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Tissue Regeneration - Science topic

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What are the different techniques used to investigate if the artificial skin tissue let say is similar to physiological tissue? Since the tissue generated might be similar in morphology, but what about the components of tissue, if those are required to be confirmed.
What are the ways to confirm those intrinsic molecules/parameters?
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Aggressing epidermis, there are many histological, protein and molecular markers of normal skin thay can be used to show structure and function of an artificial skin construct. These include layer specific keratins, intercellular junction associated proteins, pH and calcium gradient associated molecules, extracellular matrix. Histological sections (or flatmounts) and immunolabeling techniques are a good place to start. I'll attach articles using a number of the markers in use.
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Silk fibroin.
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yes;
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I want to calculate Cellular proliferation along with the population doubling time of MSCs cultured onto scaffolds for bone tissue regeneration. Can anyone suggest to me the required procedures and techniques for the aforementioned problem?
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Hey Sambit! It depends on the fact, which method is the most convinient one for you (and also available at the lab :D). You can for example directly count the cells (by staining their cell nuclei with PI or DAPI, taking the pictures from at least 10 fields of view via fluorescence microscope and manually or via software like MATLAB count them). The other option is to measure their metabolic activity via metabolic assay (MTT, CCK-8,...).
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Project is based on biodegradable materials.
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Actually in order to know that one of the important things is the properties of polymer you use. And as you said you are asking it for "bone regeneration" you cannot know it without doing preliminary experiments either in vitro or in vivo or both. Your question is is so wide and "unknown"
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Specifically, we are looking to decalcify a rabbit mandible. We will be staining for factors such as VEGF, BMP-2, TGF-beta1, etc. 
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10% neutral buffered EDTA and 5% nitric acid are the best decalcifying agent for IHC staining
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I am trying to 3d print this material for bone tissue regeneration.
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We are reputated supplier of Medical /pharmaceutical grade PLGA with attractive price.
Our capacity for PLGA production is 100kg per month.
Our price is 2540USD /1kg of PLGA
Ask for quotation
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I am doing my research on electrospun scaffolds for tissue regeneration and my team will have it them tested for biocompatibility and cell proliferation soon. I'm not yet sure which cell line will be used but I came across several papers that use cancer cells for such tests. I don't have a background on cell testing but I do know that cancer cells can proliferate indefinitely in a culture without the need for additional growth factors unlike normal cells which makes things easier. But how can it provide evidence of compatibility when my application is solely for normal cells (i.e. scaffolds for skin cell regen.)? I mean will the results be the same for normal version of the cell?
Please enlighten me about this. Thank you
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Dear Jean Raynell Bello
Cancer cells are immortal. Therefore, their use is unsuitable for assessing biocompatibility. According to the international standard, fibroblast cells are used for this task such as L929, SNL and etc. All biological tests must conduct according to ISO standards 10,993–5:1999 (Biological evaluation of medical devices; Part 5: tests for in vitro cytotoxicity). You can read the following articles:
Flexible magnetic polyurethane/Fe2O3 nanoparticles as organic-inorganic nanocomposites for biomedical applications: properties and cell behavior
Preparation and evaluation of polyurethane/cellulose nanowhisker bimodal foam nanocomposites for osteogenic differentiation of hMSCs
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Calcium phosphate (Ca-P) ceramics are extensively regarded as excellent bone grafts due to their good biocompatibility, osteoconductivity is it possible to endow biomaterials with osteoinductive ability by optimizing the material characteristics rather than by adding living cells or growth factors to induce tissue regeneration.
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Calcium phosphate may be used as hydroxyapatite
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if an osteoconductive bone graft is mixed with platelet rich fibrin does it come under the category of osteopromotive bone grafts ?
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No osteoinduction.
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I have a project on tissue regeneration by a medicinal plant, in experimental skin damage.
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Dear Nikolay, thanks a lot.
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I checked some papers on CNCs based materials for tissue engineering, but less of these papers shown in vivo results.
Someone told me there is a review paper talk about the problem, but I cannot find it.....
Anyone read it before or have some information on this question?
Thanks!!!
