Science topic

Cartilage - Science topic

A non-vascular form of connective tissue composed of CHONDROCYTES embedded in a matrix that includes CHONDROITIN SULFATE and various types of FIBRILLAR COLLAGEN. There are three major types: HYALINE CARTILAGE; FIBROCARTILAGE; and ELASTIC CARTILAGE.
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whole cartilage gag content should be estimated. Suggest me some overview of protocol from whole tissue to gag estimation.
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Hi Jaishree,
There are some kits available from different companies also.
Here I am enclosing a lab protocol, a student was using in our lab, He was able to conduct GAG assays successfully. I myself have not used it personally. I am sure if you can follow this, it should work. See the enclosed attachment,
He did use for rabbit cartilage. That is better, you will use in cartilage also.
Good luck,
Subhash
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What is the physics and search stiffness measurements in industry however, do not forget that you are going use that technique on human tissue.
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Good morning Eylül. Impossible to judge the stiffness of the cartilage in life, it is possible to judge mediocrely with the help of MRI.
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Hello,
I have question regarding the use of Abaqus.
My goal is to make an extensive FEA of the pelvic region to investigate fracture patterns (big plans I know). To do this I would like to model the contact between the pelvis bone (E=17GPa), cartilage (E=10.25MPa) and the femur head (E=20GPa), this is where I struggle loads. In this model the hemipelvis will be fixed at the sacroiliac joint and the pubis. The loads will be applied via the Femur head.
So I decided to take a step back, to investigate the contact properties, therefor I've made a simplified the contact model (see attachment) by making a simplified box (named bot) with half a sphere (R=12), then a layer of cartilage (named kraakbeen) with outer R=12mm and inner R=10mm. and then the femur head is simplified as a sphere with R=10mm. The inner surface of the bone is tied with outer-surface of cartilage. The contact between the cartilage and the femur is supposed to be friction-less.
The FEA is done in 2 steps. The first step (step-contact) is displacement step, where the head of the femur is displaced 0.01mm to ensure contact. (displacement is placed with a BC)
The second step (step-load) is the step in which a load (same direction as the displacement) of 2500N is applied.
Preferably I would like to have the displacement-BC removed in the second step and only have a influence of the load that's applied on the femur. However my model does not solve that way.
If I continue the displacement BC into the second step, my model is able to fully solve, however the results are not correct. Since the stresses do not chance during the second step (in which 2500N) is applied. Meaning the application of the load doesnt seem to have any influence.
Can anyone help me how I correctly apply the load to the model and have proper boundary conditions of the pelvic-cartilage-femur?
Thank you.
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I propose my explanation: when you impose displacements in Step 1, the upper sphere is fully constrained, so the solver is able to converge. When, instead, you remove displacements, the sphere is not constrained anymore – in fact, you have no friction at the contact, so the sphere is not prevented to rotate (this happens also you have a vertical load because the program does not know that the sphere is in equilibrium).
If you apply displacements and, then, also the load, such a load has no effect on the lower region because the applied displacements “dominate” the sphere response; in other words, you impose a displacement that behaves like a constraint that cannot be changed by the further load in Step 2, which perhaps will only modify the stress in the upper portion of the sphere.
A final remark: your geometry can be modelled by a symmetric 3D model, or even by an axisymmetric model with a significant reduction of computational time.
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Currently I use Leica BOND Plus slides to mount 5 micon thick FFPE sections of mouse knee joints. These have been fixed in PFA and decalcified in EDTA by experienced researchers, and have been embedded using the following programme in an automated tissue processor:
70% EtOH – 2h
95% EtOH – 2h
100% EtOH – 2h (x3)
Xylene – 2h (x3)
Histowax – 1h (x3)
I dry the sections overnight at 37-40 C on a drying rack, then bake at 65C for 3h and leave the sections to set at RT.
I dewax using the following protocol:
Xylene - 5 min (x3)
100% EtOH - 5 min (x2)
95% EtOH - 5 min
70% EtOH - 5 min
50% EtOH - 5 min
1x PBS - pause point until steamer reaches 99C
Then I proceed with antigen retrieval at 99C in a steamer:
dH2O - 10 sec dip
Tris-EDTA, pH 9.0 - 10 min
1x PBS - pause point at RT.
After the steamer steps, bone and cartilage seem to have fallen of the slide, folds are introduced, etc.
As for the other tissues, the muscle seems to have some folds, while the marrow is mostly intact and does not detach.
I've tried in parallel a different buffer (Citrate buffer, pH 6.0), an overnight 60C incubation in a water bath, as well as a pressure cooker. In all cases the same effect is observed, including if I just stick my tissues in water at 99C for 10 min. This leads me to believe the bone and cartilage tissue is not sticking properly to the slides.
Do you have any tips/recommendations on what I could be doing wrong?
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Thanks a lot, Subhash C. Juneja !
The antigen retrieval step I've been trying to optimise is part of a larger protool for digital spatial profiling by Nnostring, they have insisted on the Tris-EDTA method, but it might be that for my tissues an enzymatic antigen retrieval is best. The protocol includes a proteinase K step, but at a milder concentration, I will see what I can do to test if this step is sufficient, perhaps trying a few different concentrations.
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Hi,
Is there any database to identifies which protein isoform is expressed in a particular tissue?
I want to design primers for real-time PCR on cartilage samples. I need to be sure which mRNA variants are specific for cartilage.
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Hmm, hard to say...
It seems that isoform uc002bna.2 (http://genome.ucsc.edu/cgi-bin/hgGene?db=hg19&hgg_gene=uc002bna.2&org=human) is expressed in most of the tissue types (see the image in the attachment) ... but there are no data for cartilage tissue type specifically
So you may risk it and proceed further with primer design, or you can try to find expression of your isoform in some cartilage-specific public RNA-seq data (laborious)
Martin
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Hi all,
I would like to visualise the cartilage wear on the surface of glenoid after friction test using a UV light (on macroscopic level). Could you please recommended me a florescent dye that I can use and won't affect the structural propertied of cartilage as I will be using the cartilage for histology after. 
Thanks in advance.
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لأن تمزُّق الغضروف الهلالي يحدث في غضروف لا يظهر على الأشعة السينية. لكن يُمكن للأشعة السينية المساعدة في استبعاد المشاكل الأخرى للركبة، والتي تتسبَّب في أعراض مشابهة.
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I have developed ideas and concepts by which we can change the paradigm of OA to Cartilage attrition arthropathy, to facilitate a change in thinking, so that arthritis in the knee can be regarded as a condition that can and should be halted rather than being an inexorably progressive condition awaiting knee replacement.
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it looks you are a medical doctor and you have made observations in the patients? is this correct
May be you should write your research paper and ask someone to read and get suggestions. Initially send that paper to your local conference for presentation so that your idea gets scruitinized that you are in the right direction and than publish a paper if you get good feedback. Good luck.
I can read the paper if you send it to me. I will not have anything to collaborate. But I can just help you only.
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Chondrocyte isolated from Osteoarthritis patient cartilage.
