungs of COPD patients may reflect an antigen-specific immune response, which could be directed against decorin, as suggested by the induction of anti-decorin antibody-producing cells in response to antigen-specific stimulation in COPD patients.

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. we have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha 2 beta 1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where 0 is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

Purpose Chronic periodontitis (CP) is a complex immuno-inflammatory disease that results from preestablished gingivitis. We investigated potential differences in salivary peptidome in health and CP. Experimental design Saliva was collected from nine CP patients and ten healthy subjects, from which five CP and five healthy were enriched following endoProteoFASP approach, separated and identified by nanoHPLC-MALDI-TOF/TOF. Protease prediction was carried out in silico with Proteasix. Parallel gelatin and collagen (I) zymographies were performed to study proteolytic activity in CP. Results An association of CP with increased gelatinolytic and collagenolytic activity was observed, which is mainly attributed to metalloproteases, remarkably MMP9. Protease prediction revealed distinct protease profiles in CP and in health. Peptidomic data corroborated the inflammatory status, and demonstrated that intact histatin 1 may play an important role in the defense response against oral pathogens. The application of the endoProteoFASP approach to study the salivary peptidome of CP subjects resulted in the identification of eight surrogate peptide markers, which may be used in multiplex to identify CP. These peptides belong to acidic PRP and to P-B peptide. Particularly, P-B peptide fragments exhibited domains with potential predicted antimicrobial activity, corroborating an antimicrobial function. Conclusions and clinical relevance The comparison between the salivary peptidome obtained by control and CP samples showed a specific association of eight peptides to CP, with remarkable predicted antimicrobial activity, which should be further validated in studies with large number of subjects.

A chromatographic method to purify decorin core protein from human lung tissue is described. The method is simple and rapid, using a combination of two-anion exchange and one reversed phase chromatography steps and the enzymatic digestion with chondroitinase ABC. Approximately 170 mu g decorin core protein were purified from 25 g of lung tissue with an enrichment factor of 1800-fold relative to the initial protein content. SDS-PAGE analysis of the final product revealed a single 42 kDa protein band, which was recognized by anti-decorin antibodies upon Western blotting and identified by mass spectrometry. Further digestion with PNGase F evidenced the presence of three N-linked oligosaccharides on the core protein. This method forms the basis for studying structural alterations of decorin related to the pathology of diseases where tissue destruction plays a role. (c) 2006 Elsevier B.V. All rights reserved.

Development of three dimensional (3D) microenvironments that direct stem cell differentiation into functional cell types remains a major challenge in the field of regenerative medicine. Here, we describe a new platform to address this challenge by utilizing a robotic microarray spotter for testing stem cell fates inside various miniaturized cell-laden gels in a systematic manner. To demonstrate the feasibility of our platform, we evaluated the osteogenic differentiation of human mesenchymal stem cells (hMSCs) within combinatorial 3D niches. We were able to identify specific combinations, that enhanced the expression of osteogenic markers. Notably, these ‘hit’ combinations directed hMSCs to form mineralized tissue when conditions were translated to 3D macroscale hydrogels, indicating that the miniaturization of the experimental system did not alter stem cell fate. Overall, our findings confirmed that the 3D cell-laden gel microarray can be used for screening of different conditions in a rapid, cost-effective, and multiplexed manner for a broad range of tissue engineering applications., United States. Office of Naval Research. Presidential Early Career Award for Scientists and Engineers, National Institutes of Health (U.S.) (HL092836), National Institutes of Health (U.S.) (HL099073), National Institutes of Health (U.S.) (DE019023), National Institutes of Health (U.S.) (DE019024), National Institutes of Health (U.S.) (AR057837), National Science Foundation (U.S.) (CAREER Award DMR 0847287), Wyss Institute for Biologically Inspired Engineering

