Collagen Degradation Products Research Articles

Metabolomics reveals elevated urinary excretion of collagen degradation and epithelial cell turnover products in irritable bowel syndrome patients.

Irritable bowel syndrome (IBS), the most commonly diagnosed functional gastrointestinal (GI) disorder in developed countries, is characterized by chronic abdominal pain, and altered bowel habits. Accurate and timely diagnosis is challenging as it relies on symptoms and an evolving set of exclusion criteria to distinguish it from other related GI disorders reflecting a complex etiology that remains poorly understood. Herein, nontargeted metabolite profiling of repeat urine specimens collected from a cohort of IBS patients (n = 42) was compared to healthy controls (n = 20) to gain insights into the underlying pathophysiology. An integrated data workflow for characterization of the urine metabolome with stringent quality control was developed to authenticate reliably measured (CV < 30%) and frequently detected (> 75%) metabolites using multisegment injection-capillary electrophoresis-mass spectrometry. Complementary statistical methods were then used to rank differentially excreted urinary metabolites after normalization to osmolality that were subsequently identified by high resolution tandem mass spectrometry and their electrophoretic migration behavior. Our work revealed ten consistently elevated urinary metabolites in repeat samples collected from IBS patients at two different time points (q < 0.05 after age and Benjamini-Hochberg/FDR adjustment), which were associated with greater collagen degradation and intestinal mucosal turn-over processes likely due to low-grade inflammation. IBS-specific metabolites identified in urine included a series of hydroxylysine metabolites (O-glycosylgalactosyl-hydroxylysine, O-galactosyl-hydroxylysine, lysine), mannopyranosy-L-tryptophan, imidazole propionate, glutamine, serine, ornithine, dimethylglycine and dimethylguanosine. A major limitation in this retrospective case-control study was significant co-morbidity of IBS patients with other illnesses, including depression and prescribed medications as compared to healthy controls. This work provides new mechanistic insights into the pathophysiology of IBS while also offering a convenient way to monitor patient disease progression and treatment responses to therapy based on a panel of urinary metabolites that avoids invasive blood sampling, colonoscopy and/or tissue biopsies.

Stabilization and cyclic utilization of chrome leather shavings.

For the sake of the safe and efficient cyclic utilization of chrome leather shavings (CLS), the temporary stabilization of CLS during the storage and transport processes was optimized. Afterwards, the large-scale stabilized CLS degradation and collagen degradation product (CDP) extraction were completed. In this research, hydroxyapatite (HAP) and calcium hydroxide (Ca(OH)2) were for the first time used as stabilizers in the stabilization of CLS. The results showed that both Ca(OH)2 and HAP displayed resistance to chrome leaching, but HAP provided better stabilization effect than Ca(OH)2. The optimized stabilization conditions giving lower chromium leaching concentration were selected: 2% HAP, temperature 300°C, reaction time 60min, and initially added water contents of 50%. The reason for HAP and Ca(OH)2 stabilization was proved by energy dispersive X-ray spectroscopy (EDS). Subsequently, the exploitation value of the stabilized CLS was described by large-scale experiment. The hydrolysis of stabilized CLS could obtain the collagen degradation product (CDP), which had 3.52% of free amino content and a molecular weight of 3.4~30kDa. The filter residue was treated by 5% sulfuric acid solution extraction and pressure filtration again. The extraction solution and final filter residue were dried separately to obtain chromium-containing organic compounds (COP) and industrial gypsum. The COP, used as a filling for tannery, contains 9.37% Cr2O3 content and 90.6% organic content. The industrial gypsum contains 68.37% CaSO4, 0.13% Cr2O3, and 31.5% residual organic or inorganic compounds, which can be used in building materials. No waste water or solid waste was discharged in this experiment process.

Cathepsin S is associated with degradation of collagen I in abdominal aortic aneurysm.