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To be honest I have not worked on chondrocytes from native cartilage. My work was to differentiate mesenchymal stem cells isolated from human bone marrow into cartilages ex vivo, that worked perfectly fine. No direct experience in chondrocyte culture. My reprints might be useful to you in your work indirectly
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Hi everyone!
I want to use condylar cartilage (from TMJ) to do some experiments.
I read some papers and they only mentioned "harvest cartilage" simply.
I want to ask that is it easy to separate cartilage from subchondral bone?
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@Ka Tou
Hi
If you are planning to release cells from the cartilage you will need to enzymatically digest the mince
You can just shave the cartilage of the bone using a 22/15 /11 scalpel blade
If it's processing of just cartilage it should be pretty easy...as in routine processing
But if your cartilage material contains bone, it will require decalcification
And decalcification can mask the antigen binding sites and based on your antibody of interest you will be required to perform different antigen retrieval methods
Hope this helps
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please advice... as I would be performing histological staining using alcian blue Haematoxylin staining on zebrafish cryosections to look at cartilage element or strs.
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No, they are not identical from the formulation. But I think you should choose a red nuclear staining as counterstain to the blue of alcian blue.
Nuclear Fast Red. This is the traditional combination and you will find a protocol in the web.
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I want to do cartilage staining on Xenopus embryos (st 45). Following the protocol, I tried to dissolve 4 mg alcian blue in 7 ml ethanol plus 3 ml acetic acid. I found alcian blue powder is very hard to completely dissolved in this solution. Is there any suggestions?
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Chandan Kumar Panda Thanks for your reply.
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respectfully , i am ensiyeh kordparijaee , a student in medical biotechnology at babol university of medical sciences- from iran. i need the article by topic The role of stromal cell-derived factor 1 on cartilage development and disease , published in MAR 2021 at science direct written by Li,j ; chen,H.;Zhang,D. et al. for my research in the treatment of arthritis , please send it to me , if possible.the picture of its abstract has been attached
best regards
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Following
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The dynamic mechanical analysis (DMA) test with frequency sweep is well known technique to characterize the viscoelastic properties.
I had gone through the different studies which had used DMA test to investigate the variation of storage and loss modulus with frequency for bone (compression mode) and soft tissue such as skin or cartilage (In tensile mode).
In literature it is reported that loss modulus decrease with frequency in bone but it increase for skin and cartilage.
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My clínical expertice it’s my opinion. However the diferents experiencies are very important. Thanks
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I am currently modelling a load being applied in the joint zone of a hemipelvis, which correspond to a person while one leg standing.
Previously, I validated my model with an already existent one from the literature by focusing on the contact area and contact pressure parameters. Nevertheless, when it comes to comparing Von Mises stress between both models, the result variance has increased.
I have asked myself this variance question without yet confirming which is the cause.
Both models have the same boundary conditions and the same magnitude of load. Additionally, they also use the same elements and order of elements(C3D4).
After comparing displacement between them, it seems to be a slight difference (higher in the literature validated model) between them. I attribute this difference to the fact that the already validated model has a smaller thickness than mine in the zone of interest where the load is being transmitted..
Since von Mises stress is calculated from displacement in nodes, it seems plausible to me that the differences in stress between my constructed and the already validated model is due to thickness difference ergo displacement and stress.
Can somebody aid me on this matter?
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Also be aware that contour plots always need inter- and extrapolation to the nodes, so what you see in the plot may not be what happens at the integrations points. Try using the quilt option and/or use probe values to see what actually happens at IPs
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I've been trying to isolate total RNA from guinea pig's knee cartilage. I've tried Qiagen RNeasy kit, miRNeasy kit, trizol method with lots of modifications and have also used the RNaqueous total RNA isolation kit from Thermo Scientific but I've not been able to achieve RNA ratio of more than 1.4 for cartilage and 1.6 for synovium. In addition to the integrity the RNA conc. is also very low because the amount of cartilage available from the knee of guinea pig is very less (<15mg).
I homogenise the samples using Bead beater (bead mill).
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Hi Dikshit..because your starting material is less so, in Trizol method u can do overnight precipitation of RNA in isopropanol at -80°C as it will improve quantity as well as quality.
Hope it will work.
Thank u.
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I need to quantify the glycosaminoglycans from articular cartilage.
I saw some protocols using the DMMB assay. Is there a similar protocol of quantification, but using alcian blue instead of DMMB?
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Hi,
You can find a simple protocol in the following paper:
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I am working with cartilages that are in contact on the hip. In order to obtain a contour plot of the contact pressure I performed a mesh sensitivity test by selecting linear tetrahedral elements (C3D4). Nevertheless, I was advised to use a finer mesh for visualizing von Mises stress.
After doing some reading, I came across the term "reduction integration scheme" which states that strain and stress in FE uses an integration point in the center of the element. Thus, since Contact Pressure is measured in nodes of elements, it makes sense to me to increase the density of the mesh to obtain better results.
Am I correct? or Is there another explanation for this?
Thanks!
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This document may help you to solve the problem:
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I’m implementing a relatively highly cited academic research paper: “Interobserver
Reproducibility of Quantitative Cartilage Measurements: Comparison of B-Spline
Snakes and Manual Segmentation”. In short, the paper outlines how you can get a
B-spline to outline knee cartilage in a 2D slice of an MRI. I am stuck at minimizing the
cost function using gradient descent. I do not know how to gracefully apply a partial derivative to a line integral where the line is a B-spline contour in a 2D image. A full explanation of the problem and my current solution is attached, however I make a poor assumption to get to that solution. If anyone could double check my math or has any ideas I would greatly appreciate it.
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Hi all, I'm searching for volumes of synovial fluid in sheep osteoarthritis (or cartilage defect) model? Any estimation through experience, publications would help. If you have references to other large animal model or human patients, that would also be helpful.
Many thanks in advanced!
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We work with synovial fluid from clinical horses with OA. The volume is very variably. Some horses with OA has minimal or is not possible to take the sample and others has a great volumen (20 ml). The average is 5 ml.
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which 3d bioprinting companies have commercial product in bone and cartilage tissues??
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A lot of companies are trying... but, in-vivo all the results are very poor.
We are working on this, with custom printers and materials....
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I am using this protocol (https://www.scientistsolutions.com/forum/cytology-histology-and-ihc-organelle-and-cytoskeletal-staining/cartilage-staining) to do cartilage staining on organoids that have been fixed in 4% PFA, dehydrated in 10,20,30% sucrose+sodium azide, frozen in tissue freezing medium, sectioned on a cryostat and kept at -30*.
Would I need to take any additional steps before re-hydrating the sections and next staining with the hematoxylin?
Thank you in advance!
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I think you need to fix fresh frozen sections in 10% formalin prior to going for staining procedure (especially H&E). I use to this for amphibian gonadal tissue for better clarity and photography purpose.
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Hello, I'd like to extract soft tissue information from CT scans, in particular a fibro-cartilagineous tissue. Would you be so kind to suggest me a proper threshold range?
Thank you
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Thank you @ jean-philippe Hastir , do you have any reference about that value to justify it?