To characterize the changes in the extracellular matrix in smoking-related pulmonary emphysema, we undertook immunohistochemical studies in lung tissues from controls (n = 7), from patients with mild (n = 11) and severe (n = 8) emphysema, and from patients with lung fibrosis (n = 6). We studied collagens, laminin, flbronectin, proteoglycans (PGs), and beta 1-integrins. The majority of the patients with severe emphysema showed diminished staining for the interstitial PGs, decorin and biglycan, in the peribronchiolar area, compared with patients in the control and fibrosis groups. Only a minority of patients with mild emphysema showed this diminished staining. In contrast, decorin and biglycan were well preserved in the perivascular area of all of the specimens from the emphysema group. Heparan sulfate PG staining was diminished in the respiratory airspace walls of patients with emphysema and fibrosis. Staining for Types I, III, and IV collagen, as well as for laminin, fibronectin, and the integrins, showed no differences between the four groups. The specific loss of interstitial PGs may be crucial for elastic recoil loss and subsequent bronchiolar obstruction, as seen in patients with smoking-related emphysema.

Tumor microenvironment (TME) is an active player in carcinogenesis and changes in its composition modify cancer growth. Carcinoma-associated fibroblasts, bone marrow-derived multipotent mesenchymal stem cells (BMMSCs), and inflammatory cells can all affect the composition of TME leading to changes in proliferation, invasion and metastasis formation of carcinoma cells. In this study, we confirmed an interaction between BMMSCs and oral tongue squamous cell carcinoma (OTSCC) cells by analyzing the invasion progression and gene expression pattern. In a 3-dimensional myoma organotypic invasion model the presence of BMMSCs inhibited the proliferation but increased the invasion of OTSCC cells. Furthermore, the signals originating from OTSCC cells up-regulated the expression of inflammatory chemokines by BMMSCs, whereas BMMSC products induced the expression of known invasion linked molecules by carcinoma cells. Particularly, after the cell-cell interactions, the chemokine CCL5 was abundantly secreted from BMMSCs and a function blocking antibody against CCL5 inhibited BMMSC enhanced cancer invasion area. However, CCL5 blocking antibody did not inhibit the depth of invasion. Additionally, after exposure to BMMSCs, the expression of type I collagen mRNA in OTSCC cells was markedly up-regulated. Interestingly, also high expression of type I collagen N-terminal propeptide (PINP) in vivo correlated with the cancer-specific mortality of OTSCC patients, whereas there was no association between cancer tissue CCL5 levels and the clinical parameters. In conclusion, our results suggest that the interaction between BMMSC and carcinoma cells induce cytokine and matrix molecule expression, of which high level of type I collagen production correlates with the prognosis of OTSCC patients.

Background - Recent reports have suggested short term changes in serum parameters of bone metabolism with inhaled corticosteroids. The relevance of these findings to the balance between bone formation and resorption during years of corticosteroid treatment remains uncertain.Methods - Two novel markers of bone turnover were first compared with conventional markers in a pilot study and subsequently measured in a long term double blind study of inhaled corticosteroids. In study I 15 patients were newly started on at least 800 mu g inhaled corticosteroids daily. At entry and after four weeks serum levels of alkaline phosphatase, osteocalcin, and PICP (procollagen type I carboxy terminal propeptide; a procollagen splice product) were measured as markers of bone formation, as well as the urinary hydroxyproline/creatinine ratio and serum levels of ICTP (type I collagen carboxy terminal telo-peptide; a collagen degradation product) as markers of bone resorption. In study II 70 patients with airways obstruction received 800 mu g beclomethasone daily in addition to terbutaline and 85 received bronchodilators only in a double blind fashion. Serum levels of PICP and ICTP were measured before and after 2.5 years of treatment.Results - In study I a decrease in osteocalcin levels was accompanied by an increase in levels of PICP and a small and non-significant rise in alkaline phosphatase. There were no changes in hydroxyproline or ICTP. In study II no differences were found in serum levels of PICP between the treatment groups; an increase in serum ICTP was found in the group treated without inhaled corticosteroids compared with the group treated with inhaled corticosteroids.Conclusions - No detrimental long term effect of inhaled corticosteroids was found with three conventional and two novel parameters of bone metabolism. The results indicate that long term changes in bone turnover during treatment with inhaled corticosteroids should not be deduced from short term studies with single serum parameters of bone metabolism, but well designed long term studies of, for example, bone densitometry should be awaited before quoting detrimental effects of inhaled corticosteroids on bone metabolism.

