Bone Biology – Science topic
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Short stature is one of the problems that some people experience, and people are looking for a solution to this problem. its been known that there are several hormonal treatments I am interested in knowing whether it helps to grow people's height by stimulating the magnetic field of bone cells without using hormonal drugs.
“I don't think so because it is influenced by genetic factors. Best wishes”
I am just wondering if anyone has successfully demonstrated in human patients or induced OA animal models if mesenchymal stem cells can repair osteoarthritis by intra-articular injections of cells?
I saw some images from micro CT analysis of density and porosity of bones, commonly used to compare patients with or without osteoporosis. I was wondering if is there significant morphological differences between bones, through these images.
Hello, I am interested to preserve bone without lost osteoinductivity, I read the best method to preserve bone is fresh-frozen, but how much can I storage without lost osteoinductivity? Thank you for your help Luz
“MICRONIC (https://www.micronic.com/ ) makes extremely durable tubes, certainly suitable for -80C. RNAse, DNAse, pyrogen-free, available sterile, external or internal threaded, and free of leachables. Check out some of their tech notes here: (https://www.micronic.com/tech-notes) The Generation R study may be of some interest to you in particular, the study has preserved biosamples from infant to adulthood, going on 15 years.”
Hi all, I am trying to make C28I2 stable cell line using G418. Initially, to see the optimal dose of G418 for C28I2 cells, I have been treating the cells with G418 ranging from 0.5mg/ml to 5mg/ml for more than one week, but it seems cells don't die. I used new G418. Is there anyone who give me some suggestions? Thank you in advance!
“those cells were immortalized via retroviral vector-mediated simian virus SV40 Large T antigen (Tag) expression. that retroviral cassette was selected for using G418, such that the cells are already resistant. You need to switch to a different selection cassette such as puromycin, hygromycin, or blasticidin resistance. check here : http://www.emdmillipore.com/US/en/product/C28%2FI2-Human-Chondrocyte-Cell-Line,MM_NF-SCC043#anchor_Biological Information”
I am a anatomist and orthopaedic surgeon. CT osteoabsorptiometry has been famous for the method to asses the subchondral mineralization as a measure of stress loading to joint cartilage (Momma, AJSM 2011, Funakoshi AJSM 2016). However, I am not sure whether these methods could be apply to measure not a loading stress but a tensile stress from tendons or ligaments around articular joints. Is there any methods or theories to asses the tensile stress from tendons or ligaments with the distribution of the subchondral mineralization around joints by using CT osteoabsorptiometry or other... more
“Am J Sports Med. 2017 Feb;45(2):394-402. doi: 10.1177/0363546516665804. Epub 2016 Oct 1. Cortical Bony Thickening of the Lateral Intercondylar Wall: The Functional Attachment of the Anterior Cruciate Ligament. Norman D1, Metcalfe AJ2, Barlow T2, Hutchinson CE1, Thompson PJ2, Spalding TJ2, Williams MA1. Author information Abstract BACKGROUND: The anatomy of the anterior cruciate ligament (ACL) has become the subject of much debate. There has been extensive study into attachment points of the native ligament, especially regarding the femoral attachment. Some of these studies have suggested...” more
Is it possible to distinguish bone fracture by using thermal images like skull?
“Thermography is a method of functional diagnostics, and for topical diagnosis has limitations. So no need to compete with the methods of structural imaging such as CT or MRI. At the same time, practical applications might be for thermography, for example, to adjust the rate of distraction, during monitoring of engraftment or to assess the effectiveness of physical therapy, especially in the area of sport medicine. See, for example, 1) Morasievicz L. et al. (2008). Use of thermography to monitor the bone regenerate during limb lengthening, 2) RU patent 2457777 (URL:...” more
Hello, someone know what is the size or dimensions of the talus and ankle cartilage in a human adults healthy. Best regards
“And by human size”
I test 2 different methods (A and B) on the count of specific bone microscopic structure. My goal is to assess that method B is reliable in the count of those microstructure. So I count the number for each of those methods in the same specific area of the bone for 30 individuals. With method A (which is the gold standard method), I counted in total for the 30 individuals 193 structures and with method B, I counted 184 structures. However, I have only 127 structures in common for both method. What statistic can I use in order to test if it's significant? Does khi carré works even if the... more
“Given that zonal osteons were originally identified using microradiography, back scatter SEM should be the best choice I think. Good luck.”
We are hoping to decalcify mouse paws for IHC with EDTA. However, with flesh still remaining around the paws, the decalcification has not been working (as determined by an ammonium oxalate test, presumably because the EDTA cannot access the bone through the soft tissue). Since the phalanges (bone of interest) are much smaller than other bones for which decalcification is done, we are worried that trying to remove the flesh by hand will damage the bone. Is there an accepted way to remove flesh chemically without disturbing molecular structure for IHC or is careful physical removal the... more
“Careful dissection is the most reliable method. Try using very small scissors to cut parallel to the phalanges, then peeling off. Alternatively, if skin is left on fixative/decal solution can be injected under the skin. ”
adipose tissue MSC culture
“I think you must follow lab protocol of Guido Krenning at University of Groningen, Groningen”
Looking for a way of assaying bone mineral densities of the femural bones of rats.
“Thanks Luis Rodrigo.”
When expressing waking sedentary time, is it better to express it as the total time spent sedentary (hours or minutes), while accounting for any non-wear time, or as a percentage (%) of device wear time? Are they one and the same (i.e. the mean will be the same), or are there advantages to expressing it in one form over the other?
