Detection of MMP-13 Activity on Intentionally Strain-Released Type-II Collagen Network in Bovine Articular Cartilage

2011 ◽  
Author(s):  
Nazli Caner ◽  
Jeffrey W. Ruberti
Keyword(s):  
Articular Cartilage ◽  
Degradation Kinetics ◽  
Type Ii Collagen ◽  
Healthy Tissue ◽  
Cartilage Explants ◽  
Cartilage Tissue ◽  
Mechanical Stimuli ◽  
Type Ii ◽  
Collagen Network ◽  
Mechanical Tension

Articular cartilage is a specialized avascular connective tissue found at the contact regions of diarthrodial joints. Cartilage has few cells (< 5% of the volume), though these cells can maintain the balance of turnover in healthy tissue, when the tissue is damaged, they are not able to repair the defects [1–3]. Extra cellular matrix (ECM) in cartilage comprises water, collagen (principally type II), proteoglycans and noncollagenous proteins. The type II collagen network, which is the dominant structural protein in cartilage ECM, constrains the expansion of the resident PGs and is generally held in mechanical tension. In osteoarthritis (OA), the balance of cartilage tissue production/degradation is thought to be affected by abnormal mechanical stimuli leading to net matrix resorption through production of excess degradative enzymes (e.g. matrix metalloproteinases (MMP) and aggrecanases) [4–8]. In OA tissue the amount of MMP-13 is thought to be increased relative to healthy tissue. OA typically occurs in older adults where, as cartilage ages, there is a marked decrease in the fixed charge density (FCD), the hydration and, consequently, mechanical tension on the collagen type II network [9–11]. We have hypothesized that loss of tension on the collagen network accelerates degradation by MMP. Detection of the effect of MMP on loaded, native cartilage could lead to insight about cartilage degradation kinetics in OA. However, it is quite difficult to controllably deliver MMP to cartilage, to activate the MMP during detensioning of the collagen network and to detect the effect on the cartilage mechanics (because cost limits the amount of MMP used). We have developed a transpirational enzyme loading method which is capable of precisely dosing bovine cartilage explants with a small, known quantity of MMP-13. Following enzyme insertion, we are able to detect the activity of the MMP on osmotically compressed cartilage (i.e. cartilage with a detensioned collagen network) via a simple hydration measurement.

scholarly journals Osthole Inhibits Proliferation and Induces Catabolism in Rat Chondrocytes and Cartilage Tissue

2015 ◽  
Vol 36 (6) ◽  
pp. 2480-2493 ◽  
Author(s):  
Guoqing Du ◽  
Yi Song ◽  
Lei Wei ◽  
Linghui Li ◽  
Xuezong Wang ◽  
...  
Keyword(s):  
Type Ii Collagen ◽  
Cartilage Explants ◽  
Cartilage Tissue ◽  
Blue Staining ◽  
Marker Genes ◽  
Dependent Manner ◽  
Type Ii ◽  
Expression Levels ◽  
Safranine O ◽  
And Cartilage

Background/Aims: Cartilage destruction is thought to be the major mediator of osteoarthritis. Recent studies suggest that inhibition of subchrondral bone loss by anti-osteoporosis (OP) drug can protect cartilige erosion. Osthole, as a promising agent for treating osteoporosis, may show potential in treating osteoarthritis. The purpose of this study was to investigate whether Osthole affects the proliferation and catabolism of rat chondrocytes, and the degeneration of cartilage explants. Methods: Rat chondrocytes were treated with Osthole (0 μM, 6.25 μM, 12.5 μM, and 25 μM) with or without IL1-β (10ng/ml) for 24 hours. The expression levels of type II collagen and MMP13 were detected by western Blot. Marker genes for chondrocytes (A-can and Sox9), matrix metalloproteinases (MMPs), aggrecanases (ADAMTS5) and genes implicated in extracellular matrix catabolism were evaluated by qPCR. Cell proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and fluorescence activated cell sorter. Wnt7b/β-catenin signaling was also investigated. Cartilage explants from two-week old SD rats were cultured with IL-1β, Osthole and Osthole plus IL-1β for four days and glycosaminoglycan (GAG) synthesis was assessed with toluidine blue staining and Safranine O/Fast Green FCF staining, collagen type II expression was detected by immunofuorescence. Results: Osthole reduced expression of chondrocyte markers and increased expression of MMP13, ADAMTS5 and MMP9 in a dose-dependent manner. Catabolic gene expression levels were further improved by Osthole plus IL-1β. Osthole inhibited chondrocyte proliferation. GAG synthesis and type II collagen were decreased in both the IL-1β groups and the Osthole groups, and significantly reduced by Osthole plus IL-1β. Conclusions: Our data suggested that Osthole increases the catabolism of rat chondrocytes and cartilage explants, this effect might be mediated through inhibiting Wnt7b/β-catenin pathway.