Purpose: Genome-wide association studies (GWAS) have identified 6q25, which incorporates the oestrogen receptor alpha gene (ESR1), as a quantitative trait locus for areal bone mineral density (BMD(a)) of the hip and lumbar spine. The aim of this study was to determine the influence of this locus on other bone health outcomes; calcaneal ultrasound (QUS) parameters, radial peripheral quantitative computed tomography (pQCT) parameters and markers of bone turnover in a population sample of European men. \ud \ud Methods: Eight single nucleotide polymorphisms (SNP) in the 6q25 locus were genotyped in men aged 40-79 years from 7 European countries, participating in the European Male Ageing Study (EMAS). The associations between SNPs and measured bone parameters were tested under an additive genetic model adjusting for centre using linear regression. \ud \ud Results: 2468 men, mean (SD) aged 59.9 (11.1) years had QUS measurements performed and bone turnover marker levels measured. A subset of 628 men had DXA and pQCT measurements. Multiple independent SNPs showed significant associations with BMD using all three measurement techniques. Most notably, rs1999805 was associated with a 0.10 SD (95%CI 0.05, 0.16; p = 0.0001) lower estimated BMD at the calcaneus, a 0.14 SD (95%CI 0.05, 0.24; p = 0.004) lower total hip BMD(a), a 0.12 SD (95%CI 0.02, 0.23; p = 0.026) lower lumbar spine BMD(a) and a 0.18 SD (95%CI 0.06, 0.29; p = 0.003) lower trabecular BMD at the distal radius for each copy of the minor allele. There was no association with serum levels of bone turnover markers and a single SNP which was associated with cortical density was also associated with cortical BMC and thickness. \ud \ud Conclusions: Our data replicate previous associations found between SNPs in the 6q25 locus and BMD(a) at the hip and extend these data to include associations with calcaneal ultrasound parameters and radial volumetric BMD.

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by bone fragility and alteration in synthesis and posttranslational modification of type I collagen. Autosomal dominant OI is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Bruck syndrome is a recessive disorder featuring congenital contractures in addition to bone fragility; Bruck syndrome type 2 is caused by mutations in PLOD2 encoding collagen lysyl hydroxylase, whereas Bruck syndrome type 1 has been mapped to chromosome 17, with evidence suggesting region 17p12, but the gene has remained elusive so far. Recently, the molecular spectrum of OI has been expanded with the description of the basis of a unique posttranslational modification of type I procollagen, that is, 3-prolyl-hydroxylation. Three proteins, cartilage-associated protein (CRTAP), prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene), and the prolyl cis-trans isomerase cyclophilin-B (PPIB), form a complex that is required for fibrillar collagen 3-prolyl-hydroxylation, and mutations in each gene have been shown to cause recessive forms of OI. Since then, an additional putative collagen chaperone complex, composed of FKBP10 (also known as FKBP65) and SERPINH1 (also known as HSP47), also has been shown to be mutated in recessive OI. Here we describe five families with OI-like bone fragility in association with congenital contractures who all had FKBP10 mutations. Therefore, we conclude that FKBP10 mutations are a cause of recessive osteogenesis imperfecta and Bruck syndrome, possibly Bruck syndrome Type 1 since the location on chromosome 17 has not been definitely localized. © 2011 American Society for Bone and Mineral Research. Copyright © 2011 American Society for Bone and Mineral Research.