I would like to stain the spines of adult born granule neurons for the purpose of density and morphology analysis. Does anyone have a positive experience with a particular fluorescent staining? I have only performed such analysis with the aid of retroviral targeting of adult born neurons. Now I would like to simply stain the spines, without resorting to viral delivery. As I doubt DCX on its own would be enough for staining spines, I was thinking of using DCX and Acting together. Does anyone have experiences with such (or any other) double staining? Thank you in advance for your... more
Before purchasing abcam's RANKL (ab129136), I contacted the company as to whether a crosslinking antibody was necessary because I didn't want to use one. They assured me that I did not need one. But despite using the protein at up to 10x's the recommended dosage, I still cannot get my Raw264.7 cells to differentiate into osteoclasts. My cells grow VERY fast. Despite splitting them at the highest recommended ratio (1:6), I still need to split them every 2 to 3 days to prevent them from growing beyond 60-70% confluence. I am trying to use them at as low a passage number as possible since... more
“Dear Tia were u able to get the mature OC? I am still struggling with this system. Can any one help? Regards”
MG-63 cells, a line derived from an osteosarcoma, produce high yields of interferon after superinduction with polyinosinic acid polycytidylic acid, cycloheximide, and actinomycin D. Studies using MG-63 cells provide some important mechanistic clues concerning the details of the amplification process in tumors. Cells can also be transfected
“Because this cell line has the ability to retain a differentiated phenotype under culturing conditions. Additionally, the scientists prefer using MG-63 because it proliferates so fast compared to normal osteoblast. However using this cell line for implant related studies is controversial. From my experience, I would not recommend using it as a model for osteoblast because it behaves differently in many conditions. It is a cancer cell line”
I read in most papers that they either use ~4% or ~8% EDTA inPBS solution when mice/rat mandible/long bone is decalcified for immuno purpose. I myself always used 8% for faster process. Last time in the conference, a senior technician said that 8% EDTA is too high. Anyone know what are the differences between 4% and 8%? Will the 4% preserve the ultrastructure or antigens better plz? Thanks a lot!
“We used 0.5 mol (EDTA) phosphate-buffered saline (pH 7.4) for the thicker bone samples for more than half year, which presented good results. The higher the concentration, the faster . But it will also influence the IHC results . ”
I can see lipid droplets in chondrocytes in ex vivo chondrogenesis from stem cell differentiation (a figure enclosed). Report does exist in literature that lipid droplets are present in human cartilage. And they increase from childhood to adult cartilage. Can any expert in this area elaborate on this topic the real significance of lipid droplets in chondrocytes?
“Hi Subhash, that is a good question. To my knowledge, it is not very common to find lipid droplets in articular and fibrocartilage, however, it is common to find large lipid droplets in chondrocytes of elastic cartilage in some species. I am going to publish a paper confirming that.”
If the limd1 gene is deleted, can it cause the tumor to develop or affect osteoblasts and osteoclasts leading to bone development?
“Dear Jinxin, The answer to your question is yes. see this reference from one of my collaborators https://www.ncbi.nlm.nih.gov/pubmed/18657804 May I ask why? Have you some interesting follow-up data? You can always e-mail me if the data is very confidential for ResearchGate firstname.lastname@example.org BW, Tyson”
need to know if there is any study about normal thickness of synovial membranes of different joints and tendons
“That is a great question. I was looking for this data for foot and ankle joints and never found any. There are articles on the wrist and knee but as of yet I have never seen or found one that compares the different healthy joint measurements”
I want to use two different color dyes to see fusion (after MCSF/RANKL stimulation) of two sources of monocytes that express two different constructs respectively. I would like to observe which monocytes fuse with which others and my readout will be the color of multinuclear osteoclasts. Can anyone who has experience with this provide feedback? Thank you!
I am cutting these samples to do FTIR studies
“Dear Dr. Nadhim, Thanks a lot for the link to the details of cutting UHMWPE but what I need was slicing the component to 200-micron thickness which is hard with big machines. Could we possibly do anything with the blade used in the microtome? Because we already have the above mentioned one at our university. ”
Patient with hip pain and normal x-ray but MRI shows early avascular necrosis of femural head.
“I think it may be an option. In early stage any type of drilling and core decompression have a good results. In future maybe some new drug delivery system can improve the results on exact necrosis site”
Specifically, we are looking to decalcify a rabbit mandible. We will be staining for factors such as VEGF, BMP-2, TGF-beta1, etc.
“I agreed with other comments, EDTA would be the best results. If you need the shorter decalcification period and reasonable results, I recommends Morse's solution. It takes about 1/8 decalcification period as compared to EDTA sol., with maintaining reasonable immunostaining results. Here are some infos. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3796219/ http://www.ihcworld.com/_protocols/histology/decalcification_solution.htm”
as the bones are irregular in shape
“First of all you need to have sufficient experimental data in order to have the physical and mechanical properties of your material. Second, due to the irregularities in the geometry of your problem you need to use 3-D eight node elements and you need to have a very fine mesh.”
We are interested in purchasing the Leica CryoJane tape transfer system to facilitate sectioning of murine bone (primarily tibia and femur). However, until we have funding for this equipment we are trying to create our own "DIY" version. We can successful cut sections which adhere to the tape. The problem is that transfer of the sections from the tape to the slide is inconsistent - sometimes giving beautiful sections and on other occasions partly or not sticking. We are trying to figure out whether the UV (source / duration) or the slide temperature (as the CryoJane has a temperature... more
“Hi Nicholas, thanks for the prompt answer! I have checked Brya suggestion, but I thought it could have been easier just to ask Leica instead of having stuff shipped from Japan xD In general, we focus more on femoral condyles and tibia, so overall the knee joints.so I reckon the BM loss is not that important to us! Anyway my mail is mario.alvarez dot qmul so you can drop me an email and we can further discuss this! Cheers, Mario”
Further Details : Ritchie RO, Koester KJ, Ionova S, Yao W, Lane NE, Ager 3rd JW. Measurement of the toughness of bone: a tutorial with special reference to small animal studies. Bone 2008;43:798–812.