Cathepsins have been described in the pathogenesis of abdominal aortic aneurysm (AAA), their exact role, especially in collagen degradation, is still unclear. The aim of the present study was therefore to analyse relevant cathepsins in human AAA tissue samples in relation to collagen I, III, and their degradation products. Samples from 37 AAA patients obtained from elective open surgical repair and eight healthy non-aneurysmatic aortas from kidney donors were included. Expression of cathepsins B, D, K, L, S, cystatin C, collagen I and III, their degraded products C-Telopeptide of type 1 and 3 collagen (CTX-I, CTX-III), cellular markers for leukocytes (CD45), T cells (CD3), macrophage scavenger receptor-1 (MSR-1), synthetic, and contractile smooth muscle cells (SMCs) (smoothelin: SMTH, collagen I and III, myosin heavy chain: MHC, embryonic smooth muscle myosin heavy chain: SMemb) were determined at messenger RNA (mRNA) level, using SYBRGreen-based quantitative PCR and at protein level using enzyme-linked immunosorbent assay (ELISA). Expression of cathepsins B, D, L, and S at mRNA level was significantly elevated in AAA compared to control aorta (1.7-fold, p = 0.025; 2.5-fold, p = 0.002; 2.6-fold, p = 0.034; and 7.0-fold, p = 0.003). Expression of cathepsin S correlated significantly with leukocytes and macrophages (ρ = 0.398, p = 0.033 and ρ = 0.422, p = 0.020), synthetic SMCs were significantly associated with cathepsins B, D, and L (ρ = 0.522, p = 0.003; ρ = 0.431, p = 0.015 and ρ = 0.467, p = 0.008). At protein level, cathepsins B and S were elevated in AAA compared to controls (5.4-fold, p = 0.001 and 7.3-fold, p < 0.001). Significant correlations were observed between collagen I, its degraded product, and cathepsin S (r = -0.350, p = 0.034 and r = +0.504, p < 0.001). Expression of cathepsin B was associated with SMCs, expression of cathepsin S with inflammatory cells. Particularly cathepsin S was associated with the degradation product of collagen I and thus might be involved in the progression of AAA. Furthermore, cathepsin S correlated with inflammatory cells.

Effect of zinc on the collagen degradation in acid-etched dentin

Background/purposeMatrix metalloproteinases (MMPs) play a crucial role in the pathogenesis of dental caries, collapse of adhesive interface, and chemical erosion of teeth. The objective of this study was to investigate the inhibitory effect of zinc on collagen degradation. Materials and methodsHuman dentin was ground and demineralized by citric acid (pH 2.0). The demineralized ground dentin was incubated in six different media: artificial saliva (AS); 5 mg/ml doxycycline in AS; 3.33, 6.82, 13.63, and 27.26 mg/ml of zinc chloride (Zn) in AS. Each group was divided into two subgroups, and active MMP-2 was incorporated into one subgroup. Specimens were incubated for 24 h, 1 week, and 2 weeks. Collagen degradation product was assessed using ELISA. The results were analyzed using repeated measured ANOVA and Duncan's post hoc analysis (α = 0.05). ResultsThe amount of collagen degradation was the lowest in Doxy group. Zn groups showed a significant inhibitory effect in collagen degradation for all concentrations (P < 0.05). In subgroups without exogenous MMP-2, zinc-mediated inhibition increased in a concentration-dependent manner with increasing zinc concentration. The amount of collagen degradation product slightly increased with increased incubation time from 24 h to 2 weeks. However, in subgroups with exogenous MMP, the inhibitory effect of zinc on collagen degradation did not depend on zinc concentration. ConclusionAll Zn groups for the four concentrations tested exhibited statistically significant inhibitory effect on collagen degradation.

Novel mechanisms of Collagenase Santyl Ointment (CSO) in wound macrophage polarization and resolution of wound inflammation

Collagenases are useful in enzymatic wound debridement. Clostridial collagenase, marketed as Collagenase Santyl Ointment (CSO), is FDA approved for such use. Building on the scientific premise that collagenases as well as collagen degradation products may regulate immune cell function, we sought to investigate the potential role of CSO in wound inflammation. We tested the hypothesis that in addition to enacting debridement, CSO contributes to the resolution of persistent wound inflammation. Wound macrophages were isolated from PVA sponges loaded with CSO or petrolatum and implanted in mice. Significant increase in pro-reparative and decrease in pro-inflammatory polarization was noted in macrophages of acute as well as diabetic wounds. Wound macrophages from CSO-treated group displayed increased production of anti-inflammatory cytokines IL-10 and TGF-β, and decreased levels of pro-inflammatory cytokines TNF-α and IL-1β. The active ingredient of CSO, CS-API, induced the expression of mϕheal /M(IL-4) polarization markers ex vivo. CS-API treatment attenuated transactivation of NF-κB and significantly induced STAT6 phosphorylation. A significant role of a novel PGE2-EP4 pathway in CS-API induced STAT6 activation and the mϕheal /M(IL-4) polarization was identified. Taken together, findings of this work reposition CSO as a potential agent that may be effective in resolving wound inflammation, including diabetic wounds.