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Dearest hive mind,
I have been trying to get a staining going for collagen type II in human specimens (de-calcified). Until now, without great results. My initial approach has been with enzymatic antigen retrieval with pronase and hyaluronidase according to Amirtham et al. 2019 (1 mg/mL Pronase for 30 min. and 5 mg/mL Hyaluronidase also for 30 mins). I had a suspicion that the enzymatic treatment would be quite rough on my sections (formalin fixed, paraffin embedded), so I did not do 37 degree during incubation with the enzyme. Of course the sections basically fell of (I guess the enzymatic treatment was too harsh). But, the most annoying thing was, that the tissue that was still on the slide, did not stain nicely (bone with clear areas of cartilage (Specimens were demineralized in 10% EDTA ). I used Dako Envision and AEC as chromogen (In my old lab we used DAB so this is a first time for me). The antibody is a polyclonal rabbit anti human Collagen II from Abcam. The incubation time at room temp with the primary antibody was 2 hours. Do any of you genious-researchers have any good ideas about what I am doing wrong. I'm used to looking at histochemistry, and boy, with a bit of Picrosirus Red you are not in doubt about what is collagen and what is not - but maybe I should not expect to see an intense staining. What I am ultimately trying to do, is to show where there is Collagen I and where there is collagen II as a way of saying "this is cartilage as opposed bone" - am I approaching this from a wrong angle?
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Dear Sune,
decalcification should not disturb COLII-IHC as far as our experience goes.
I also work with human cartilage specimen and know what it fells like when the sections just float off the slides during IHC.
The most important thing is that you use coated slides! Either silanized slides (it is cheap, but more work for you) or you use slides from commercial sources (i.e. "super frost" slides; these are more expensive). For antigen retrieval, we perform a digest with pepsin (1 mg/mL in 0.5 M acetic acid) for 30 min at 37°C and a primary antibody from Acris (AF5710; incubation over night at 6 °C). Afterwards we stain with the Dako LSAB2 System-HRP kit (advantage: biotin & avidin-based -> signal amplification).
If you just want to differentiate between cartilage and bone, you might also perform a Safranin-O/ fast green staining: this stains the proteoglycans of the cartilage nicely red, while the bony parts appear in green/blue. It is much easier!
Attached you will find a publication in wich we used both staining methods in an in vivo cartilage-trauma model after decalcification (rabbit model; but we used the same COLII-antibody as for human specimen).
Best wishes
Jana
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I am trying to stain bone tissues from human knees however I am having a hard time with antigen retrieval. I use citrate buffer and a water bath set at 85 ° for 20 minutes and I leave the tissue in the solution until it reaches room temperature. I still have some tissue left but they become so fragile that they come off after a few washes during blocking step.
I do use positive charged slides too.
Can anyone suggest a good method for unmasking antigens when using bone and/or cartilage?
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Try 80 degrees for 20 min using autoclave and let them cool down slowly. It worked for my samples.
Best Regards
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I have followed the advice on this site, Penn State research papers and numerous www.ncbi.nim.nih.gov articles. A man was diagnosed with osteoarthritis of the knee due to a severe motocross accident. I've attached his x-rays from before and after the plates were finally removed from his leg which was one year after his crash. I need advice. Does the second set of x-rays reveal a total healing, or are we just seeing the effects of numerous chondroprotective supplements which were given to him in the hopes of opening the space in his knee? I ask because it seems to soon to experience this level of healing. I've also attached the patient's supplement list with dosages and dates. Please understand that the supplement list was created for my benefit and not necessarily to be read by others. By this, I mean I was only attempting to keep my research straight.
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On A/P radiograph measurements between the medial femoral condyle and the midpoint from medial tibial spine to medial border of the medial tibial condyle can provide Joint space measurements. Using the Baumgaertner score can help to determine clinical results with radiographs. (Bae et al J Korean Orthop Assoc 2008). However, if one is looking to determine cartilage healing, MRI, biopsy (histological assessment), arthroscopy, immunohistochemical staing and Western blotting test for type II collagen after microfracture can be used.
(Bae et al, J Arthroscop & Rel Surg 2006; Systematic Analysis microfracture technique, Mithoefer et al Amer J Sport Med, 2009). I hope this helps.
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Dear All,
I have to paraffin embed shavings of cartilage ...super small less than 1X1 mm in size for H&E staining. I will have a scoop of these shavings that will be glued together during culture using Evicel. After 4 weeks in culture I'll have to pick up the blob of glue with the cartilage and proceed for histology. Any pointers as to: duration of fixing in 4%PF - dehydration - embedding and sectioning? I will have two groups with and without glue. The scoop of cartilage without glue will be a challenge as the tiny pieces will be all over -floating around in media.Thanks-Anu
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Thank you. Any suggestions as far as the orientation of the tissue is concerned and the sectioning?
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We are making a 3D mechanical model of the thorax and we need the properties of the materials therein. The density of bone is about twice that of water, and I found unreliable affirmations that cartilage is less dense than bone. Does anyone have more reliable information? We are interested in costal cartilage, but would be satisfied with any old type of cartilage for initial, order-of-magnitude simulations.
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Thank you. I found a reference for it!!!
Emely Bortel,Max Langer,Alexander Rack,Jean Baptiste Forien,Georg Duda, etal.
.Combining Coherent Hard X RayTomographies with Phase Retrieval to GenerateThree-Dimensional Models of Forming Bone. Frontiers in Materials, 2017,4(1),pp.36-47.10.3389/fmats.2017. 00039hal-01886993
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Tendons, ligaments, cartilage are composed of collagen. There are articles saying that collagen supplementation can help with tendon injuries. Where is the scientific evidence to support these claims? How is ingestion of collagen linked to an increase in collagen synthesis in the body?
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I thought I would describe in a bit more detail the nature of my tendon injury that I referred to early on in the discussion so as to give a bit more context and hopefully provide information that may be useful to others experiencing similar injuries.
How did my injury occur? I am not entirely sure. I keep racking my brain to remember where I was and what I was doing and the only thing that comes to mind is that I was walking home one morning after having gone to get a coffee and I thought I will sprint home.
I started to run and then something didn’t feel right, I’m not really sure what or how to describe it but I felt like I couldn’t run to the end of the street and I had to stop and walk home. I didn’t think anything of it, I just thought I am not fit, I am not warmed up, I probably have tight muscles.
In the subsequent weeks I had pain in the left thigh that felt like muscular pain, similar to when you exercise and get sore muscles, only that weeks passed and the pain didn’t go away and so I became suspicious that perhaps there was something more to it and lo and behold I go to see my GP and she orders an ultrasound and the report indicates a partial thickness defect in the deep fibers of the quadriceps tendon measured over 1.5cm in width, approximately 8cm above the insertion of the quadriceps tendon from the patella. Associated hyperemia is seen. A complex suprapatellar effusion is also noted.
This first ultrasound was done on the 11th of April 2019 and was maybe between 4-8 weeks after the injury. I followed my GPs advice and went to see a physiotherapist and this was a good move, the physio is much more knowledgeable in musculoskeletal injuries and adept at communicating and made me feel more at ease and that it was not the end of the world. I am grateful for meeting Chris.