Biomaterials used in human bone fixations
“Bio-compatibility (non antigenic and easy integration) , biodegradability and good bio mechanical strength are the requirements. It should also be biomimetic. As regards to the part of fixation techniques you can think of well fabricated cortical screws derived from cortical bone which needs no removal and also bone plates made out of synthetic absorbable like polymers already in use in suture materials which should be fabricated in the way that the biodegrability should be slow till the bone healing is over. and later on it will be resorbed. ”
Hello, I'm new in the field of osteoclast and when you look in the publication for osteoclast in TRAP assay you can find everything and nothing. I want to be able to recognize for sure osteoclast because I want to make a real quantification of them as I'm working on different protocol of differentiation. Thank you so much in advance to all that will help. I'm attaching some pictures that I took here. Jen
“Adding to above question Is this picture taken at 100X? also do we need to counter-stain with hematoxylin? thanks in advance ”
In hyaline, fibro and elastic cartilage have been characterizated diferent types of proteins (collagen II, I, V, VI and elastin) depending of cartilage type. But, respect to the proteoglycans composition (aggrecan and decorin) I am not sure that if elastic cartilage also contain aggrecans.
“elastic cartilage composition: elastin, fibrilin, glycoproteins, collagen type 2, -9, -10 und -11 and aggrecan Composition depends on the source of the cartilage. Regards”
Caplan's mesenchymal stem cells express CD105, CD123, and CD166 cells surface markers, conform to Hayflick's limit, and differentiates into cartilage, fat, and bone. The mesodermal stem cells we work with express CD90 and CD13 cell surface markers, expresses telomerase, has essentially unlimited proliferation potential, and can differentiate into skeletal muscle, smooth muscle, cardiac muscle, unilocular white fat, multilocular brown fat, hyaline cartilage, articular cartilage, growth plate cartilage, elastic cartilage, fibrocartilage, compact bone, cancellous bone, tendon, ligament,... more
“Dear Carlos, Were the MSCs you mentioned derived from a single cell or were they tested from a mixed population.of cells? Do they have the same characteristics in all situatios: cryopreservation, progression factors, inductive factors, growth on plastic, same appearance, etc. Only when cells share EXACTLY the same characteristics can one assume they are the same cell.”
We have project concerned to biomechanical testing of fresh pig bones that are glued together. Bone adhesive that we are using has setting time 72 hours. During 72 hours fresh bones in temperature 37° start to be under putrefaction. We are interested how to stop or to slow down this process.
“Thank you David :-)”
In vivo studies in mice have shown promising results of endochondral ossification using upscaled cartilage models. Clinical trials have to factor the size of the models and the soft tissue coverage. Are any studies being done in this matter?
“LLLT iradiation have good effect on cartilage healing.”
can you use image j to plan for spine or long bone deformity osteotomies. if so, how? thanks much, Ayo
“J/Fizi images are now considered as more accurate determination of size of a cam lesion or spincer defect , in FAI syndrome , this technique can be utilised for determining the extent of osteotomy on long bones and correction of kypho scoliotic deformities of spine, which need to be taught to us for proper utilization in all surgical orthopedic procedures for precision planning to correct deformities by osteotomy in any bones mainly maxillofacial bones, spines and long bones. I am looking for papers related thisand will add in near future”
Can anyone tell or clarify that is there any effect of bone density over the range of motion of spine ? ROM will increase, decrease or remain constant?
“@Ebrahim Bani Hassan Sir..this is from mechanical point of view and for consideration of motion.”
Laser (NextEngine) or white light (Breuckmann)? This will mainly be for teaching purposes (building a comparative database of 3D .pdfs for students). I won't need any internal geometry - surfaces are all I'm after. Ideally, I would like to find a scanner/method that can also be used for research purposes (eg, collecting landmark data, dental topographic analysis).
“White light over a nextEngine any day.The terms 'white light' and 'structured light' are often used interchangeably (although some structured light scanners use blue or green light-they tend to be aimed at industry). The time difference is huge (>5 minutes scanning per object with a good workflow). Breuckmann scanners output very nice data. Good alternatives to Breuckmann are Steinblicher, Mephisto, LMI and Artec. As Luciano said above, photogrammetry is also worth a try, although you need a more powerful computer than for scanning to make it time efficient. It gives very good results...” more
An obvious advantage of fixation is the anatomical fit of the fragment. But loosening is part of the disease and bone as well as cartilage has biologically changed. Although the successful fixation is recommended and described it might be better to remove the fragment and treat by bone grafting possibly in combination with cartilage repair procedures.
“Juvenile OD dissecats should be debrided and refixed. Smart nails and chondral darts could be used. My experience with biocompression screw is not successful, every case the implant broke. My interpretation: there is too much instability during healing process. OD is a pseudarthrosis under vitamin D3 deficiency. ”
“I'm looking into the effectiveness of caffeine, especially when ingestion after creatine supplementation on anaerobic power and isoenzyme creatine kinase in male athletes. is there anyone that could enlighten me on this subject?”
This is for all bone biologists. If so, we can not do on paraffin section, but only on frozen and plastic sections.
“I will put my previous question this way, are there some bone molecular targets (protein and RNA) which has been better detected by undecalcified bone rather than decalcified bone? If there is research DATA to provide evidence on above statement? Much gratitude.”
For paraffin sections, whats better for decalcification of bones, I target the complex vertebra of catfish.
“Dear Gloria The centrum is hollow, the thickness is about one millimeter, I target the complex vertebra that lodged a part of the webberian ossicles (fused first to fifth vertebrae of clarias garipenus, standard length of fish about 30 cm). From your answers and other reports, I think that nitric acid 5% for 3 hours , no neutralization will be helpful, what is your opinion?”