scholarly journals The Effect of Varying Concentrations and Application Periods of Chondroitinase ABC on Tissue-Engineered Cartilage

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Eric Tong ◽  
Grace D. O'Connell ◽  
Terri-Ann N. Kelly ◽  
Clark T. Hung
Keyword(s):  
Mechanical Properties ◽  
Articular Cartilage ◽  
Treatment Option ◽  
Type Ii Collagen ◽  
Treated Group ◽  
Cartilage Tissue ◽  
Type Ii ◽  
Chondroitinase Abc ◽  
Engineered Cartilage

Osteoarthritis, a chronic malady characterized by joint pain and swelling, is caused by damage to articular cartilage and is perpetuated by low-grade inflammation.  Treatments for osteoarthritis do exist, but many treatments focus on coping with the disease rather than curing it.  Surgical options that replace damaged cartilage tissue with that of donor cartilage tissue or cartilage tissue from other parts of articular joints face complications especially when the tissue is not of the correct size or does not have native-like properties. A more suitable treatment option for osteoarthritis is to develop an in vitro tissue-engineered cartilage construct that can be grown using the patient’s own cells and to surgically remove the patient’s damaged cartilage and replace it with the tissue-engineered cartilage. A challenge in developing such a treatment option is producing tissue-engineered cartilage with mechanical properties akin to those of native human articular cartilage. This challenge may be overcome by maximizing the production of type II collagen by the chondrocytes in vitro. One way to maximize collagen production is through the application of chondroitinase ABC, an enzyme which temporarily suppresses proteoglycans in the cartilage matrix to create more space for type II collagen to develop. In this study, two two levels of cABC treatment were applied (“high” and “low”) to cartilage tissue constructs. The “low” cABC treated group received daily feeding of 0.075 U/mL from day 14 to 21 followed by a replacement of chondrogenic media without cABC.  The “high” cABC treated group received a single addition of 0.15 U/mL from day 14 to 16 followed by a replacement of chondrogenic media without cABC.  At the end of 42 days, the constructs were subjected to mechanical testing and biochemical analyses. These analyses showed that the high cABC treatment yielded more native-like mechanical properties when compared to the low cABC treatment and the control results.  Biochemical and histological analyses confirmed that the proteoglycan and collagen II content were higher in the low and high cABC treated groups when compared to the control. All analyses show that the most efficient application of chondroitinase ABC is through a two day duration treatment of a higher concentration (0.15 U/mL).

scholarly journals Inhibition of activity of collagen degradation in cartilage of patients with osteoarthrosis byactivation of glycolysis

2011 ◽  
Vol 14 (1) ◽  
pp. 8-12
Author(s):  
E. V. Chetina ◽  
E. V. Chetina
Keyword(s):  
Articular Cartilage ◽  
Dna Synthesis ◽  
Articular Chondrocytes ◽  
Type Ii Collagen ◽  
Cartilage Destruction ◽  
Cartilage Explants ◽  
Type Ii ◽  
Cleavage Activity ◽  
Articular Cartilages ◽  
Elisa Inhibition