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CNC in general may trigger oxidative stress and result in tissue damage or inflammation,
You may check these reviews
The Potential of Cellulose Nanocrystals in Tissue Engineering Strategies
Biomacromolecules, 2014, 15 (7), pp 2327–2346
DOI: 10.1021/bm500524s
Bionanocomposites from lignocellulosic resources: Properties, applications and future trends for their use in the biomedical field
Progress in Polymer Science (2013), 38 (10-11), 1415-1441CODEN: PRPSB8; ISSN:0079-6700. (Elsevier Ltd.)
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I make a defect in the rat bone and i want to put stem cell in this defect as a topical application but i dont know how to implant cells without losing in blood stream ( how to inject the bone )
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s
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I want to fabricate hydroxyapatite hydrogel scaffold for bone tissue regeneration purpose.
I want to know the suitable cross linking agent. will poly e-caprolactone useful on it.
Any researcher who working on this area pls give me appropriate protocol papers and idea
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Thank you for everyone for the valuable suggestions.
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Advanced Platelet-Rich Fibrin (A-PRF) - A new concept for cell-based tissue
engineering by means of inflammatory cells (rpm 1500, 14 minutes) 
this changes the distribution of inflammatory cells through the clot and may influence bone and soft tissue regeneration. Is this advancement may add a benefit in development of immature teeth?
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Hi, until Jan 2016 it has not been used.
Del Fabbro M, Lolato A, Bucchi C, Taschieri S, Weinstein RL. Autologous
Platelet Concentrates for Pulp and Dentin Regeneration: A Literature Review of
Animal Studies. J Endod. 2016 Feb;42(2):250-7.
Lolato A, Bucchi C, Taschieri S, Kabbaney AE, Fabbro MD. Platelet concentratesfor revitalization of immature necrotic teeth: a systematic review of the
clinical studies. Platelets. 2016 Jul;27(5):383-92
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I wish to do the histological analysis of my silk sponges. For that I need to slice my scaffold through microtome but I am not getting the results. My protocol is like i put the scaffold in 4% formalin fro 24 hours, then pass through ethanol grades to dehydrate, transfer to xylene and embed in paraffin. But when i slice it through microtome so it slice the paraffin part but is unable to slice the scaffold part. Kindly help me out that what's the issue and what i am missing? Picture of the sliced scaffold is attached where the mid round unsliced area is the scaffold. 
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ok.Your material is inside of a histological cassette? If your answer is yes. You must leave at least 50 minutes in 90% ethanol and more 3 times of 50 minutes each in pure ethanol (absolute ethanol P.A.). After this, you must put your cassettes in xylene (absolute = pure) at the same time (50 minutes) in 3 differents xylenes (named I, II and III respectively) as you did with the ethanol. Then, you must put you material inside the hot wax (65 Celsius degrees ) in 3 differents pure wax (named I,II and III), for 60 minutes each. Only after this steps you "block" the material. Before slice you must put the block in the refrigerator for 50 minutes, to cool the wax.
Your material is with holes because you didn't left the time enough in each solution.
Try this protocol, I think that will work. But if there are salts or sand  within your material you will not be able to slice. You have to be sure that all salt/ sand was removed before.
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Can anyone please provide me any reading material/links to articles that explain use of the extracted tooth as autogenous bone graft?
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Fully formed adult teeth do indeed provide a rich source of cells for a variety of graft/tissue engineering procedures including a variety of stem cells from cementum and PDL, as well as pulp cells and some from developing stages of teeth, too. As regards autogenous bone graft, yes the tooth enamel is essentially hydroxyapatite (HAp) and the same chemical composition as that of bone. However, please note, the crystallinity and arrangement/packing of the HAp crystals is different in a tooth compared to bone HAp, and this may dictate optimal use of such a graft.
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Most of the naturally available biodegradable materials such as agarose, chitin, chitosan, collagen, hyaluronan, gelatin etc. have already been explored. What else can I try? How about pectin?