Hello, I'm trying to do a TRAP staining of mouse femora embedded in paraffin. The bones were fixed for 1 week in 4% buffered paraformaldehyde before they were embedded. After deparaffinization I first incubated the slides in TRAP staining buffer (40mM sodium actetate, 200mM di-sodium tartrate-dihydrat, pH 5,0) for 30min and then in TRAP staining solution (0,5mM Naphthol-AS-MX-phosphate, 3mM Fast Red Violet LB Salt, 2% di-methyleformamide, 1% Triton X-100) for 2,5h. Now I can see a lot of red structures and it looks more like a smear than like osteoclasts. Can anybody help my with the... more
“Dear Jirko and Anja, Could you help me with TRAP staining. I would like to know the role of Pararosaniline Dye in TRAP staining. As it is not included in TRAP kit A from Sigma?(procedure No 387) Thanks”
Hello everyone, I would highly appreciate if someone could give me an advice regarding plastic embedding. I am working with the bone tissues, and I am trying to establish embedding process in Spurr resin in order to analyse dynamic histomorphometry. Bones are from 6 months old mice. First, I dehydrate them in 70%, 95%, 100% ethanol and 100% acetone (every step takes 2 days) and then start infiltration process with Spurr resin 50%, 75% (mixed with acetone) and 100% (also steps take 2 days). Dehydration and infiltration processes take place in desiccator connected to vacuum pump. At the end... more
“Does anyone know silver technique to visualize nerves in Undecalcified rat Bones?”
I need some help with a bone tissue homogenization protocol for protein extraction. My purpose is to extract proteins, both from the extracellular matrix and (hopefully) from osteocytes, for running SDS-PAGE. I’m following a protocol from Buckley et al (Journal of Archaeological Science, 37(1), 13–20) with the following steps: 1. Clear all surrounding soft tissue and wash the bone marrow 2. Pulverize bone sample with a mortar and pestle in liquid nitrogen 3. Collect some bone powder (approx 1g) to an eppendorf tube 4. Add 10 volumes of 0.6M HCl and incubate 4 hours at 4oC 5. Centrifuge at... more
“Dear Anthony Asmar! You seem work with biglycan? is would be great if you can send me the detail of protocol you collected protein form mouse bone. My email is: email@example.com Thank you very much in advance!”
I have been culturing RAW264.7 mouse macrophages and differentiating them successfully using R&D Systems RANKL for about 7 months now. The cells are multinucleated and I can see a ruffled border, but when I look at the ivory or hydroxyapatite wafers in SEM I see no resorption pits. For continuous culture of RAW264.7 cells I use DMEM-High Glucose with 10% FBS and 1% Pen/Strep. For differentiation: alpha-MEM with 10% FBS, 1% Pen/Strep, 75 ng/mL RANKL, 2.5ug/mL cross linking antibody (required for the RANKL that I am using), and I have tried with and without 10nM vitamin d3. The... more
“ I agree with David. Also you can try it by using BD BioCoat Osteologic Bone Cell Culture System. It is simple, you only need a light microscope. Check the pictures from my paper.”
I tried fixing with 4% PFA and sucrose 10% O/N 30% O/N. when I put the bone in the sucrose solution, it sank immediately and did not float. When sectioning the bone detached from the oct immedialty when it touched the slide the best sections I had was with an unfixed and undecalcified bone, bet those sections were not good enough. I am only interested in the structure and direction of the collagen, using polscope, polarized light microscope. Thanks Efrat
“If you are interested in the collagen, why don't you decalcify? It is very difficult to get nice undecalcified sections without some sort of tape transfer, but is probably more doable with decal. It just takes longer and you have to fix the bones. I have attached a link that has some protocols too. http://bonebase.org/bonebase/”
Does anybody know an explicit reference where someone showed that systemic and local RANKL/OPG ratio can be different? I found a lot of reviews about the RANK/RANKL/OPG system but none of them answered these questions properly. Thanking you in anticipation, Anja.
“I can not answer this question Sorry, Piet Hein jongbloet”
- Ahed Jumah Alkhatib
I need to grind bone for initiating a new study. Who is familiar with theses techniques? what are the proper instruments? thanks in advance
“In this link you can find the same instrument many people have used to grind bone in order to get protein or RNA. https://www.fishersci.com/shop/products/kinematica-polytron-pt-1300d-homogenizers-2/p-327007 https://www.fishersci.com/shop/products/kinematica-polytron-pt-1300d-homogenizers-2/p-327007”
For numerical analysis of the double porosity consolidation model of cancellous bone, I need the following constants: Young’s modulus, Poisson’s ratio, fluid viscosity, macroscopic intrinsic permeabilities associated with matrix and fissure porosity, cross-coupling permeabilities for fluid flow at the interface between the matrix and fissure phases etc.
“Young;s Modulus: http://www.boneandjoint.org.uk/content/jbjsbr/84-B/6/900.full.pdf Poisson ratio, viscoelasticity: https://www.researchgate.net/publication/12420588_Critical_evaluation_of_known_bone_material_properties_to_realize_anisotropic_FE-simulation_of_the_proximal_femur Permeability: http://www.issres.net/journal/index.php/cfdl/article/viewFile/73/23 Young's modulus, poisson ratio, Permeability, porosity: http://www.sciencedirect.com/science/article/pii/S0895717709001216”
I have animal bones fixed in formalin , is it possible to do gene expression analysis - mRNA expression studies using these samples. ..