On the road to recovery I began muscle strengthening exercises and approximately 3 months after the first ultrasound, I was curious to know what was going on with my tissue so I went back for a second ultrasound on the First of July 2019, 80 days after the first ultrasound, almost 3 months, to check the progress of my injury to know if it was improving or worsening. Thankfully the report came back saying that there is a partial thickness tear of the quadriceps mechanism at the musculotendinous junction which has decreased in size from the prior examination and that there is no new injury or fluid. In my case ultrasound was useful in showing that there was a reason for the pain I was experiencing.
Musculotendinous junction
The myotendinous junction (MTJ) is a complex specialized region located at the muscle-tendon interface that represents the primary site of force transmission.
Overall, I have normal range of motion, I am not debilitated in any way, I do not have any change in movement, however I am aware of my left quad tendon now and that it feels different and has changed. I would also like to mention that tendons heal slowly from what I have been reading in the literature and that healing doesn’t just stop it keeps on going. The various phases of healing have been documented scientifically and consist of inflammation in the early stages, followed by repair and tissue remodeling and this is another area that I have been reading about and that prompted me to look at tendon dynamics and nutritional intervention. In addition to the basic muscle strengthening exercises and walking that I do I have started using a skipping rope.
As Dr Jill Cook articulates exercise and provoking the tendon with load is the best intervention.
Loads in different parts of the tendon
Energy storage acts like a spring
Compressive load – tendons compressed against the bone
Friction load between tendon and surrounding tissue peritendon
The exercises I have been doing have mainly been strengthening the muscle and not really loading the tendon so this is where consulting with a physio is of benefit because they can help with exercise programs and recovery.
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I'm doing AP IHC of collagen I and II on cartilage frozen sections.
The results look quite different than on paraffin sections. I tried different fixations methods (formalin, EtOH) and the antibodies should work on frozen sections according to the datasheet. It seems as if the collagen that is in the lower part of the cartilage (closer to the bone, higher proteoglycan content) does not stain as it should. Biggest difference between the paraffin and frozen sections is the lack of antigen retrieval in the protocol for frozen sections. Could it be that I still need to do antigen retrieval on the frozen sections?
protocol in short:
frozen sections, non fixated
- fixate in 3.7% formalin for 10 minutes
- wash in PBS-T
- block with 1% BSA/5%HS in PBS-T 30 minutes
- primary AB @ 4 degrees overnight
- wash with PBS-T
- secondary antibody conjugated with biotin 1 hour
- wash with TBS-T
- ABC-AP complex 1 hour
- wash in TBS
- incubate with AP substrate solution 15 minutes
- stop reaction by washing in TBS
Thanks for your help!
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Dear Meike Kleuskens,
You may try the antigen retrieval for short time but you need to have good salinized coated slide to do that. Otherwise, you also can play around with the tween-20..good luck
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Hi,
I'm having trouble with facial cartilage staining in zebrafish (120hpf).
except that I had fixed in PFA overnight at 4 degrees, and had to bleach for much longer (a few hours).
I followed the protocol through but saw no staining!
There are a few issues that I think could be the problem.
This protocol makes up alcian blue at pH7.5 but I have seen others which state that it is active at pH 2.5... Any experience with this? I've seen that at different pHs it seems to stain different things though, so not sure what is best for facial cartilage then?
Second, my alcian blue (0.04%) appeared to have precipitated, and appeared to go back into solution upon shaking - but maybe it didn't really and requires heat? Or another step to avoid precipitation?
Did I over-fix or over-bleach?
Any help would be much appreciated!
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I have not noticed less staining with longer fixation. In fact, I could argue that your specimen may not be completely fixed after the short amount of time listed. I have successfully stained that have been stored in fixative for days or weeks.
See below for the protocol we use in our lab. You will notice that we use an Alcian blue solution that contains glacial acetic acid (which you don't need to warm) - you can argue either way for this component as to whether or not you get better staining. With the solution I use, I see very little to no precipitate and have never needed to check pH. With the recipe you are using, it says to warm it but not to what temperature. From the protocol, it looks like you really have to check very closely that there are no suspended particles at any time during the warming process - hence their mention of checking under a microscope. Is it completely dissolved and then precipitates when it cools? It is possible that the presence of undissolved particles causes a chain reaction, forming more precipitate upon cooling.
Assuming that your Alcian blue solution is fine, are your specimens staining? After the overnight stain they should be completely blue and very dark, almost black. It is possible that you are over bleaching. This is a tough step (regardless of protocol) as there is no real set time to it; you kind of have to keep checking periodically and stop when it looks good. In the below protocol, you will also notice there is an enzymatic digestion step before bleaching that helps to get the unwanted blue out and clear surrounding tissues. Basically, the enzyme punches little holes in soft tissue to help release the stain. I notice that I also get better infiltration and later clearing of the Alizarin stain too with this step. You may be able to add/modify this step for the protocol you are using.
I follow Method 3 in this protocol:
I have used it several times with both medaka and zebrafish larvae with very good results. Over time, various lab members have added detail to the original protocol and rearranged a couple of steps to get something that is very reliable.
Here’s how we do the staining:
Fixing Fish
1. Fix the specimens in 10% neutral buffered formalin overnight (16+ hours) at room temperature overnight on an orbital shaker and then move to 4°C until processing.
  • Euthanize fish in ice water bath rather than using MS-222
  • Use a 7.5 mL disposable transfer pipette to move the fish to a 2 mL Eppendorf tube; remove as much water as possible
2. Wash the specimens with 1X PBST twice.
  • 50 mL 1X PBST = 5mL 10X PBS + 500uL 10% TWEEN20 + 44.5mL double-distilled water
3. Go to the Method 3 Alcian blue cartilage stain protocol, starting at Step 2.
Method 3: Alcian blue staining for embryos or young larvae
1. Fix the specimens in 10% buffered formalin overnight at room temperature or 4% paraformaldehyde (PFA) at 4°C overnight.
2. Wash the specimens in 1X PBST twice.
3. Stain in ‘Alcian blue solution’ overnight at room temperature.
  • Recipe: 0.1g Alcian blue + 70 mL ethanol + 30 mL glacial acetic acid
4. Wash in 100% ethanol (EtOH) one time.
5. Transfer through an ethanol series to 1X PBST.
  • 90% EtOH --> 80% EtOH --> 70% EtOH --> 1X PBST (twice)
  • Each step sits for 20 mins to 1 hour.
6. Digest in ‘Enzyme solution II’ at room temperature for less than 1 hour (minimum 40 minutes).
  • Make a new Enzyme solution II each time you stain
  • Recipe for 30% Saturated aqueous sodium borate solution (10mL): 7 mL MilliQ + 3 mL saturated sodium borate
  • Recipe for enzyme solution II: 0.1g (=100mg) trypsin in 10 mL 30% saturated aqueous sodium borate
  • There is no exact time for this step. Periodically look at specimens, if leave too long you will lose eyes and skin.