Bone is a composite material in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals. [1,2] In the periodic 67 nm cross-striated pattern of the collagen fibril3, [4, 5], the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow. [6, 7, 8, 9] This process is believed to be directed by highly acidic non-collagenous proteins; [6, 7, 9, 10, 11] however, the role of the collagen matrix [12, 13, 14] during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography [15] with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleation.

An increase in bone turnover markers in patients with prostate cancer may predict bone metastases but it can also reflect the effects of androgen deprivation treatment. To assess the diagnostic efficacy of early detection of skeletal metastases we retrospectively performed serial measurements of a bone formation marker (PINP) and a bone resorption marker (ICTP) in serum of patients with prostate cancer.Residual serum samples from 64 patients with prostate cancer treated between 1999 and 2004 were selected from our prostate specific antigen serum archive, and divided into 3 groups of patients with no metastases (N0M0), with lymph node metastases only (N1M0) and with skeletal metastases (M1). In the M1 group the T(1) sample was collected near the first positive bone scintigraph.The N1M0 and M1 groups showed increased PINP levels (ANOVA T(0) p = 0.035, T(1) p0.001). The PINP levels in the M1 group increased further (paired t test p = 0.028), while no increase was found in the other groups. There was no significant difference between the number of patients receiving androgen deprivation therapy in the N1M0 and the M1 groups. Increased PINP levels in the M1 group were detectable 8 months before the first positive bone scintigraph. The increase in ICTP in the M1 group differed significantly from the other groups (the Student t test in 45 patients p = 0.029). The increases in PINP and ICTP differentiated between patients with or without skeletal metastases (AUC 0.71, p = 0.002 and AUC 0.64, p = 0.045, respectively).Followup measurement of serum PINP and ICTP is useful in the early assessment of skeletal metastases in patients with prostate cancer regardless of the confounding role of androgen deprivation treatment. The bone formation marker is the most indicative.

The use of yeast as a host for heterologous expression of proteins that are normally derived from animal tissue is a promising way to ensure defined products that are devoid of potential harmful animal side products. Here we report on the production and secretion of a custom-designed gelatin, Hu3-His8, by the yeast Hansenula polymorpha. We observed that Hu3-His8 was poorly secreted by the heterologous Saccharomyces cerevisiae invertase secretion signal. In contrast, the S. cerevisiae mating factor alpha prepro sequence efficiently directed secretion into the culture medium. However, at higher copy numbers, intracellular accumulation of Hu3-His8 precursors occurred. Overproduction of Erv29p, a protein required for packaging of the glycosylated pro-alpha factor into COPII vesicles, did not improve gelatin secretion in the multicopy strain. Previously, H. polymorpha was reported to hydroxylate proline residues in gelatinous sequences. In contrast, we were unable to detect hydroxyprolines in the secreted Hu3-His8. Also, we failed to identify a gene encoding prolyl-4-hydroxylase in the H. polymorpha genome.

Changes in the ECM and increased airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle (BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction (Emax) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I-induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin-induced depression of contractility was blocked by the integrin antagonist Arg-Gly-Asp-Ser (RGDS) but not by its negative control Gly-Arg-Ala-Asp-Ser-Pro (GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM-induced changes in Emaxin BTSM strips and their proliferative responses in BSTM cells and for Emaxand contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM.