“Thank you for your suggestion..I have read about this kit... .Is it ok, if i try the kit for non paraffin embedded formalin fixed samples. Also please let me know the yield of RNA, from formalin fixed samples, because my bone samples have been in formalin for quite a long time. Formalin degrades RNA, so what all precautions should i take before I start the isolation. ”
We're trying to age Alpine tree frogs. We've decalcified in 10% formic acid for 24hrs, then cut 10um sections from the 3rd phalange and stained with meyers haemotoxylin for 1hr. We can see the lines of arrested growth (LAG's) but they are very faint. How could we make the LAG's darker and therefore easier to count? thanks!
“well, thanks, I do have written a few protocols myself (I wrote my post because I hadn't realized this question had been asked back in 2014...) but as I should start fairly soon a new skeletochronology project one more to read will be welcomed ;-)”
I just found out I have it. The Dr. States that if you find out you have this disease it is probably too late.
“I was also diagnosed with AVN of femoral heads and went thru a a double hip replacement in September of 2014 my left hip was detected and and no sign of AVN in right, but thru recovery my right hip hip began to display the same senstations that my left hip did, and was immediately taken back in for a THR with a massive amount of deterioration in only 5 months. Feb 2015. More than happy to share research and facts of my case. The AVN was attributed to 15 ESI's "Epideral Steroid Injections" using Corticosteroids The ESI procedures were given within a years timeframe in 2009-2010 with my PCP...” more
Concerns has been expressed about some unwanted effects that could develop after years of treatment with bisphosphonates . Please add your knowledge , opinion and experience .
“Once bisphosphonate therapy is started, it should continue for at least 3 years (in the absence of any adverse events. For alendronate, there is benefit in improving bone density and reducing vertebral fractures when treatment is continued for 10 years, and similarly for IV zoledronate for 6 years. The other bisphosphonates do not have such long-term data, both risedronate and ibnadronate do not suppress bone turnover markers for long after stopping treatment, and they can start to lose bone by 6 months of stopping. So, it is important to take into consideration which bisphosphonate the...” more
I'm trying to stain blood vessels in murine periosteum (whole mount), tried antibodies against CD31, SMA and lectin and in different bones (scal, iliac crest femur). The protocol works well but staining is not deep enough - about 15 um with formaldehyde fixation for 30 min incubation and ~35 um with a mixture of formaldehyde and glutaraldehyde. My goal is to image as deep as ~100 um (periosteum) with confocal microscope.
“Hi Dinesh. CD45 is pam-leukocyte marker. For neoangiogenesis you can use von Willebrand Factor antibody. I also used CD31 (endothelial marker) and lectin.”
I want to use FITC, which stain intestetial fluid to find osteocytes. Is there any way I could acid etch the surface and still make the stain work? The samples I am working with have already been embedded for some time and I am afraid that any method of staining will not work at all for the miniscule details I am trying to map.
“Looks like a very useful method. Thank you for sharing it.”
Which one is more capable in early detection of osteoporosis?
“Dear Dr Hui, Please let me copy to you the answer I have just given to a similar question asked by Dr Alkhaishani. "Osteoporosis" was first defined by Pathologists (prior to X-ray discovery) as just "the presence of an exaggerated number of pores in the bones" as a result of which the mineralized mass of the bones is impaired with no necessary alteration of its intrinsic "quality" (composition, mechanical properties, etc). Then, X rays allowed detection of that "excess of pores" by (subjectively) appreciating a reduction of the natural "radio-opaque" characteristic...” more
Kindly, explain your answer. Thanks in advance.
“The golden method to measure the percentage of calcium absorptions is to meaure calcium intake and fecal calcium excretion associated or not with calcium stable isotope intake. However, one can have a rapid idea about calcium absorption (comparative) by measuring 24 hours urinary calcium after ca intake.”
Or it´s only transitional stage of chondrocytes formation which is separated into a individual cell of chondrocyte later? And why should it separate, whereas it differentiates from a single chondroblast? Couldn´t it just differentiate from a single chondroblast? I mean, if it´s give rise to isogenic group, what´s the purpose of this formation? What it benefits? Why not stay still in form of an individual cell? Sorry to keep asking such basic stuffs that an undergraduate should´ve already known well.
“Several others, above, have responded to your question from the perspective of "how" cartilage differentiates and grows -- but not "why". I'd like, instead, to comment on the assumptions that seem to underlie the way you chose to articulate your question, i.e., that biological structure and function are consequences of design focused on serving a predetermined purpose. Needs and purposes cannot determine their own means of fulfillment. Such teleological thinking was abandoned by biologists very long ago. The only meaningful answer to "why ... ?" in biology is evolution. Cartilage...” more
In China, ITS+premix can not be imported because of mad cow disease, and other ITS from SIGMA or THERMO SCIENTIFIC manufacturers have different formulas. So could I directly make a recipe like:insulin (6.25ug/ml), transferrin (6.25ug/ml), selenous acid (6.25ug/ml), and linoleicacid (5.35ug/ml), with bovine serum albumin (1.25mg/ml))? or why people always bought ITS+premix instead of making ITS by themselves? I mean people write this formula in their papar, they know the formula, but they don't make ITS themselves. Are there something else behind the formula? I will really appreciate your... more
“Yes you can do it, we did it several times when the ITS+ was back ordered (which happens from time to time)”
Hey guys, I wanted to know if anyone has any suggestions. I am currently trying to help someone with IHC-P on rat tissue for a specific marker (RAGE) that is SUGGESTED to be on osteoclast membrane. So the tissue was fixed in 10% non-buffered formalin for 5 days, and decalcified in EDTA for over a month ( I am new to the lab, but formalin fiixation for over 48 hours is known to create strong crosslinks). Anyways, the student has tried multiple methods for antigen retrieval and point is- Heat retrieval does NOT work, the tissue just slides off. Is it possible to do a successful enzymatic... more
“Hey guys, so just wanted to update on the protocol for this in case anyone runs into the same issues We were able to successfully resolve the problem The antigen retrieval was done at 65C overnight ( 12 h) in a citrate buffer and then followed with the appropriate quenching of peroxidase activity, blocking, primary and secondary antibody incubations and Vectastain ABC developer kit While the leg of the bone ( Rat Tibia) was falling off slightly, the region of interest ( the growth plate of tibias head remained intact) Thanks”
I have seen papers which measure femur length etc. using whole body scans, however I am unable to locate any tools to do this during analyses. I have noted that these scans have been on an iDXA whereas I am using a Prodigy scanner. Is anyone aware of how to do this, or if this in an add-on to the software that will have to be purchased?