Aim. To study the effect of glycolysis activators deferrioxamine (DFO), CoCl2, V(SO4)2 and mimosine on collagen cleavage activity by collagenase in osteoarthritic (OA) articular cartilage explants. Materials and methods. 32 OA articular cartilages obtained after arthroplasty were examined in the study. Cartilages were cultured in the presence of 10-50μM DFO, CoCl2, V(SO4)2 or mimosine. Collagen cleavage activity was measured by ELISA. Inhibition of protein or DNA synthesis in the presence of [3H]-labeled proline or thymidine, respectively, was used for evaluation of examined agent toxicity. Results. Glycolysis activators DFO, CoCl2, V(SO4)2 or mimosine were capable of inhibiting type II collagen cleavage activity in OA articular cartilage explants. The examined agents have shown no toxic effect in the concentrations used. Conclusion. Glycolysis activation in articular chondrocytes may offer a means of inhibiting articular cartilage destruction in OA patients.

scholarly journals Intermittent Dynamic Compression Confers Anabolic Effects in Articular Cartilage

2021 ◽  
Vol 11 (16) ◽  
pp. 7469
Author(s):  
Amalie Engstrøm ◽  
Frederik S. Gillesberg ◽  
Solveig S. Groen ◽  
Peder Frederiksen ◽  
Anne-Christine Bay-Jensen ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Mechanical Loading ◽  
Kinase Inhibitor ◽  
Dynamic Compression ◽  
Compressive Loading ◽  
Type Ii Collagen ◽  
Cartilage Explants ◽  
Type Ii ◽  
Bovine Articular Cartilage ◽  
Collagen Formation

(1) Background: Mechanical loading is an essential part of the function and maintenance of the joint. Despite the importance of intermittent mechanical loading, this factor is rarely considered in preclinical models of cartilage, limiting their translatability. The aim of this study was to investigate the effects of intermittent dynamic compression on the extracellular matrix during long-term culture of bovine cartilage explants. (2) Methods: Bovine articular cartilage explants were cultured for 21 days and subjected to 20 min of 1 Hz cyclic compressive loading five consecutive days each week. Cartilage remodeling was investigated in the presence of IGF-1 or TGF-β1, as well as a TGF-β receptor 1 (ALK5) kinase inhibitor and assessed with biomarkers for type II collagen formation (PRO-C2) and fibronectin degradation (FBN-C). (3) Results: Compression of cartilage explants increased the release of PRO-C2 and FBN-C to the conditioned media and, furthermore, IGF-1 and compression synergistically increased PRO-C2 release. Inhibition of ALK5 blocked PRO-C2 and FBN-C release in dynamically compressed explants. (4) Conclusions: Dynamic compression of cartilage explants increases both type II collagen formation and fibronectin degradation, and IGF-1 interacts synergistically with compression, increasing the overall impact on cartilage formation. These data show that mechanical loading is important to consider in translational cartilage models.

The Effect of Crosslinking Agents on the Properties of Type II Collagen Biomaterials

2021 ◽  
Vol 57 (4) ◽  
pp. 166-180
Author(s):  
Maria-Minodora Marin ◽  
Madalina Georgiana Albu Kaya ◽  
George Mihail Vlasceanu ◽  
Jana Ghitman ◽  
Ionut Cristian Radu ◽  
...  
Keyword(s):  
Research Work ◽  
Cartilage Tissue Engineering ◽  
Chemical Properties ◽  
Cartilage Regeneration ◽  
Type Ii Collagen ◽  
Biomechanical Properties ◽  
Cartilage Tissue ◽  
Cross Linking ◽  
Type Ii ◽  
Crosslinking Agents

Type II collagen has been perceived as the indispensable element and plays a crucial role in cartilage tissue engineering. Thus, materials based on type II collagen have drawn farther attention in both academic and research for developing new systems for the cartilage regeneration. The disadvantage of using type II collagen as a biomaterial for tissue repairing is its reduced biomechanical properties. This can be solved by physical, enzymatic or chemical cross-linking processes, which provide biomaterials with the required mechanical properties for medical applications. To enhance type II collagen properties, crosslinked collagen scaffolds with different cross-linking agents were prepared by freeze-drying technique. The present research work studied the synthesis of type II collagen biomaterials with and without crosslinking agents. Scaffolds morphology was observed by MicroCT, showing in all cases an appropriate microstructure for biological applications, and the mechanical studies were performed using compressive tests. DSC showed an increase in denaturation temperature with an increase in cross-linking agent concentration. FTIR suggested that the secondary structure of collagen is not affected after the cross-linking; supplementary, to confirm the characteristic triple-helix conformation of collagen, the CD investigation was performed. The results showed that the physical-chemical properties of type II collagen were improved by cross-linking treatments.