Fib3-3 as a Biomarker for Osteoarthritis in a Rat Model with Metabolic Dysregulation.

ObjectiveFibulin-3 is a glycoprotein highly expressed in osteoarthritic cartilage and inhibits angiogenesis and chondrocyte differentiation. Recent studies have indicated that fibulin-3 has potential value as a biomarker in osteoarthritis. The aim of the present study is to examine the role of 3 fibulin-3 peptides (Fib3-1, Fib3-2, and Fib3-3) and a type II collagen degradation product in a rat osteoarthritis model with systemic metabolic alterations combined with local cartilage damage.DesignForty, 12-week-old male, Wistar rats were randomly divided over 2 groups: a standard or a high-fat diet inducing metabolic dysregulation. After 12 weeks, articular cartilage damage was induced on the femoral condyles (groove model), in 1 knee joint in 14 rats of each diet group. At endpoint, blood was collected and serum was isolated. Enzyme-linked immunosorbent assay on all selected fibulin-3 fragments was performed from serum samples in addition to immunohistochemical analysis for Fib3-3.ResultsSerum concentrations of Fib3-3 were increased by 29.9%, when cartilage damage was induced in addition to a high-fat diet. Fib3-3 was also associated with an increased histological total joint degeneration (r = 0.435) and cartilage degeneration (r = 0.435). Immunostainings demonstrated increased Fib3-3 in the superficial cartilage of animals with high-fat diet and/or cartilage damage.ConclusionsIn the rat groove model combined with high-fat diet–induced metabolic dysregulation an increased Fib3-3 concentration was observed systemically, which is associated with local joint degeneration. This suggests that systemic Fib3-3 concentrations can indicate the status of joint degeneration and function as a biomarker in osteoarthritis.

Dust pollution and control with leather waste

Dust is a serious worldwide pollution, notably for nanoscale particles that are both toxic and difficult to control. Dust suppression can be done by adsorption on solid wastes, such as collagen from by-products of leather manufacturing. Here we review dust pollution and dust control, notably using collagen degradation products. Simulation experiments show that dust suppression using modified collagen degradation products reaches 68.2% for particulate matter 2.5 (PM2.5) and 78.7% for particulate matter 10 (PM10). The advantages and disadvantages of the current dust suppressors are discussed.

High Levels of Circulating Type II Collagen Degradation Marker (CTx-II) Are Associated with Specific VDR Polymorphisms in Patients with Adult Vertebral Osteochondrosis.

Both vitamin D and collagen have roles in osteocartilaginous homeostasis. We evaluated the association between the circulating 25-hydroxyvitamin D (25(OH)D), type I and II collagen degradation products (CTx-I, and CTx-II), and four vitamin D receptor gene (VDR) polymorphisms, in Italian males affected by low back pain (LBP) due to herniation/discopathy and/or vertebral osteochondrosis. FokI, BsmI, ApaI, and TaqI VDR-polymorphisms were detected through PCR–restriction fragment length polymorphism (RFLP), and circulating 25(OH)D, CTx-I and CTx-II were measured by immunoassays in 79 patients (of which 26 had osteochondrosis) and 79 age-, sex- and body mass index (BMI)-matched healthy controls. Among all 158 subjects, carriers of FF and Ff genotypes showed lower 25(OH)D than ff, which suggested a higher depletion of vitamin D in F allele carriers. Higher CTx-I concentrations were observed in TT versus Tt among controls, and Tt versus tt among LBP cases, which suggested a higher bone-cartilaginous catabolism in subjects bearing the T allele. Higher CTx-II concentrations were observed in patients with osteochondrosis bearing FF, bb, TT, or Aa genotypes in comparison with hernia/discopathy patients and healthy controls. Vertebral osteochondrosis shows peculiar genotypic and biochemical features related to vitamin D and the osteocartilaginous metabolism. Vitamin D has roles in the pathophysiology of osteochondrosis.