7. Wash in 1X PBST twice.
8. Bleach with hydrogen peroxide (3% H2O2 in 1% KOH) for 1 hour at room temperature or until eyes of specimens become transparent (or very light grey).
  • Recipe (100mL): 20 mL 5% KOH + 10 mL of 30% H2O2 + 70 mL MilliQ
  • Make new each time because H2O2 bubbles some out over time.
  • Keep the caps on the Eppendorf tubes open during this hour or the hydrogen peroxide case will cause tubes to pop open and specimen will fly out.
  • Then centrifuge at 2500g for 1-2 minutes (cap open) to remove bubbles.
9. Wash with 1X PBST twice.
10. Store in 70% glycerol.
  • Skip this step if you want to go to bone staining (Alizarin red – Method 2 – start at Step 2)
  • Store at 4°C
Method 2: Alizarin red staining for embryos or young larvae
1. Fix the specimens in 10% buffered formalin or 4% paraformaldehyde with 1% 5N NaOH overnight at room temperature.
  • Skip this step if you have already stained with Alcian blue.
2. Wash the specimens in 1X PBST.
3. Stain in ‘Alizarin red solution II’ ‘Alizarin red solution I' at room temperature for several hours or overnight.
  • Recipe for Saturated Alizarin red S: Add a small amount of Alizarin Red S powder in 100% ethanol and vortex well -- Want it saturated so it will precipitate out after vortexing; if it does not add more powder and vortex again
  • Recipe for Alizarin red solution 1: 2 mL of saturated Alizarin red S solution in with 8 mL 0.5% KOH (avoid getting precipitate)
4. Stain specimens overnight; cover container in foil or store in dark place.
5. Wash specimens in 0.5% aqueous KOH two times.
6. Transfer in graded series of glycerine (15%, 30%, 60%, 70% in 0.5% KOH).
7. Store in 70% glycerine at room temperature.
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Has anybody ever used azole antifungals (fluconazole etc) in their cell/tissue culture? I work with primary human bone/cartilage tissue and I struggle with amphotericin B resistant fungal infections that most likely come from the surgery theatre. Are there any alternative antimycotics that would be safe for the cultured cells?
Thanks in advance,
Kasia Styczynska-Soczka.
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Dear Stefan Zimmermann
Thank you very much!
Kasia.
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Hi,
I work with congenital disorders of bone and cartilage. I send my proband samples for exome sequencing and validate the variant by doing sangers in lab. Recently I got a result with heterozygous duplication inframe (c.2026-1_2042dup p.Ala680_Gly685) I am facing a difficultly in capturing these variants by sangers sequencing! How do you design a primer for inframe duplication of 18bp with 6 residue change?
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You might be able to see this on Sanger as a "messy' region starting at the known 2026bp location. Design primers that are at least 100 bp upstream and 100 bp downstream of the duplication.
Or you could use melt-curve based genotyping. Design primers that closely flank your known duplication site, include a full set of controls (known wt variant, known heterozygous variant, water, etc.). Include a fluorescent dye into your PCR and amplify like normal. Then, instead of an agarose gel, use a qPCR machine to run a melt-curve on your product. With an 18-bp difference, you could probably use a fast 0.5*C step melt and not high-resolution melting.
Good luck!
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I am actually using bat embryos, but they should behave similarly to mouse or other mammals. We have tried the staining before, but to no success. We think that the PFA may have created a barrier at the skin which prevents Alcian blue from entering the sample, because the stain works perfectly when it is sectioned.
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Subhash C. Juneja Thanks for the response! I am going to try some more conditions in the upcoming weeks (assuming my mice breed) to troubleshoot. Once I figure it out, I will post it here.
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Hello,
I need to ship some mouse long bones and I don't know how they should be stored since I want to analyse the bone and the cartilage after the shipment. I've seen that I can stored them with dry ice but I haven't found a specific procedure.
Thank you in advance.
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In 70% ethanol, at room temperature.
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I am interested to collaborate with any researcher with suitable animal model for osteoarthritis. I have a new therapeutic approach for repairing damaged cartilag that requires serious investigation. Please get in touch, my e mail: morcsk2@aol.com
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Are you taking equine/horse subjects with cartilage damage?
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we face many patients, females, young and complaining from cartilage softening and only known while doing knee arthroscopy .
the alignment angles was within accepted range.
what to do to eradicate this lesions
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The most optimal method for working with young female patients with anterior knee pain is the development of an individualized rehabilitation program based upon a thorough subjective and objective assessments. SALTYCHEV Saltychev et al. in a review article concluded that there is no single treatment that works for all patients with patella-femoral pain J Rehabil Med 2018. Galloway et al. reported that females prepubescent may have maladaptive hip mechanics during landing and that may contribute to patella-femoral pain AJSM 2018. A systematic review by Lankhorst et al determined that weak knee extension strength is a risk factor for anterior knee pain JOSPT 2012. I have found that a strategy that can utilize dynamic control, manual therapy, strengthening, flexibility if an issue, along with proper shoewear and compliance with good home ex program and utilization of a good physical therapist has been successful.
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I am trying to do indentation test for my cartilage sample, but I am curious the boundary between compression and indentation, (like the image shows). I was wondering is there any standard use to define the size relationship between indenter and sample?
If I already know my indenter size is 3 mm diameter, what is the minimum size for my tested sample?
May I use FEA to check the deformation at sample edge, if the indentation process do not bring any deformation to the edge which means it is indentation process.
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Victor J. Аdlucky i done the FEA to check the sample size effect on the result. as you said the mechanical properties is hard to setting (this time i use young's modulus and passion ratio). the result shows there is exist affect. i am still looking for more reliable cartilage mechanical properties data.
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Osteoarthritis is characterised as the deterioration of the articular cartilage which then causes bone-to-bone contact. Glucosamine is a primary constituent of cartilage proteoglycans, which is why there are claims that glucosamine could possibly be an anti-arthritic. Despite this, there has been no current and impressive evidence supporting this claim. Has there been any current advances on this claim and if so, what are they?
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Many of the products used for arthritis like glucosamine etc are more of neutraceuticals than valid pharmaceutical agents. In early arthritis many may improve symptoms but we'll established cartilage degeneration is not repaired by any of drugs at present.
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Hello
I'm from Argentina and i'm working whit genetic expression of degenerative joint disease in dogs. So we're trying to isolate RNA from cartilage to further NGS analysis. But till now we haven't get good results. We need some advices about which is the best kit or method to isolate RNA from cartilage tissue. Could you tell me some about it please?
My best regards
Gabriela Rudd
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Hi Gabriela,
RNA isolation can be done by simplly by Trizol method and purifying by RNAeasy kit.
Key point is homogenization of cartilage. Our lab colleague clipped cartilage pieces from rabbit knee with scalpel and placed all the pieces in stainless steel grinding jar containing some trizol (jar placed in LN2), and vibrated on the Tissue Lyser II with some specific speed for 30 secs or so. The information of Tissue Lyzer II and stainless steel jars is as below:
After that he isolated RNA using Trizol protocol and purified by RNAeasy kit. He got the best RNA all the times, I was super impressed.