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Dear Saha, 
Skin regeneration requires scaffolds of well defined characteristics, in terms of tensile properties, micro-structure and morphology, surface modifications by specific cell adhesive proteins, etc. The choice of the polymer is not enough to let you conclude that it works or not, unless you explore the impact of the aforementioned characteristics which I listed above. The micro-fabrication of your scaffold impact the biological performance, the internal structure of the scaffold counts. How did you produce your scaffolds? thin films; non woven mesh, oriented porous scaffolds, filaments deposition? did you explored the impact of these parameters using ONE promising polymer, as gelatin or collagen for example?
I think that the development of scaffold for skin regeneration goes beyond the simple screening of polymers.
Bests,
Irini Gerges, PhD 
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Dear Researchers,
    Has anybody tried protoplast culture and got plant regeneration from that? I want to try it in cotton if possible. Experts please share your experiences and ideas. Thanks in advance.
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Yes, I have done that on tobacco. See attached paper. Pictures of the results from different steps are shown in p.160 of the paper. However, I have not tried that on cotton. You can use the paper as your reference.
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How can one analyse the results of a wound healing assay using Wimasis WimScratch software? 
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Dear Vincent,
you can see a detailed description of how to interpret the results in our website, in the following link:
Thanks for your interest
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I want to know a protocol for making fibrin,but in my research i can't access to it yet.can you help me to find that?
thanks a lot
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Dear Zahra,
Thank you for your thank you.  I am glad that my answer was quite useful to you.  Please keep up the good work.
Omer
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Want to study graft uptake biology?
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I think porcine will be better than rabbit for ACL reconstruction studies.
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Regarding fistula-in-ano we have all been traditionally taught that it is imperative to curette out the epithelial lining of the tract and granulation tissue, to prevent recurrence (as part of an advancement flap/LIFT/fistulotomy etc).
I am interested to know that exact pathophysiological mechanism by which the partial epithelialisation of the tract (esp at the internal opening) impairs wound healing. 
More so, I am keen to know the mechanism by which the fistula tract closes if we are removing granulation tissue (the primary vascular tissue of healing)? I accept that epithelialisation at the internal opening or granulation tissue can cause obstruction which then continues the vicious cycle of obstruction/discharge etc - but this doesn't explain how it it will eventually heal i.e. what replaces it that doesn't obstruct the tract?
The evidence is a little mixed and I wonder what people think? Seems like a paradox - removing granulation tissue to then let the wound heal secondarily without problem?
Many thanks,
JB.
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The pathophysiology has not changed, nor is it unusual, particularly in any focus of chronic granulation. It relates to the quality of the granulation tissue I.e. The amount of oxygen it provides and carbon dioxide it removes which play a major role in all healing. So, in chronic inflammations - particularly granulomatous ones where the inflammatory response is not a hyperaemic one - the granulation tissue itself becomes a source of infection. We all know the reasons that a fistula remains patent - ongoing sepsis, particularly granulating variety, foreign body/neoplasia/ epithelialisation of the track I.e. Anything that prevents enough oxygen reaching the ulcer or causes a physical obstruction. hence, removing ineffective granulation tissue is a necessary first step when attempting to get a track to heal. In fact, in a couple of diabetic patients with nasty ccomplex pilonidal sinuses around the natal cleft, I have curetted them out till they bleed well, have then passed a wick through, and have got them to heal with a few change of dressings, without any excisions or flaps. I had planned to plug both, but this was not required eventually. Turns out that vascularity is important in surgery - who would've thought!
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I tried to encapsulate hMSC into PEG-based Maleimide-Thiol hydrogels. However, I could not see any alive cells after preparing the 10% gels (gelation within 2-5 min). 
Could anyone have any idea or reference about cell viability of encapsulated cells within PEG-based Maleimide-Thiol hydrogels?
Thank you.
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Looking for best scaffold for skin cell regeneration
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Integra is probably the best known. We have also developed a skin substitute which perfectly promotes skin regeneration in burns, called Suprathel:
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Dear friends, please help me to find a reference to the healing of the bone tissue of the skull. Dates trepanation healing I need. Thank you!
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Привет, Наташа! Конечно! Но как то нет ничего...
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Dear users,
I would like to use survival models to test differences in the total time (in days) for tissue regeneration from a specific type of wound. 