“Thank you for your help!”
I'm trying to do flow cytometric analysis of the inflammatory cell infiltrate in the arthritic joints in mice. I've been targeting the ankle and "wrist" joints. I've tried to wing it (flushing with 1 mL PBS/2 mM EDTA/10% FBS using a 25G needle) but the variability from mouse to mouse is very high. I haven't found an article that describes the precise methodology for flushing the joints for flow or cytokine analysis. Does anyone have a protocol they trust? With thanks, Michael
“Hey Mike :) What you describe is exactly what I did earlier - however I believe I went with 3 separate flushes rather than just one - typically helps to get everything out when you flush more than once (a bit like a BAL). I also remember using joints which were cut out right before and after the ankle so I had access to the joint quite easily and the liquid that came out was pretty clear. It was a bit messy but I got plenty of inflammatory cells and not much blood. The results we got then were pretty consistent. I'll send you the PDF by email. ”
Hi, I am working with isolation of primary osteoblasts from different sources to study their interaction with biomaterials. I recently isolated cells from neonatal mice (i day old) calvaria. I am not sure if they are preosteoblasts? and do they require ostegenic medium (i.e alpha MEM supplemented with ascorbic acid, glycerophosphate and dexamethasone) to become functionalised osteoblasts (which can form bone nodules)?
“wow, Thanks heaps for the information! ”
we are trying to differentiate MG-63 pre osteoblasts into osteocytes using the differentiaing media (containing Dexamethasone, . Sodium L - ascorbate and Beta-Glycerophosphate disodium salt hydrate) but the cells are detaching as they continuously proliferate. is their any solution for the same? we are doing it in 12 well plate, seeding 60,000 cells in each well. we are letting the cells grow in normal MEM media till 70% confluency, then replacing the media with the above mentioned differentiating media, and changing the media on every 2nd day. cells are suppose to stop proliferation and... more
“Maybe you can give us an idea? Safety of bioabsorbable implants in vitro Mehmet Isyar · Ibrahim Yilmaz · Gurdal Nusran · Olcay Guler · Sercan Yalçin · Mahir Mahirogullari ·”
I am trieng to preform a calorimetric TRAP assay in RAW264.7 cells using RANKL for differentiation. I plate 5000c/w in 96w plate using DMEM with 10% FCS as assay medium. Cells at passage 3-15. Nice differentiation with 50-100ng/ml RANKL after 4-5 days but high background in TRAP stain. Can anyone help?
“Did you know your microplate reader filters has an maximum and optimal wavelength? Because it is important when you have low amount of cells and weak enzymes activity. Filters that differ from the optimal wavelength more than +/- 20 nm could affect detecting the absorbance and or fluorescence emission of dyes and reagents, the further they are from the peak the weaker the signal will be, which can lead to higher background and noise.”
Hello everyone. We have FFPE decalcified mouse tibiae, and would like to stain for osteoid, woven and lamellar bone. Can you suggest a staining protocol that would enable us to distinguish between these types of tissue? Also, if anyone would be so kind to share a recipe, that would be appreciated. I did find a reference to a modified tetrachrome method by Ralis and Watkins, however, since I do not have access to their full text from 1992, I also have no recipe. Some help with this or another method would be great. Thanks in advance!
“Dear William Thank you for your answer. Impressive inadvertence, if I may say so! :)”
A student I mentor is planning on an experiment to check the role of natural ingredients in bone growth or restoration.
“Thanks Asiri wijienayaka. Can we do same procedure (that you mentioned) with an animal bone (pig?)?”
Dear all, I am running some test on human femoral heads using the hip simulator against different material cups to workout which material is the best, could you please tell me what is the best way to preserve the samples during the test, which serum works best? Most studies use bovine serum but I have real human samples and am not sure if I can use that. Also is it okay to run the samples for 1 million cycles (or more)? Is it necessary to adjust the environment temperature in order to avoid any damage to the samples? Wouldn't its properties change during the test? Are there any groups that... more
“Many years ago (more than 12) I had been in Switzerland in a hip simulating lab and they were using for the tests of ceramic on ceramic articulations bovine serum. Their argument was that it was cheap enough and easy to be found. Friction must be similar to human anyway. About the cycles you have to experiment on this. I believe that you have to careful as the heads may not have the same bone density and they may not all "afford" to be stressed with the same number of cycles. A bone density of each head may help you understand the limitations. Sorry that I cannot be more helpful. I wish...” more
I am culturing osteoclast from bone marrow. After flushing bone marrow, and culture overnight to get non-adherent cells. Treat the cells with M-CSF to expand cell number. But sometimes before the cells reaching 70% confluence, most of them become detached and dying. When RANKL is used, the cells become round without pseudopods. Also after differentiation using M-CSF and RANKL for 4 days, sometimes cells become detached. But sometimes I can get good osteoclasts. I don't know why. Does anyone ever experience such dying cells? I need a lot of cells, and I don't want them to be dying before... more
“I suggest fresh bone marrow not frozen. The cell density is crucial for this experiment. Try a few different densities in a 96 well plate”
I need a human osteoblast cell line for in vitro study of inflammation following pro-inflammatory and anti-inflamatory stimuli. Could someone suggest me a good cell line for studying inflammatory response?