scholarly journals Boosting the Intra-Articular Efficacy of Low Dose Corticosteroid through a Biopolymeric Matrix: An In Vivo Model of Osteoarthritis

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1571
Author(s):  
Matilde Tschon ◽  
Francesca Salamanna ◽  
Lucia Martini ◽  
Gianluca Giavaresi ◽  
Luca Lorenzini ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Pain Sensitivity ◽  
Type Ii Collagen ◽  
Male Rats ◽  
In Vivo Model ◽  
Type Ii ◽  
Chemical Induction ◽  
Local Pain ◽  
Gait Parameters

The purpose of this study was to verify the efficacy of a single intra-articular (i.a.) injection of a hyaluronic acid-chitlac (HY-CTL) enriched with two low dosages of triamcinolone acetonide (TA, 2.0 mg/mL and 4.5 mg/mL), in comparison with HY-CTL alone, with a clinical control (TA 40 mg/mL) and with saline solution (NaCl) in an in vivo osteoarthritis (OA) model. Seven days after chemical induction of OA, 80 Sprague Dawley male rats were grouped into five arms (n = 16) and received a single i.a. injection of: 40 mg/mL TA, HY-CTL alone, HY-CTL with 2.0 mg/mL TA (RV2), HY-CTL with 4.5 mg/mL TA (RV4.5) and 0.9% NaCl. Pain sensitivity and Catwalk were performed at baseline and at 7, 14 and 21 days after the i.a. treatments. The histopathology of the joint, meniscus and synovial reaction, type II collagen expression and aggrecan expression were assessed 21 days after treatments. RV4.5 improved the local pain sensitivity in comparison with TA and NaCl. RV4.5 and TA exerted similar beneficial effects in all gait parameters. Histopathological analyses, measured by Osteoarthritis Research Society International (OARSI) and Kumar scores and by immunohistochemistry, evidenced that RV4.5 and TA reduced OA features in the same manner and showed a stronger type II collagen and aggrecan expression; both treatments reduced synovitis, as measured by Krenn score and, at the meniscus level, RV4.5 improved degenerative signs as evaluated by Pauli score. TA or RV4.5 treatments limited the local articular cartilage deterioration in knee OA with an improvement of the physical structure of articular cartilage, gait parameters, the sensitivity to local pain and a reduction of the synovial inflammation.

Finite Element Modeling of Osmotic Swelling in Tissue-Engineered Cartilage

2008 ◽  
Author(s):  
Liming Bian ◽  
Terri Ann N. Kelly ◽  
Eric G. Lima ◽  
Gerard A. Ateshian ◽  
Clark T. Hung
Keyword(s):  
Articular Cartilage ◽  
Potential Role ◽  
Swelling Pressure ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Osmotic Swelling ◽  
Fixed Charges ◽  
Central Regions ◽  
Significant Research ◽  
Engineered Cartilage

Proteoglycans and Type II collagen represent the two major biochemical constituents of articular cartilage. Collagen fibrils in cartilage resist the swelling pressure that arises from the fixed charges of the glycosaminoglycans (GAGs), and together they give rise to the tissue’s unique load bearing properties. As articular cartilage exhibits a poor intrinsic healing capacity, there is significant research in the development of cell-based therapies for cartilage repair. In some of our tissue engineering studies, we have observed a phenomenon where chondrocyte-seeded hydrogel constructs display cracking in their central regions after significant GAG content has been elaborated in culture. A theoretical analysis was performed to gain greater insights into the potential role that the spatial distribution of proteoglycan and collagen may play in this observed response.

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