Collagen Type I as a Ligand for Receptor-Mediated Signaling

Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B and Lair-1 of the leukocyte receptor complex and mannose family receptor uPARAP/Endo 180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

Urinary collagen degradation products as early markers of progressive renal fibrosis

BackgroundRenal fibrogenesis is associated with increased ECM remodeling and release of collagen fragments in urine in progressive renal disease. We investigated the diagnostic value of urinary collagen degradation products in a proteinuria-driven fibrosis rat model with and without anti-fibrotic S1P-receptor modulator FTY720 treatment.MethodsProteinuria was induced in male Wistar rats by Adriamycin (ADR) injection (n = 16). Healthy rats served as controls (n = 12). Six weeks post-injection, all underwent renal biopsy, and FTY720-treatment started in ADR-rats (n = 8) and controls (n = 6). Others remained untreated. Rats were sacrificed after 12 weeks. Collagen type I (C1M) and III (C3M) degradation fragments were measured in blood and urine using ELISA. Kidneys were stained for various inflammatory and fibrotic markers.ResultsSix weeks post-injection proteinuria increased (versus controls, P < 0.001) and although no accumulation of interstitial renal collagen type III (iColl3) was observed at this time, urinary C3M (uC3M) and C1M (uC1M) were significantly increased (both P < 0.001). At 12 weeks, uC3M (P < 0.001) and uC1M (P < 0.01) further increased in ADR-rats versus controls, just as fibronectin, PDGF-β receptor, hyaluronan (all P < 0.01), iColl3, PAS, myofibroblasts, macrophages and T-cells (all P < 0.05). FTY720-treatment reduced accumulation of immune cells, α-SMA+ myofibroblasts and PAS-score, but not iColl3 and uC3M. Correlation analyses indicated that uC3M and uC1M reflected and predicted tubulointerstitial fibrogenesis.ConclusionsThese data displayed urinary collagen breakdown products as sensitive early markers of interstitial fibrosis, preceding histological fibrotic changes, which might replace the invasive renal biopsy procedure to assess fibrosis. Anti-fibrotic FTY720 intervention reduced some fibrotic markers without affecting collagen type III metabolism.

N-Acetylated Proline-Glycine-Proline Accelerates Cutaneous Wound Healing and Neovascularization by Human Endothelial Progenitor Cells

Human endothelial progenitor cells (hEPCs) are promising therapeutic resources for wound repair through stimulating neovascularization. However, the hEPCs-based cell therapy has been hampered by poor engraftment of transplanted cells. In this study, we explored the effects of N-acetylated Proline-Glycine-Proline (Ac-PGP), a degradation product of collagen, on hEPC-mediated cutaneous wound healing and neovascularization. Treatment of hEPCs with Ac-PGP increased migration, proliferation, and tube-forming activity of hEPCs in vitro. Knockdown of CXCR2 expression in hEPCs abrogated the stimulatory effects of Ac-PGP on migration and tube formation. In a cutaneous wound healing model of rats and mice, topical application of Ac-PGP accelerated cutaneous wound healing with promotion of neovascularization. The positive effects of Ac-PGP on wound healing and neovascularization were blocked in CXCR2 knockout mice. In nude mice, the individual application of Ac-PGP treatment or hEPC injection accelerated wound healing by increasing neovascularization. Moreover, the combination of Ac-PGP treatment and hEPC injection further stimulated wound healing and neovascularization. Topical administration of Ac-PGP onto wound bed stimulated migration and engraftment of transplanted hEPCs into cutaneous dermal wounds. Therefore, these results suggest novel applications of Ac-PGP in promoting wound healing and augmenting the therapeutic efficacy of hEPCs.

Phagocytosis of Collagen Fibrils by Fibroblasts In Vivo Is Independent of the uPARAP/Endo180 Receptor.