“You could use either MG-63 cells or the SaOS-2 cell model. Both are easy to grow and keep their phenotype for a number of passages.”
I want model bone remodeling by titanium nano tubes in comsol multiphysics software, and I need to model osseointegration between bone regeneration and Implant contains TiO2 nano tubes. What is required boundary conditions?
“Thanks Oleksiy, I considered that bone structure in cortical and spongy bone with image processing in mimics software, then I extract cortical bone and spongy bone geometry from these model. afterward I insert cortical bone in Catia software and instead spongy bone, I modeled that spongy bone geometry to solid body which bone remodeling start from that. and next when I Imported this Geometries to Comsol multiphysics software I don't know what boundary conditions must give to this geometry (spongy bone which is modeled in solid body) that considered bone and Interaction Implant contain TiO2...” more
Can anyone please help me to know what volume of liquid alcoholic diet should be given per animal per day to induce osteopenia or to check the adverse effects of alcohol?
“Dear Monika, There are many alcohol-dependent rat strains so you can read how much alcohol these rats were given.”
Both PRP and bone marrow injection gives results in fracture nonunion. Which one is better for the old age with nonunited fracture??
“I am totally agree with all comments about the gold standard cancellous bone autograft. for several years I have more than one research about the use of Autogenous bone marrow Injections with very satisfied results. I will try to submit my work with its detailed results.”
Calcium citrate and calcium carbonate benefits in mechanism, kinetics, dynamics and patient related any benefits...
“Rupa, You may want to read this latest publication! Reid IR, Bristow SM, Bolland MJ (University of Auckland, Auckland; and Auckland District Health Board; Auckland, New Zealand). Calcium supplements: benefits and risks. (Review). J Intern Med 2015; 278: 354–368. Issue published online: 4 SEP 2015 Article first published online: 14 JUL 2015 http://onlinelibrary.wiley.com/enhanced/doi/10.1111/joim.12394/ http://onlinelibrary.wiley.com/doi/10.1111/joim.12394/pdf Abstract "Calcium is an essential element in the diet, but there is continuing controversy regarding its optimal intake, and its...” more
I am trying to obtain machined bone specimens for tension, compression or three-point bending test, but I have little idea how to obtain these specimens. Can anyone give detailed information about how to machine whole bone into regular bone specimens(see attachment)? Thanks.
“Veterinary Transplant Services is a professional, commercial animal tissue banking service that can provide bone (or other tissue) grafts processed to your customized specifications for research purposes. Everything from non-sterile benchwork to clean room-processed implantable graft materials from many animal models. Please contact us to discuss details of your project needs. You can view our website www.vtsonline.com or visit the link below.”
here is a pic - the area in question is at the arrow......ty all
“You have been presented with and article that give you directions by Milos Vater. We thank him for that. However, the mandible of the vertebrate animals have dicriptive features that are similar. That side is the vertical ramus of the mandible here with two notches for muscle attachments. You cannot immagine the ventral part of the mandible to be articulated to another bone for flexibility. I would rather say that circled part is the caudo-vetral ramal notch. Check out for its nomenclature and claim its copy right ownership. Check out for this "Caudofrontal depresion of olopade" and...” more
25 D, if low is associated with many illnesses, including osteoporosis. My question is respect to supplementation of a low 25D when there is a high 1,25D with high urinary Calcium excretion. High 1,25 D is known to be catabolic and increase Calcium resorption from bone. If 1,25D is high ( taken in dark, transported on ice) with excess urinary Ca loss in a patient with low 25D , what concerns are there to supplement 25D ?
“Thanks for your comment. Vitamin D deficiency can be an issue with older African Americans but not overly common. I am making house calls to older African Americans age 70 to 105 and only a very few of these women have sustained a minimal trauma fracture. Michael Kleerekoper”
I am trying to build a scalable (based on anthropometry) multi-body model of adult human leg and thigh. Femur and tibia are represented by a set of segments (3-4) connected together with torsional springs to capture the bending of bones in Medio lateral and anterio posterior mode. The model is mainly aimed to capture and assess the injury risk in the medial lateral bending in case of pedestrian to automotive vehicle crashes. 3 point bending data of bones is available and there is considerable variation among the responses owing to the difference in length, geometry and other biological... more
“I found the works from the following authors quite helpful as it deals with the data from cadaveric specimen or volunteers rather than archeological specimens. Miller et. al. 1980 (C) "The Geometrical Properties of Human Femur and Tibia " Minns et. al. 1975 (C) "The geometrical properties of the human tibia" Piziali et. al. 1976 (C) "An extended structural analysis of long bones--application to the human tibia" Capozza et. al. 2010 (V) "Structural analysis of the human tibia by tomographic (pQCT) serial scans" Cristofolini et. al. 2012 (C) "Shape and function of the diaphysis of the...” more
Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica.
“1 Dip the bone sample in propanol for 24 hours. 2 drain out remanants of alcohol. 3 Keep the sample at minus 20 degree centigrate for 7 days. 4 Macerate the sample in tissueliser for complete breaking of bone material. 5 Add DEB (500-500 micro litre) and protenase K 60-80 MICROLITRE,KEEP OVERNIGHT IN WATERBATH at 56 degree centigrate.Vortex intermitently. further follow extraction procedure till end. You will find this method better than others.”
I am wanting to freeze some whole bones with bone marrow intact so that I can isolate the bone marrow at a later stage for DC culture. Is this possible?
“I had the same question. I found a publication on PLoS one. Basically, 10% DMSO and 90% FBS turns the best number of viable cells after thawing http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015263”
In the secondary healing of bone fractures, is the formation of hyaline cartilage ideal for bone formation? What is the role of fibrous tissue and what are the possible reasons and consequences of improper tissue differentiation?