If you do not have this Tissue Lyser II, you can crush your cartilage to very fine powder in the presence of LN2 by using a mortar and pestle (your hands will feel cold due to LN2 by doing that, take it easy), that will work too (we have done with human cartilage)
Good luck
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I am trying to detect senescent cells in OA cartilage. For this I have tried SA b gal staining but the results are not good with very little staining . Since enzyme activity is very tricky to detect and fixation and freeze thawing has adverse effect, I am stuck with the experiment. I am not sure whether doing immunostaining with b gal antibody would make any difference. Fluorescence substrates may be another option as well. persons with first hand experience with these please give your opinion. Thanks
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doi: 10.1002/cbin.10619, doi: 10.1002/cbin.10561
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Do you prefer to use cartilage for reconstruction or bony plate ?
Any available literature regarding this?
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Thank you.
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Cartilage has neither blood supply nor innervation. However, patients with cartilage lesions typically present with pain. The question does not aim at advanced osteoarthritis, which is associated with inflammation and expression of pain-mediating cytokines. The main emphasis is the focal damage after the acute phase.
Who plays the predominant role, mediators or biomechanics?
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Hi Hagen,
I address your points in the following:
- often MRI allows diagnosis of subchondral edema, which is not pain-related,
SCB edema lesions detected by MRI are simply tissues with high proton density and nothing else. In other terms increased vacularisation (resorption by osteoclasts) or development of microfractures and haemorrhage/edema would have the same sign of increased proton density in the SCB. Hence “SCB edema lesions” are non-specific terms to describe a variety of causes for focal water accumulation that can be a benign or severely pathologic.
- the same with subchondral bone cysts,
SCB cysts to my limited experience are usually osteoclast filled spaces at the early stages, that later are filled with fibrotic tissue if the bone formation is not successful. We still do not know what causes this repair mechanism to be hindered mid-way, but lack of vscularisation is a good candidate, as endothelium or peri-endothelial cells provide the osteoblast precursors which are supposed to repair bone. For any reason, if the vascularisation is impaired (e.g. due to sclerotic SCB or continuous fragmentation of SCB because of continued heavy loading- I have seen both) the cysts will not be repaired and become permanent. However, if such cysts are under significant loading the peripheral bone fractures and the subchondral bone gives in and the articular surface collapses and pain may ensue. Otherwise, the cysts themselves if not too big, are not the source of pain.
- there are shear injuries, which primarily affect cartilage, not bone, which also can cause pain.
I hope I understood your question.
Honestly, I have not personally seen many such cases. In most of the cases that cartilage had some injuries, the synovial reaction was far more significant to my experience and better correlated with SCB injury that changes in the cartilage. Synovium is well-vascularised and well-innervated tissue that is ignored in many research endeavours.
Chemical induction of OA in animal models also relies on this fallacy too. The acute chemical injected to the joint not only injures cartilage, but also damages the synovium, and induces inflammation, which in many cases is not scrutinised by the naïve researchers focused solely on cartilage.
Could you perhaps comment on this and perhaps include mediators and biomechanics in your evaluation?
Can you please be more specific?
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I would like to obtain mitotic figures of chondrocytes, so I am staining cartilage tissue embedded in paraffin with crystal violet. I would be very thankful if anyone kindly shares his protocol or experience.
Also, there are some confusing terminology between crystal violet and cresyl violet in the literature sometimes. Are they totally different or share common characteristics/related structures?
Thank you in advance for your help.
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Probably washing takes care of that, if you add concentrated, that will be washed away,
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can someone guide me to a paper please?
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Hello Usama.
There should be i) a or more sources of stem cells that can generate cartilage cellular and extracellular components, ii) guiding scaffolds that can make a preferable shape in vitro, and iii) other proliferative or inductive growth factors to get sufficient number of differentiated chondrocytes.
Start with the following reviews to grasp a full understanding of current status and limitations.
Best wishes.
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I have a lipid emulsion for topical application that I want to test on the cartilage cells. I wanted to know if the different components of the emulsion (oil and surfactant) can cross all the barriers of the skin and the muscles to reach the cartilage?
I need publications or references please .
Thanks
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Dear Fadel,
Ok I got ur point now. I'm not verse in skin drug delivery systems. I hope some other researchers in the field will respond to ur question appropriately.
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Hey all,
I'm using two different types of PI ( Photoinitiators )
VA-086 and IRGCURE 2959 to Photo-curable my polymers (methacrylated Alginate and methacrylated Gelatin) .
- Could you please suggest the proper concentration of each PI to form hydrogel (Bioink) for Cartilage applications?
- what is the proper UV intensity (xx mW/Cm^2) that I can use for less time please ?
Thanks
Hussein
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Hussein,
Have you considered using Ruthenium? It uses lower concentrations than irgacure, and you can use visible light (405-450nm). I can send you the links to buy the product from Sigma and the protocol if you want.
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I have tried to stain rabbit cartilage using different hematoxylin stains (Harris and Mayer (very old one)). However, I can not see the nucleus clearly.
The tissue was fixed 2 days in formalin after it was decalcification and embedded in paraffin for 5 days. After dewaxing, the slide was stained with hematoxiline for 16 min.
Would you please let me know if you have any proper protocol for cartilage H&E staining?
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HCl is a strong acid and can destroy chromatin-stainability. You should use weaker acids like 5% hno3 or 10% formic acid. Decal solutions with EDTA take longer, but don't influence stainability.
Washing does not solve the problem.
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Can someone advice on a protocol to detach MSCs from the surface of scaffolds for PCR analysis.
I am working with decellularised trachea scaffolds ( Pig) seeded with MSCs. Cells only attach at the surface. I want to avoid grinding the tissue because it contains genomic DNA remanants and dense ECM cartilage. and I got very low yeild.
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Great thanking you , I will definitely try it
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We found that sliding of cartilage against an artificial meniscus could increase friction during the swing phase and long term elevated friction could cause cartilage wear.
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Again the biomechanics differ from young to old age. Older people suffer from high friction and we should adjust the flexibility
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I am doing IHC with bone and cartilage samples (eg. growth plate).
For antigen retrieval, I usually heat the samples in Citrate buffer (pH 6.0) using pressure cooker (for 1 min at max. temp and pressure and cool down for 20 min.)
But, bone and cartilage part are peeled off from the slide in high temperature.
In case of proteinase K - induced antigen retrieval, tissue is okay, but it doesn't work, no staining.
Any better antigen retrieval method for bone or cartilage sample?
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Hey there,
we also work with cartilage and decalcified bone samples. These samples even detach by room temperature during non-IHC staining (safranin-O etc.). We found improved attachment on silanized slides, a coating which can be easily made in the lab and is a cheap solution. However, some samples were still washed off during IHC. Now, we use "Superfrost Plus" slides, which are quite expensive but result in successful outcome.
Best regards.
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I have tried to take a part of the vertebral column and dissolve the surrounding tissue with 6% potassium hydroxide but after one day I found that the cartilage has broke into very small pieces. do you have any tips on determining ray fish age?