As a crucial step in ECM remodeling, collagen degradation occurs through different processes, including both extracellular and intracellular degradation. The extracellular pathways of collagen degradation require secretion of collagenolytic proteases, whereas intracellular collagen degradation occurs in the lysosomal compartment after uptake, involving either pre-cleaved or intact fibrillar collagen. The endocytic collagen receptor uPARAP/Endo180 plays an important role in internalization of large collagen degradation products, whereas its role in the phagocytosis of fibrillar collagen has been debated. In fact, the role of this receptor in regular collagen phagocytosis in vivo has not been established. In this study, we have studied the role of uPARAP in the phagocytosis of collagen fibrils in vivo by analyzing different connective tissues of mice lacking uPARAP. Using transmission electron microscopy (TEM), we found that fibroblasts in the periosteum of tibia and calvaria, as well as in the periodontal ligament of molar and incisor, phagocytosed collagen fibrils independently of uPARAP. Quantification of phagocytosed collagen in the periodontal ligament of uPARAP-deficient mice and controls revealed no difference in the amount of fibrillar collagen taken up by uPARAP-deficient mice. The findings show that under in vivo conditions uPARAP does not play a role in the phagocytic uptake of collagen fibrils by fibroblasts. Consequently, the cellular uptake of collagen fibrils and collagen cleavage products probably occurs through fundamentally different pathways. J. Cell. Biochem. 118: 1590-1595, 2017. © 2016 Wiley Periodicals, Inc.

Anti-MMP-9 Antibody: A Promising Therapeutic Strategy for Treatment of Inflammatory Bowel Disease Complications with Fibrosis.

Despite medical treatments or surgical options, more than one-third of patients with Crohn's disease suffer from recurring fistulae. Matrix metalloprotease 9 (MMP-9), a type IV collagenase that cleaves components of the extracellular matrix leading to tissue remodeling, is upregulated in crypt abscesses and around fistulae suggesting an important role for this enzyme in fistula formation. Our aims were (1) to correlate serum levels of MMP-9 degradation products in patients with CD with the presence of fistulae and (2) to investigate the impact of selective MMP-9 inhibition in a mouse model of intestinal fibrosis. Serum MMP-9 degradation products were quantified in subjects affected with nonstricturing and nonpenetrating CD (n = 50), stricturing CD (n = 41), penetrating CD (n = 22), CD with perianal fistula (n = 29), and healthy controls (n = 10). Therapeutic efficacy of anti-MMP-9 monoclonal antibodies was assessed in a heterotopic xenograft model of intestinal fibrosis. C3M, an MMP-9 degradation product of collagen III, demonstrated the highest serum levels in patients with penetrating CD and differentiated penetrating CD from other CD subgroups and healthy controls, P = 0.0005. Anti-MMP-9 treatments reduced collagen deposition and hydroxyproline content in day-14 intestinal grafts indicating reduced fibrosis. The serologic biomarker C3M can discriminate penetrating CD from other CD subgroups and could serve as marker for the development of penetrating CD. Anti-MMP-9 antibody has therapeutic potential to prevent intestinal fibrosis in CD complications.

Assessment of bone mineral density and bone metabolism in young male adults recently diagnosed with systemic lupus erythematosus in China.

Objective Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease. However, the exact mechanism underlying SLE-related osteopenia and osteoporosis in patients newly diagnosed with SLE remains unknown. Methods 60 male subjects with SLE aged 20-30 years were enrolled. Serum osteocalcin was examined as a marker of bone formation and type I collagen degradation products (β-crosslaps) as markers of bone resorption. Lumbar spine (L1-L4) and total hip bone mineral density (BMD) were determined by dual energy X-ray absorption (DXA). Results Among the 60 subjects with SLE at the time of diagnosis, the cohort showed a significant reduction of osteocalcin (12.62 ± 2.16 ng/mL), and serum β-crosslaps level (992.6 ± 162.6 pg/mL) was markedly elevated. Univariate correlation analyses revealed negative correlations between osteocalcin and SLEDAI, dsDNA antibody and β-crosslaps. A positive correlation was also observed between osteocalcin and C3, C4, 25-OH vitamin D, BMD L1-L4 and BMD total hip (see Table 3). Osteocalcin and β-crosslaps were strongly associated with SLE disease activity by multiple stepwise logistic regression analysis. Conclusion Osteocalcin was negatively associated with SLE disease activity, and β-crosslaps was positively associated with SLE disease activity, suggesting SLE disease activity itself directly contributed to the development of SLE-associated osteopenia and osteoporosis.

The Effect of Long-Term Exercise on the Production of Osteoclastogenic and Antiosteoclastogenic Cytokines by Peripheral Blood Mononuclear Cells and on Serum Markers of Bone Metabolism.