“In bone formation and bone healing process, The collagen fibril production in tension led to fibrous tissue, which could then differentiate to intramembranous bone while compression, led to cartilage formation, which could then differentiate to endochondral bone. In his conception bone could only form from cells protected from intermittent stretching by either the fibrous tissue or cartilage. Please check this reference : BONE DEVELOPMENT AND ITS RELATION TO FRACTURE REPAIR. THE ROLE OF MESENCHYMAL OSTEOBLASTS AND SURFACE OSTEOBLASTS. European cells and materials. Vol 15; 2008”
I just have started my project. My project focuses on the BMP-2 and VEGF role in osteogenesis and vascularization. I am doing survey looking for enzymes that cleaving proBMP-2 and proVEGF. I am interesting in finding the sequences that make BMP-2 and VEGF inactive initially and active ultimately by enzymes secreted from osteoprogenitor cells. If you could help me in that I will appreciate that
“Very nice question and idea:)”
If you do mma plastic bone for histomorphometry, do you use the same percentage of benzoyl peroxide se in last infiltration and embedding mixture? do you vacuum the bone after embedding?
“We used an additional gram of dried benzoyl peroxide (per 905mL of solution) in our embedding mixture than our last infiltrating mixture. Using a vacuum to eliminate bubbles is definitely necessary.”
Hello. I am having some experiment about differentiation periosteal-derived cells to osteoblasts in 2D culture. I cultured cells in OM (osteogenic medium) and additionaly certain amount of BMP-2 was added in medium. After 7, 14, 28 days I checked ALP activty and calcium deposition. ALP was increased until 14days, but calcium was not detected until 28 days. Plus I did RT-PCR, but late marker (osteocalcin, osteopontin) was not detected. Has anyone eperienced about osteogenic undifferentiation, please answer. Thank you for reading and sorry for my bad english.
“There are several things to be concerned. Are the cells freshly isolated and had no problem during culture? Some cells detach easily by mechanical stress. And if you lost cells during culture, you won't get much (or any) mineralized nodules. If you didn't see any calcium deposition, it may also difficult to see any upregulated OCN by RT-PCR. In my experiences (but not the same cell type as you have), 7-10 days culture for ALP and 14-20 days for calcium deposition. Additional BMP2 will boost osteogenic differentiation. You can test different concentration (usually 50ng/ml or 100ng/ml). ”
Patient might be on bisphosphonate which is associated with osteomyelitis of the jaw and delayed wound healing.
“Wound healing and bone healing does not delay but perioperative blood loss is increased and peroperative cell savers should be considered.”
Hi everyone. I am working with MC3T3 cell line and I tried to see the osteopontin levels in supernatant, 4,7 and 14 days after seeding, using elisa. the absorbance numbers are identical with the coating buffer.I used ascorbic acid and β-glycerolphosphate acid in order to differentiate pre-osteoblasts. I also used in cell elisa and the levels are surprisingly high, 8days after seeding. Did anyone know how can I solve this problem?
“Alizarin RedS staining is for checking the extent of mineralization. It mainly forms orange-red colour complex with Calcium, which is deposited during the mineralization process.”
Hi, I read the article Fluorescence of Bone, Nature 1965. And they talk about preparation of the bone that have been given in a preceding article. But they don't say which one. I think it's Photoelectric Effects in Human Bone, but I'm not sure. And besides I can't get that article from free. Can anyone help me?
“Can help you but literature is in russian. Need it?”
I´m working with osteoclasts, and once they come from macrophages, I need to add M-CSF in the media. But M-CSF by itself is very expensive and I got some L929, but I am a little bit lost about how to work with them. I saw some protocols that don´t do the selection step and some that do. Does anybody could help me? I´d appreciate a lot.
“Hi Marilia, here you are the protocol I'm using: Plate 4,7 105 L929 cells in a 75 cm2 flask containing 55ml of L929 medium (DMEM, 10% FBS, 1% HEPES, 1% P/S) Grow cells for 7 days Collect and filter supernatant. Store at -20°C. Good luck”
I would like to see where orthopaedic community stands with diagnosis of PJI and according to surgeons, what is the best diagnostic test when it comes to PJI?
“Early PJI have sign and symptoms like other site infections. But i agree with Dr Tanchev that in late onset infections and microorganisms with low pathogenesis clinical suspicion in patient with sustained pain is better”
Structural allograft is devitalised bone after all- why not keep the devitalised bone in Acute fractures (excluding infected non-unions) as bone graft rather than have a bone defect?
“Avascular contaminated small bone fragments, special cortical fragments in open fractures it is very important to be removed.”
It's also a very tricky scenario when deal with a moderate deficient acetabulum as for choosing the proper bone coverage and abduction of the cup in THA., in case of avoiding bone graf, does anyone have a practical technique and surgic philosophy in this aspect to share with me?
“One of the best ways is to use an allograft for reconstruction of the acetabulum roof with screws. Another method is to us a neck/head- component with a varus-constructed angle, so that lateral dislocation is not so easy. In several cases of recurrent dislocations I have used a thick resolvable cord with which the femoral neck/head is held medially in the cup. You may have to fix it through either the hip capsule or the lower acetabular rim. The result is, that dislocation cannot occur during the first few weeks during which the scars get mor tight to hold the head into the cup. For...” more
Many methods were used to induce bone by the use of PRP but the exact mechanism still unclear.
“Thank you Priya Rajendar for your information ”
Tarlov cysts--another cause of sacral insufficiency fractures?
“In spite of the "bone erosion" in case of a Tarlov cyst enlargement, pathological fractures of the sacrum are very rare, if ever.”