In this case the event would be 0 (not fully regenerated) and 1 (fully regenerated). The problem is that I know the interval in which regeneration occurs, but in some situations I am sure that regeneration occurred much earlier than observed.
For example, if I check an individual 800 days after the wound was first observed, it will be fully regenerated but regeneration happened before day 800. However, (I think) survival models will consider that regeneration happened at day 800. 
How should I proceed? In my case study, all individuals observed after 250 days had their wounds fully regenerated. I thought that maybe I could truncate days after 250, but there's no way I can tell for sure that all individuals were fully regenerated after that period, so probably this is not the most correct approach.
I also tried to use left censored intervals, where the lower limit would be 0 (the time at which wound is first observed) and the upper limit the time when regeneration is observed).
Any advice?
Thank you in advance
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1)What is your criterium for full regeneration or what is the general accepted critrium (nice is criteria).
2) Whether you like or not, in regeneration research steps of checking should be as small as possible.
Thus recalculating via a model is a bad idea, unless you know the time behavior of the criteria.
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Do children have the ability to regrow their fingertips? If they do, up to what level?
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Depending of the age the small defect of the skin can be healed nicely but as already mentioned in case of bone loss the loss is permanent
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Hi
I need to select some drugs that are important for regeneration and repair. Is there any database that helps me to select drugs based on this criterion?
Best,
Maryam
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http://www.drugbank.ca/ is a drug database that you can search for drug of interest on browse tab. 
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i am working on rat model of in situ tissue regeneration. do i have to inject substance P systemically before or after implant placement?
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I think you may inject SP after implantation, so it can specifically induce repair/regeneration processes.
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Hello! I am using Platelet Rich plasma in different concentrations for tissue regeneration, in-vitro. The media turns into a thin gel with cells embedded in it. Can anyone kindly suggest , how to avoid his. And if it has happened then how to retrieve the cells from the culture ?Thanks for the support.
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Hi Priya,
You can avoid gelification of platelet rich plasma by adding heparin (I use 2U/ml) to PRP before its contact with culture medium (calcium from culture medium is what causes clotting).
I haven´t been able to retrieve cells from the fibrin clot, but I have successfully extracted RNA from cells embeded in the PRP clot using Trizol. I also have performed other techniques such as immunofluorescence directly in the clot.
Hope my answer can be of any help. If you need anything  do not hesitate to contact me.
Kindly,
Eva
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I'm trying to develop an alternative assay for tissue collagen based on previous and commercially available kits. The protocols do not give the formula for the extraction buffer.
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Dear Heins,
to quantitate collagen in cell cultures, 0.1 M NaOH was used to extract picrosirius red to be read at 540 nm in a spectrophotometer. I did not test this staining on collagen-rich tissue, but, according to Sircol instructions, 1N NaOH has to be used. I think you have to test your experimental system to find what is the best. Good work!
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I'm currently producing thin film bio-composite membrane. Before doing the cell culture for my membrane, I need to sterilize it first, thus I would like a suggestion on the best/ simplest sterilization method for the bio-composite membrane that can be done in lab besides gamma irradiation since it is not available in my uni.
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UV alone can also work, depending on thickness
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I have seen various articles regarding the major amino acid source is glycine and alanine in case of silk fibroin from B.mori. I would like to know about the possibility of the residual functional groups in the fictionalization of our fibroin with other polymers? Are there any ways?
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Thank you for ur kind concern professor!!!
I mentioned Grafting in the sense??? like functionalisation??? coupling with other proteins or polymers by some chemistry??? 
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I am looking for a good relaible and efficient software to analyse wound healing assay. I found one of them is the TScratch Assay. 
Please whoever is expert on this help me get the software and protocol to use this.
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Does anyone have a suggestion for a proper housekeeping protein that IS NOT cleaved in necrotic tissue AND its levels are more or less constant during wound healing/tissue regeneration process?
I noticed that GAPDH and alpha-Tubulin are rapidly degraded in the samples where the caspase activity is high. Beta-Actin is more stable, but it is also a target of caspases, and it is upregulated during wound healing stages. I would appreciate any feedback from those who work with animal tissues.