Although it is recognized that the mechanical stresses associated with physical activity augment bone mineral density and improve bone quality, our understanding of how exercise modulates bone homeostasis at the molecular level is lacking. In a before and after trial involving 43 healthy adults, we measured the effect of six months of supervised exercise training on the spontaneous and phytohemagglutinin-induced production of osteoclastogenic cytokines (interleukin-1α, tumor necrosis factor-α), antiosteoclastogenic cytokines (transforming growth factor-β1 and interleukins 4 and 10), pleiotropic cytokines with variable effects on osteoclastogenesis (interferon-γ, interleukin-6), and T cell growth and differentiation factors (interleukins 2 and 12) by peripheral blood mononuclear cells. We also measured lymphocyte phenotypes and serum markers of bone formation (osteocalcin), bone resorption (C-terminal telopeptides of Type I collagen), and bone homeostasis (25 (OH) vitamin D, estradiol, testosterone, parathyroid hormone, and insulin-like growth factor 1). A combination of aerobic, resistance, and flexibility exercises done on average of 2.5 hours a week attenuated the production of osteoclastogenic cytokines and enhanced the production of antiosteoclastogenic cytokines. These changes were accompanied by a 16% reduction in collagen degradation products and a 9.8% increase in osteocalcin levels. We conclude that long-term moderate intensity exercise exerts a favorable effect on bone resorption by changing the balance between blood mononuclear cells producing osteoclastogenic cytokines and those producing antiosteoclastogenic cytokines. This trial is registered with Clinical Trials.gov Identifier: NCT02765945.

220 Specific protein degradation products of collagen and vimentin as well as fibronectin are released from the tumor core and adjacent tissue in a human colorectal tumor ex vivo model - biomarker potential

220 Specific protein degradation products of collagen and vimentin as well as fibronectin are released from the tumor core and adjacent tissue in a human colorectal tumor ex vivo model - biomarker potential

Matrix Metalloproteinase Mediated Type I Collagen Degradation — An Independent Risk Factor for Mortality in Women

Chronic fibro-proliferative diseases are associated with nearly 45% of all deaths in the developed world. Matrix metalloproteinase (MMP) mediated remodeling of the extracellular matrix (ECM) plays an important role in disease development. Degradation of type I collagen is considered having a major role in this matter. C1M is a biomarker measuring type I collagen degradation fragments in blood. The aim of the current study was to investigate whether MMP mediated type I collagen degradation (C1M) was predictive of mortality in a large prospective cohort of Danish women aged 48–89 (n=5855).Subjects with high serum C1M showed significant increased mortality. The adjusted three year HR was 2.02 [95% CI: 1.48–2.76] for all-cause mortality, 2.32 [95% CI: 1.51–3.56] for cancer and 1.77 [95% CI: 0.98–3.17] for cardiovascular diseases. The adjusted nine year HR was 1.50 [95% CI: 1.28–1.75] for all-cause mortality, 1.49 [95% CI: 1.16–1.90] for cancer and 1.69 [95% CI: 1.27–2.24] for cardiovascular diseases.High MMP-mediated type I collagen degradation was associated with increased mortality. Subjects with high C1M had a 2-fold increase in mortality compared to subjects with low levels of this collagen degradation product.

Development of a Serum‐free, Three Dimensional Cell Culture Model for Primary Human Articular Chondrocytes to Test Potential Therapeutics

The objective of this study was to establish a culture system of primary human articular chondrocytes with which to test novel drugs for the treatment of osteoarthritis (OA). Articular cartilage was obtained from OA subjects undergoing total knee arthroplasty (TKA) or above knee amputation (AKA) for pathology unrelated to OA. Chondrocytes were isolated by protease digestion and cultured in alginate at 2, 5, 10 x 106 cells in serum‐free medium. Production of collagen degradation products was measured in days 2 and 5 culture medium and day 5 cell‐associated matrix. The optimal plating density was 5x106 cells/culture, as determined by Lowry protein analysis. Type I collagen telopeptide breakdown products were higher in cultures established from the femoral condyles than from the patella, and in OA cultures compared to AKA controls. Type II collagen telopeptides were also detected in both OA and TKA cultures; however, the amounts of these telopeptides were not significantly different in the two types of cultures. These results demonstrate that human articular chondrocytes survive and are metabolically active when cultured in this serum‐free, 3D culture system. The femoral condyles and tibial plateau were the optimal sites for tissue harvesting. With this model system we can effectively measure clinical endpoints for testing new drugs that may reduce tissue destruction in OA.&#x0D;This work was funded by PCOM, CMSRU, Cooper Foundation and Cooper Bone and Joint Institute.