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Several techniques have been described to highlight growth rings in ray fish.
The following three techniques are based on alizarin S affinity for calcium (Du Buit, 1977):
"1 - Fixation of vertebral bodies by formalin at 10% for 48 h. Treatment with potash at 5% for 15 to 60 minutes; potash at 2% + alizarin for 24 to 48 hours; potash at 2% for 15 to 60 minutes; potash at 2% + glycerin for 24 to 48 hours. Storage in glycerine for 24 to 48 hours. Conservation in glycerine.
2 - Fixation of vertebral bodies to alcohol at 95 ° for 48 to 96 h. Treatment with potash at 1% for 24 to 48 hours; 1% potash + alizarin for 24 hours; 1% potash + glycerine (50 + 50). Conservation in glycerine..
3 - Treatment with sodium hydroxide at 0-2% for 24 h. Staining with sodium hydroxide + alizarin (9 + 1) for 24 to 72 hours; washing with running water for 15 min; Differentiation with oxygen water at 3% for 24 to 72 hours". Conservation in alcohol at 70 °.
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Hello I am performing the chondrogenic differentiation of adipose-derived stem cells in bidimensional cultures.
I would like to ask which one is the best protocol for the staining and the semi-quantification by absorbance at 630 nm.
I read several protocols... I have Alcian Blue 8GX (powder) from sigma (A5268). I saw in short, two kind of staining with Alcian blue in Acetic acid (pH - 2.5) and Alcian blue in 0.1 M HCl overnight after 20 min of fixation in 95% methanol (also I have doubts with the fixation... formalin, PFA 4%...?). The second one seems to be the best for cell layers in 2D, however I am a little afraid of the 0.1 M HCl (Did anybody tried it?).
Finally for the extraction of Alcian blue in order to make the quantification I read 6h of incubation in Guanidine-HCl 6M, correct?
Thanks in advanced,
Sergio.
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We've had good luck with the following protocol on fixed samples in 6-well plates:
1) Rinse wells 3x with PBS.
2) Rinse 1x with 1% acetic acid. 3) Apply enough Alcian blue solution to cover wells. Incubate 15mins.
4) Rinse well repeatedly with PBS until clear. Dry plates in hood.
5) Measure absorbance at 630 nm
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Hi! I am trying to analyze cartilage using Micro-CT. Does anyone know where I can buy such contrast agents? Thank you.
best regards
carl
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Dear Carl,
Phosphomolybdic acid (PMA) has been mentioned as a good contrast agent for cartilage structures.
You can also check this publication:
Pauwels, E., Van Loo, D., Cornillie, P., Brabant, L., & Van Hoorebeke, L. (2013). An exploratory study of contrast agents for soft tissue visualization by means of high resolution X‐ray computed tomography imaging. Journal of microscopy, 250(1), 21-31. doi:10.1111/jmi.12013
Best regards,
Niki
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To be used for Energy dispersive X-ray spectrophotometry
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Good software comes with EDS spectrometer, so you already have it. In addition you need a good standard (certified hydroxyapatite will do) and a good EDS operator. Set your software to analyze Ca and P and as a "ballast" element you can set O or some combination of O and C if your software allow it. If you want to analyse not only Ca and P, but light elements also, you'll need more standards and really excellent operator.
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Someone here recently posted a question about counterstaining Oil-Red-O.
I have a similar question. Nuclear staining with Hematoxylin would be ok, but are there other stains (that don't use ethanol) that I could try? I was hoping for something purple or blue that I could use to stain cartilage or cell membranes. I am using the Oil-Red-O to stain adipocytes so cell membranes are not as critical (the red cytoplasm is what will inform me).
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That would be alcian blue, I gess. It is a watery stain. No personal experience, you have to try.
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I am currently having problems with IHC(immunohistochemistry) of a nuclear protein. I am working with human osteoarthritis cartilage tissue(damaged region-undamaged region) and mouse cartilage tissue(DMM-SHAM and young-aged). I am using peroxidase-based methods while blocking endogenous peroxidase with 0.3% H2O2 for 10 minutes. I also have a negative IgG control(there is non-specific staining here as well). I used both the trypsin and heat antigen retrieval method(they both have the same problem), blocked the samples with 1% BSA in PBS for 15 minutes. Also, I added a permeabilization step because I am working with a nuclear protein. The problem is that there is non-specific binding and high background where it is not supposed to have a signal and no signal where there should be staining according to my hypothesis. Can anybody help me with this problem?
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I would also try with the serum of the secondary Ab produced animal and with BSA.
Also diluting the primer Ab with PBS containing BSA %2.5.
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I am currently having problems with IHC(immunohistochemistry). I am working with human osteoarthritis cartilage tissue(damaged region-undamaged region) and mouse cartilage tissue(DMM-SHAM). The problem is that there is non-specific binding and high background where it is not supposed to have a signal and no signal where there should be staining according to my hypothesis. Can anybody help me with this problem?
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Hi Yoo Jin, we would need some more info to help you, are you using peroxidase-based methods? If so, are you blocking endogenous peroxidase and inspecific antibody binding? Do you have positive and negative controls?
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Hello,
I have a sample of human articular cartilage that has been freeze-dried last year and kept in a freezer until today. I'm not sure that it can be used for a new experiment because of the possibility of changes in its biological and mechanical properties. I'd be so grateful if anyone who experienced the same situation or has the knowledge, shares his/her information with me.
P.S.: Is there any test or characterization method that can validate the consistency of the mentioned sample properties?
Regards,
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Agree W Pillai
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Hi. I am currently working with new materials that should be tested on a scarified skin.
I am employing the widely-accepted wound model using the ventral surface of a rabbit ear, which was originally suggested by professor Mustoe. In brief, this model involves full thickness excision of rabbit ear skin and perichondrium, leaving the bare cartilage to heal itself and form a scar wound.
However, i am having some drawbacks. From some samples, the hypertrophy of the rabbit ear cartilage, rather than the dermal portion, is too prominent. This makes the analysis of the dermal portion very difficult.
My guess is that there had been some random microdamage to the cartilage portion during the removal of the perichondrium, but that leaves me no choice but to leave the perichondrium, which will accelerate the healing and make the wound UN-hypertrophic.
Is there a way to maximally prevent cartilage hypertrophy, but only achieve dermal hypertrophy using this model? Thank You.
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kindly apply keloid (scar) preventing medicament. It is my guess only. Thanks
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Hi All,
I am trying to do a 3D cell culture for MSC cells and condrocyte Cell lines for cartilage application using different concentration of photocrosslinking alginate and/ or gelatin hydrogels?
I was wondering if anyone can refer me to a step-wise protocol for culturing the cells using those hydrogels please?