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Could thioredoxin pathways shed any light? also acts as a signalling pathway for ROS levels
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I am planing to subcutaneously implant cells into mice using Matrigel. Does anyone have any guidance regarding the concentration on Matrigel to be used with cells, or indeed any tips on using Matrigel. Any help would be greatly appreciated by this first timer to Matrigel. 
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For colon cancer cell line no need to inject with matrigel ,you can inject the cells only. 
find attached the article.
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Enamel or Blood?
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Formaldehyde fixation and EDTA-G decalcification are quite popular methods , however, I got a bad experience with this protocol. Anyone has a successful routine method to fix and decalcified bone samples for immunohistochemistry?
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As Nickolay says, the final test is X-ray or calcium in the bone.
According to my experience 7 days are enough for rat long bones. Simple test is just twisting the bone. If it is flexible it is decalcified. However, fix them well before decalcification.
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Acemannan is an extract from Aloe vera
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Chantarawaratit P, Sangvanich P, Banlunara W, Soontornvipart K, Thunyakitpisal P. Acemannan sponges stimulate alveolar bone, cementum and periodontal ligament regeneration in a canine class II furcation defect model. Journal of Periodontal Research. 2014;49(2):164-178
Other studies are also available in the llt. mostly animal experiments
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The chicken eggshell and its membranes are an inexpensive and abundant waste material which exhibit interesting characteristics for many potential applications. The eggshell is formed mainly of calcium carbonate (CaCO3) and is used widely as an animal feed, lime (Ca(OH)2) substitute or a fertilizer. Moreover, the associated eggshell membranes have a high content of bioactive components, as well as properties of moisture retention and biodegradability which have potential use for clinical, cosmetic, nutraceutical and nanotechnology applications. The eggshell membranes have been also used for biosorption of heavy metals and dyes and as a template to synthesize metal nanoparticles. The combination of nanosized calcium phosphate (Ca3(PO4)2) biomaterials synthesized from eggshell and eggshell membrane show promise to develop drug delivery system and nanowires for electronic devices. In addition, a derived product, the soluble eggshell membrane protein (SEP) has applications in tissue engineering.
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An interesting and logical idea. Some herbal medical experts recommend ground egg-shell as a dietary supplement for the excellent bone minerals and other proteins required for healthy cell function and bone physiology. They, do however, insist on utilising eggs produced by organic-bred chickens to avoid some of the antibiotics and synthetic supplements given to farmed/caged chickens, and to boil them first prior to use.
Chemically the mineral of human bone is a composite of (essentially) calcium phosphate whereas the main component of egg shell is calcium carbonate. There, will, of course be structural, chemical and mineral phase differences between the two, but I see no reason why egg-shell proper and it's associated proteins from membranes should not be considered for synthesising artificial tooth or bone composite materials for tissue repair/regeneration.
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Over the past three decades great strides have been made in the field of periodontal regeneration. Reviews of the literature identify many surgical techniques and materials that have been used successfully to obtain new clinical and histological attachment. Although to date the goal of complete, predictable regeneration has not been attained, the literature has clearly demonstrated the clinical feasibility and histological possibility of periodontal regeneration with many of the procedures. But now the question arises: are those true?
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Dear Saurav
Now there are lot of techniques to assess the regeneration,among which MRI cell viability test which uses gadolinium can tag regenerated tissue.
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There are different polyols for fabrication of biodegradable scaffolds. What parameters are important for selection of a specific polyol? Which polyol is the best candidate for both in vitro and in vivo applications?
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Sincerely dear Dr. Matar
I believe that the Science has gained its progress with respect to the great efforts of honourable researchers like you. Surely, devoting time for the synthesis of novel polyesters with unique mechanical properties is worthwhile.
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The cell proliferation capacity of a cell line is rendered by many factors in in-vivo conditions in comparison to in-vitro.
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I agree that greater cell numbers are needed for in vivo applications than in vitro. When cells are implanted in vivo, a significant portion (I believe over 75%) of implanted cells will die, so the larger cell number allows for a greater population on the scaffold than a lower cell number that might be suitable for in vitro.
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I would like to evaluate new bone formation in response to a biomaterial. TRAP (Sigma kit) seems to be a good option for osteoclasts but I am not finding a histochemistry method for osteoblasts (such as ALP). Should I perform immunohistochemistry for ALP? Has someone experience with any antibody?