Use of time-resolved fluorescence spectroscopy to evaluate diagnostic value of collagen degradation products.

The concentration of collagen degradation products (CDPs) may reflect the process of left ventricular remodeling (LVR). The aim of this study was to evaluate the potential diagnostic usefulness of time-resolved fluorescence spectroscopy (TRFS) in assessment of CDPs. The preliminary experiment was designed to establish if CDPs’ characteristics might be visible by mean fluorescence lifetime (FLT) in determined conditions. The in vitro model of CDPs was prepared by conducting the hydrolysis of type III collagen. The FLT of samples was measured by the time-resolved spectrometer Life Spec II with the subnanosecond pulsed 360-nm EPLED diode. The FLTs were obtained by deconvolution analysis of the data using a multiexponential model of fluorescence decay. In order to determine the limit of traceability of CDPs, a comparison of different collagen/plasma ratio in samples was performed. The results of our study showed that the increase of added plasma to hydrolyzed collagen extended the mean FLT. Thus, the diagnosis of LVR based on measurements using TRFS is possible. However, it is important to point out the experiment was preliminary and further investigation in this field of research is crucial.

Abstract 4852: Collagen degradation products measured in serum can separate ovarian and breast cancer patients from healthy controls

Abstract Background: The hallmarks of breast and ovarian cancer include increased matrix metalloproteinase (MMP) activity, increased collagen deposition (desmoplasia) and breakdown, as well as invasion of the basement membrane and extracellular matrix. The combination of these processes result in the release of specific protein (e.g. collagens) fragments to the circulation, which when measured may reflect disease pathogenesis. Aim: To investigate if biomarkers measuring MMP-degraded collagen could differentiate ovarian and breast cancer patients from healthy controls when measured in serum. Methods: The levels of markers reflecting MMP-mediated degradation of type I (C1M), type III (C3M) and type IV (C4M, C4M12) collagen were assessed in serum from female ovarian cancer patients (n=10, stage I-III, age 37-74), breast cancer patients (n=14, stage II-III, age 41-79) and healthy controls (n=49, age 31-83) using characterized and validated competitive ELISAs. The levels of the individual markers were compared using one way ANOVA on log transformed data and AUROC was calculated. Results: The levels of the markers (table) were significantly elevated in serum from ovarian cancer patients (p&amp;lt;0.0001) and breast cancer patients (p&amp;lt;0.04-0.0001) compared to healthy controls. All the markers were able to differentiate ovarian (AUROC 88%-95%) and breast cancer patients (AUROC 70%-88%) from healthy controls, with C1M being the strongest differentiator of disease vs. controls. Conclusions: Four serum biomarkers measuring altered MMP-mediated collagen turnover were able to differentiate ovarian and breast cancer patients from healthy controls in this small cancer cohort study. As these markers reflect altered disease pathogenesis they may increase the understanding of disease mode of action and, if validated in larger clinical studies, provide an improved and additional clinical tool for stratifying and monitoring patients according to subtype/ disease severity. Serum levels of C1M, C3M, C4M and C4M12 in healthy control, ovarian and breast cancer patients.Controls (n=49)Ovarian (n=10)Breast (n=14)Median, ng/ml (range)Median, ng/ml (range)ANOVA (P-value)AUROCMedian, ng/ml (range)ANOVA (P-value)AUROCC1M56.0 (3.0-194.4)372.6 (51.9-451.4)p&amp;lt;0.000195252.2 (20.5-345.5)p&amp;lt;0.000188C3M11.7 (6.0-28.9)29.1 (11.3-40.3)p&amp;lt;0.00018922.0 (10.6-37.2)p&amp;lt;0.000181C4M51.7 (20.5-180.4)108.1 (43.4-223.2)p&amp;lt;0.00018975.8 (40.4-142.5)P=0.000483C4M1238.8 (18.3-95.2)114.6 (31.0-282.6)p&amp;lt;0.00018860.6 (20.2-154.7)P=0.0470 Citation Format: Cecilie L. Bager, Nicholas Willumsen, Diana J. Leeming, Victoria Smith, Morten Karsdal, Anne-Christine B. Jensen. Collagen degradation products measured in serum can separate ovarian and breast cancer patients from healthy controls. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4852. doi:10.1158/1538-7445.AM2014-4852