Cytokines are considered a promising immunotherapy for chronic diseases, because of their potency and fundamental roles in pathological processes. However, their therapeutic use is limited because of their poor pharmacokinetics and pleiotropic effects in various organs. These problems may be overcome by cell-specific delivery of the cytokine. This approach involves chemical modification of the protein with homing devices that recognize receptors on target cells. The cytokine interleukin-10 (IL10) may be valuable as a therapeutic cytokine for patients with liver cirrhosis. However, its rapid renal elimination and general immunosuppressive activities limit therapeutic use. We therefore aim to target this cytokine in the liver, in particular to fibrogenic hepatic stellate cells (HSCs). We show that IL10 is successfully modified with mannose 6-phosphate (M6P), which is a homing device for the mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptor expressed on activated HSCs. Chemical modification did not diminish IL10 efficacy with regard to in vitro anti-inflammatory (lipopolysaccharide-stimulated tumor necrosis factor alpha release) and antifibrotic (collagen deposition and degradation) activities. Biodistribution studies with radiolabeled M6P-IL10 and IL10 in rats with liver fibrosis showed that modification with M6P groups induced a shift in the distribution from the kidneys (IL10) to the liver (M6P-IL10). Hepatocellular binding of M6P-IL10 occurred via M6P/IGFII receptors and scavenger receptors, indicating that not only HSCs but also Kupffer and endothelial cells are target cells. IL10 did not bind to these receptors. We conclude that we prepared an active and liver-specific form of the cytokine IL10 that can be evaluated for its efficacy to treat liver diseases.

Bu çalışma, 16-20 Eylül 2001 tarihleri arasında Rome[İtalya]’de düzenlenen 9. Congress on ESPRAS’da bildiri olarak sunulmuştur. New cartilage formation originated from perichondrium has previously been researched in many clinical and experimental studies.(1-6) After these studies, the use of perichondrium for the repair of cartilage defects has been used clinically when enhancing neochondrogenesis has been found to be of great value in decreasing the recovery period. Collagen is the major protein of whole connective tissue, and in vitro positive effects of collagen matrices on neochondrogenesis have also been studied before.(7-9) In this experimental study, in vivo effects of heterologous collagen sponge in perichondrial neochondrogenesis were examined in an animal model, and acceleration and enhancement effects were observed.

To characterize the changes in the extracellular matrix in smoking-related pulmonary emphysema, we undertook immunohistochemical studies in lung tissues from controls (n = 7), from patients with mild (n = 11) and severe (n = 8) emphysema, and from patients with lung fibrosis (n = 6). We studied collagens, laminin, flbronectin, proteoglycans (PGs), and beta 1-integrins. The majority of the patients with severe emphysema showed diminished staining for the interstitial PGs, decorin and biglycan, in the peribronchiolar area, compared with patients in the control and fibrosis groups. Only a minority of patients with mild emphysema showed this diminished staining. In contrast, decorin and biglycan were well preserved in the perivascular area of all of the specimens from the emphysema group. Heparan sulfate PG staining was diminished in the respiratory airspace walls of patients with emphysema and fibrosis. Staining for Types I, III, and IV collagen, as well as for laminin, fibronectin, and the integrins, showed no differences between the four groups. The specific loss of interstitial PGs may be crucial for elastic recoil loss and subsequent bronchiolar obstruction, as seen in patients with smoking-related emphysema.

Background - Recent reports have suggested short term changes in serum parameters of bone metabolism with inhaled corticosteroids. The relevance of these findings to the balance between bone formation and resorption during years of corticosteroid treatment remains uncertain. Methods - Two novel markers of bone turnover were first compared with conventional markers in a pilot study and subsequently measured in a long term double blind study of inhaled corticosteroids. In study I 15 patients were newly started on at least 800 mu g inhaled corticosteroids daily. At entry and after four weeks serum levels of alkaline phosphatase, osteocalcin, and PICP (procollagen type I carboxy terminal propeptide; a procollagen splice product) were measured as markers of bone formation, as well as the urinary hydroxyproline/creatinine ratio and serum levels of ICTP (type I collagen carboxy terminal telo-peptide; a collagen degradation product) as markers of bone resorption. In study II 70 patients with airways obstruction received 800 mu g beclomethasone daily in addition to terbutaline and 85 received bronchodilators only in a double blind fashion. Serum levels of PICP and ICTP were measured before and after 2.5 years of treatment. Results - In study I a decrease in osteocalcin levels was accompanied by an increase in levels of PICP and a small and non-significant rise in alkaline phosphatase. There were no changes in hydroxyproline or ICTP. In study II no differences were found in serum levels of PICP between the treatment groups; an increase in serum ICTP was found in the group treated without inhaled corticosteroids compared with the group treated with inhaled corticosteroids. Conclusions - No detrimental long term effect of inhaled corticosteroids was found with three conventional and two novel parameters of bone metabolism. The results indicate that long term changes in bone turnover during treatment with inhaled corticosteroids should not be deduced from short term studies with single serum parameters of bone metabolism, but well designed long term studies of, for example, bone densitometry should be awaited before quoting detrimental effects of inhaled corticosteroids on bone metabolism.