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Osteonectin is a IHC marker for human osteoblasts. Maybe it's also reactive with rat osteoblasts.
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We are working on an osseointegration project of a new resorbable biomaterial. We implant the biomaterial samples in rabbit condyle and we need to evaluate the local biological effects after implantation following ISO 10993-6 standard (Annex E). We work with undecalcified bone samples embedded in PMMA and cut them with a microtome at 5-10 microns. We would appreciate if you could help us to select the best staining technique/s to identify different cell types/responses: polymorphonuclear cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis, neovascularisation, fibrosis, and fatty infiltrate.
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You can do it if you use a tungsten carbide knive and a motorised microtome. We usually obtain 5-7 microns slice. Diamond saw is needed if your samples contain metal implants and, sometimes, when a TCP scaffold is implanted.
It is very important to use an adequate tissue processing procedure to get good PMMA infiltrated samples. In our lab we usually cut bone samples up to 2 cm diameter.
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I am starting C. elegans research and would like to source some OP50.
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Hi Dan,
Have a look at the CGC (http://www.cbs.umn.edu/research/resources/cgc). They provide lots of nematode strains, but also E. coli OP50 at low costs.
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During cell culture on scaffolds, most of the cells migrate to the bottom and attach to the bottom of the tissue culture plate. How can one prevent this? I have read about a method which calls for coating the well bottom with 2% agarose, is this a reliable method?
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1. Use low-attachment dish for cell seeding. Normal petri dish (ie., non-treated dish) will also do.
2. The volume of cell suspension for seeding should be no greater than the volume of the scaffold to be seeded. For instance, 80 ul suspension would be OK for a scaffold of 100 mm^3.
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Which one will be provide more reliable results and minimal errors during procedure?
I need to perform either of these to show there is increased protein/mRNA expression because of scaffold which I incorporated in rat skull bone.
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Hi,
qPCR will give you quantitative data (if designed properly, it is very robust, especially when comparisons are needed), and this is extremely important, as most biological analyses are only semiquantitative, if at all. But qPCR is not enough, as you don't know how the protein behaves, so next thing will be doing Western, that will give you semiquantitative data on the protein of interest. I think immuhistochemistry is the least reliable method, the interpretation of the results could be subjective, and besides nice pictures, in many cases it will not give you really useful data, even w/o going into deeps like rounds of optimizations that could be required to get optimal staining (such as fixation procedures, antigen demasking, ab concentrations etc.). Good luck!
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The preparation conditions will vary dependent on the antibody you are looking at, which may require some optimisation in terms of antigen retrieval steps or the fixation of the tissue. Can I ask if your slides were from tissue fixed (in say formalsaline) and wax embedded, or were cut as frozen sections?
A good starting point for fixed and wax embedded sections is to dewax with Zylene for 20 mins, followed by washing with graded ethanols (100%, 100%, 90%, 70%, 50% ) for 2 mins each and into water. An antigen retrieval of heating in citrate buffer (I use 0.1M at ph6) is probably the most commonly used, and the one I would try. Most of my antibodies work on skin tissue with citrate retrieval heating on full power in a microwave for 2-3 mins, and leaving to cool for a further 10 mins. Alternative retrieval methods using enzymes such as proteinase, EDTA or acids are possible. After the retrieval you follow the instructions for whatever kit you are using - I find the vectorstain ones are good. You would have to just try and see what works for your particular experiment.
Good luck!!
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For several years we are investigating hyaluronic acid and related polysaccharides with the aim to develop modern scaffolds for the treatment of poorly healing wounds. For the continuation of this work we are looking for cooperation partners in academic institutions or pharmaceutical industry.
The first step should be the development of a research concept to find a sponsoring with public funds followed by the development of a research project.
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We are working in the field of scaffolds for tissue engineering via non conventional fabrication techniques (solid freeform manufacturing a.k.a. rapid prototyping) since several years. So far we've been looking at the properties of scaffolds made of PCL and hydroxyapatite. In any case, we will be very interested in a cooperation in the proposed field. Regards Alida