Thanks
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Thank very much Yong-Can
No, I will start with MSC only!
thanks again
Hussein
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I need suggestions for how to properly homegenize cartilage tendon. Right now, I use a pulverizer dipped in liquid nitrogen and then take the powerderd pulverized sample and put it in a RIPA buffer. I have tried multiple types of RIPA buffers and all leave at least some amount of sample out of the solution. Usually, even if the sample is completely powder, after using a motorized homogenizer most of the parts congeal back together. I am working in a lab where mostly muscle homogenizations occur, and no one understands why my samples re-congeal into a ball after pulverization. I think the fibrous tissues are hard to break down and want to stick back together, but I am unsure of how to make them stay in solution. I tried a mortar and pestle over dry ice once, and the same thing happened- actually it was worse than when I pulverized it into a powder. Maybe I should find a different RIPA recipe(?) but I have already tried a couple I made and one I bought and it's not a whole lot of diffrence.
RIPA recipe 1, 50 mL: .302 g Tris, .48 g NaCl, 0.05 g SDS, 0.25 g Sodium deoxycholate, 0.5 mL Triton X 100, pH 7.4
RIPA recipe 2: 100 mL: .242 g Tris, .8775 g NaCl, 0.29 g EDTA, 2% SDS, 1% Triton, pH 7.5
*** RIPA recipe 2 has worked the best so far
RIPA from Sigma-Aldrich:150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0
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Has this problem been solved, because I'm experiencing the EXACT same problem :)?
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Hello All,
 This is the image of Total RNA non- denaturing gel electrophoresis. I performed a total RNA extraction from dog (cartilage, blood, synovial fluid and knee ligament) tissue. Can someone please tell me what are the two bands intercalated between the 28s and 18s bands showing in the 3rd lane? Thanks
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Hello Gabriela,
Firstly i think if you further want to process your RNA then try to optimize your isolation procedure so that your band (28s and 18s) intensities correspond to 2:1 ratio. That will ensure you a good quality of your RNA. Also for RNA quality assessment you can use RIN.
Now as it appears from your gel image the 2 bands might correspond to 5s and 5.8s.
There are articles where you will find that it is being stated that when eukaryotic RNA isolation is done then along with 28 and 18s one might also observe 5 and 5.8s.
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After digesting cartilage tissue in a collagenase solution, I always observe tiny oil-like droplets floating the digested suspension. Has anyone made a similar observation? Is the tissue a source of the oil droplets or is it the collagenase?  
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^^ Thank you everyone for the response.
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Hello! I would like to ask about the quantification of sGAG after the chondrogenesis of mesenchymal stem cells.
My principal doubt is how to extract the sGAG to performs an assay with Dimethylmethylene Blue Assay (DMMB) because I have found protocols for that and they talk about the complex sGAG-DMMB but they do not mention how to obtain the sGAG in solution... Maybe with the application of the reagent the sGAG are realising to the medium as sGAG-DMMB complex directly after following the protocol?
Another issue, if I do Alcian blue/nuclear fast red stain. I cannot use them for the abovementioned assay right? I should have two pellets (one for the histology and one for the quantification)
Thanks a lot,
Sergio.
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Alternatively, you could use an Alcian blue based assay. But be forewarned they stopped making Alcian blue back in 1979 due to its highly carcinogenic activities during manufacture (large number of employees making stain were getting cancers). So any Alcain blue purchased after 1979 is not anywhere near to being 100% stain, it is mostly fillers. Alcian blue assay protocols can be found in:
Young HE, Dalley BK, Markwald RR. Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma. Anatomical Record, 223:231-241, 1989.
Young HE, Young VE, Caplan AI. Comparison of fixatives for maximal retention of  glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated radiolabeled [35S]sulfate. Journal of Histochemistry and Cytochemistry, 37:223-228, 1989.
Young HE, Carrino DA, Caplan AI. Histochemical analysis of newly synthesized and resident sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg. Journal of Morphology, 201:85-103, 1989.
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I would like to digest the entire porcine glenoid cartilage to create some Osteoarthritis like damage using MMP-1 and mechanical test the glenoid with the joint simulator to see the effect of the damage. how can I determine how much MMP-1 I need for the whole glenoid? I have attached the joint that I would like to digest. Could you please recommend me groups that might do similar work or recipes that I could use.
Many thanks for your help in advance. 
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Try "Tergazyme" from Alconox.  Their cocktail is not public information, but its properties may be appropriate for your project.
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Different type of scaffolds are used for regeneration cartilage, bone and several other tissues. Every researcher at the end of the day and at the bottom of the paper suggest that his scaffold is the best so please help me out which one is really the suitable and best as far as cartilage is concerned.   
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There are lot of bio materials available for cartilage regeneration. But the selection of suitable biomaterial is the main challenge in this filed. Each researcher may think in different angle such that regenerated tissue should mimic the native tissue. The main criteria for selection of material is its mechanical property , biocompatibility and biodegradability. Each material behaviour is different for example PCL has mechanical property suitable for cartilage TE, minimum 2 years of degradation period, good compatibility and FDA approved. But if you have to achieve these properties the concentration of polymer and the preparation methods also have an important role.
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I want a positive control to compare to native cartilage and i want to induce cartilage ( Collagen Type II  or GAG lysis, denaturation or degradation? what is the best way to do this?
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Hey, this depends on the grade of degradation you want to achieve. Do you plan to simulate osteoarthritic cartilage degradation? Then you could add cytokines (TNF-a or IL-1b). These cytokines strongly induce the expression of ECM-destructive enzymes (collagenases and aggrecanases). You could also subject the cartilage to a mechanical impact  (using a drop tower or the like). This also induces catabolic processes, but is less intensive in matters of proteoglycan-/ collagen-release and destruction. Additionally the trauma causes high cell loss (which might be unwanted in your experiments). It is also possible to add ECM-destructive enzymes (MMP-1/ -3/ -9/ -13, ADAMTS-4/-5, and so on) directly into your media. This depends on your aims of this study.
If you plan to destroy the entire cartilage structure, enzymatic digest as used for chondrocyte isolation is an easy way. 
... actually there are like a million ways to induce cartilage degradation :)
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I would like to have just the whole costal cartilage for my work. Most of the article I have found involves hand dissection and washing in phosphate buffered saline or using trypsin. Does anyone know of any chemical methods that could be used for cleaning a large number or thoracic cages?
Regards
Sam
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cooking them in boiling water for long time by adding sodium bicarbonate
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While studying M. electricus, my co-author and I were observed that, the skull of M. electricus was consisted of both bones and cartilage, as the frontal, parietal,
supraoccipital, postparietal, sphenotic and pterootic were remained cartilages in mature fish (J. Morphol. Sci., 2014, vol. 31, no. 3, p. 162-170). I wonder is this observed by researchers worked on other electric fishes, as we suggested that, these cartilage may play a role in protecting the electric fish brain from electric shocking.
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Dear Gloria, your note is reasonable, if you have an article describing embryo-genesis of skull in M. electrics, kindly send it to me. I did not think that the roof of skull was over-stained. However, I have little success to find articles about  developing of bones in M. electrics, so we could not confirm their chondral origin.  In my opinion, each fish skull is a separate puzzle and nothing stable. I am trying to test our findings by reviewing other researches as it is hard to study the fish embryo-genesis. Thank you very much for your notes.