Tissue Regeneration - Science topic

Thanks for your response, it really helped.

I guess partial decay of lymphocytes in blood sample (in vitro) makes plasma more effective. Cortisone or radiation are useful tools to provoke a decay. Thanks for question. See attachment, please.

Regards

2018; 22: 36.

Published online 2018 Dec 19. doi: 10.1186/s40824-018-0148-4

PMCID: PMC6299977

PMID: 30598836

Recent advances in stem cell therapeutics and tissue engineering strategies

Seong Gyu Kwon,1 Yang Woo Kwon,1 Tae Wook Lee,1 Gyu Tae Park,1 and Jae Ho Kim📷1,2

Author information Article notes Copyright and License information

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.

yes;

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,...).

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"

10% neutral buffered EDTA and 5% nitric acid are the best decalcifying agent for IHC staining

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

Calcium phosphate may be used as hydroxyapatite

Thanks Chandravati, your information are helpful.

Dear Nikolay, thanks a lot.

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Thank you for everyone for the valuable suggestions.

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

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.

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.

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 

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.

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

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

I think porcine will be better than rabbit for ACL reconstruction studies.

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!

dear Sir I hope you will get benifit from these papers:

regard

Integra is probably the best known. We have also developed a skin substitute which perfectly promotes skin regeneration in burns, called Suprathel:

Привет, Наташа! Конечно! Но как то нет ничего...

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.

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

http://www.drugbank.ca/ is a drug database that you can search for drug of interest on browse tab. 

I think you may inject SP after implantation, so it can specifically induce repair/regeneration processes.

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

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!

UV alone can also work, depending on thickness

Thank you for ur kind concern professor!!!

I mentioned Grafting in the sense??? like functionalisation??? coupling with other proteins or polymers by some chemistry??? 

For colon cancer cell line no need to inject with matrigel ,you can inject the cells only. 

find attached the article.

It may be usefull http://www.jendodon.com/article/S0099-2399(14)00962-5/abstract

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.

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

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.

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.

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.

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.

Osteonectin is a IHC marker for human osteoblasts. Maybe it's also reactive with rat osteoblasts.

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.

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.

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.

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!

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!!

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