In primary human melanoma, the membrane-type matrix metalloproteinase, MT3-MMP, is overexpressed in the most aggressive nodular-type tumors. Unlike MT1-MMP and MT2-MMP, which promote cell invasion through basement membranes and collagen type I-rich tissues, the function of MT3-MMP in tumor progression remains unclear. Here, we demonstrate that MT3-MMP inhibits MT1-MMP-driven melanoma cell invasion in three-dimensional collagen, while yielding an altered, yet MT1-MMP-dependent, form of expansive growth behavior that phenocopies the formation of nodular cell colonies. In melanoma cell lines originating from advanced primary or metastatic lesions, endogenous MT3-MMP expression was associated with limited collagen-invasive potential. In the cell lines with highest MT3-MMP expression relative to MT1-MMP, collagen-invasive activity was increased following stable MT3-MMP gene silencing. Consistently, MT3-MMP overexpression in cells derived from less advanced superficially spreading melanoma lesions, or in the MT3-MMP knockdown cells, reduced MT1-MMP-dependent collagen invasion. Rather than altering MT1-MMP transcription, MT3-MMP interacted with MT1-MMP in membrane complexes and reduced its cell surface expression. By contrast, as a potent fibrinolytic enzyme, MT3-MMP induced efficient invasion of the cells in fibrin, a provisional matrix component frequently found at tumor-host tissue interfaces and perivascular spaces of melanoma. Since MT3-MMP was significantly upregulated in biopsies of human melanoma metastases, these results identify MT3-MMP as a matrix-dependent modifier of the invasive tumor cell functions during melanoma progression.

Background Osteogenesis Imperfecta (OI) is a heritable connective tissue disorder mainly caused by mutations in the genes COL1A1 and COL1A2 and is associated with hearing loss in approximately half of the cases. The hearing impairment usually starts between the second and fourth decade of life as a conductive hearing loss, frequently evolving to mixed hearing loss thereafter. A minority of patients develop pure sensorineural hearing loss. The interindividual variability in the audiological characteristics of the hearing loss is unexplained. Methods With the purpose of evaluating inter- and intrafamilial variability, hearing was thorougly examined in 184 OI patients (type I: 154; type III: 4; type IV: 26), aged 3-89 years, with a mutation in either COL1A1 or COL1A2 and originating from 89 different families. Due to the adult onset of hearing loss in OI, correlations between the presence and/or characteristics of the hearing loss and the underlying mutation were investigated in a subsample of 114 OI patients from 64 different families who were older than 40 years of age or had developed hearing loss before the age of 40. Results Hearing loss was diagnosed in 48.4% of the total sample of OI ears with increasing prevalence in the older age groups. The predominant type was a mixed hearing loss (27.5%). A minority presented a pure conductive (8.4%) or pure sensorineural (12.5%) loss. In the subsample of 114 OI subjects, no association was found between the nature of the mutation in COL1A1 or COL1A2 genes and the occurrence, type or severity of hearing loss. Relatives originating from the same family differed in audiological features, which may partially be attributed to their dissimilar age. Conclusions Our study confirms that hearing loss in OI shows a strong intrafamilial variability. Additional modifications in other genes are assumed to be responsible for the expression of hearing loss in OI.