Wound Healing - Science topic

Good evening,

As far as I know, ESWT extracorporeal shock waves can improve wound healing, especially in diabetics. The ISMST (a learned society on ESWT) could provide you with more detailed information on this subject. ESWT are known to act on macrophages.

Kind regards.

Grégory VIEQUE

Dear Dr. Roghayeh Mokhtari Asl,

In the wound healing assay, the scratching process may involve mechanical injures to the cells at the wound edges. It exerts high shear from the wound edges influencing cell-cell as well as cell matrix interactions, where the fibroblasts need to re-establish through cell-surface interactions prior to accelerating migration into the defective cell-free area. Some damaged cells and cell debris can also get attached to the wound margins, perturbing the motility of other cells.  Additionally, the migrating surface can be damaged in the scraping process, leading to preferential paths in cell movements. The leading cells disrupt their site of adhesion, protrude, and contract the cytoskeleton to move.

So, you will observe some changes in cell morphology, but they maintain the original shape, with round pseudopodia, and the group of cells move forward uniformly making the wound margin look dim. Please note that depending on the cell type, they can migrate as individual cells (for instance, fibroblasts) or collectively as sheets of cells (for example, epithelial and endothelial cells). Usually, 8-24 hours is needed for cells to close the scratch depending upon the cell type used.

From the image it seems to me that the wound has not healed even at 24 hrs after the scratch.

I have a few questions.

1. Do you have a control image (cells without treatment)?

2. How do you introduce the scratch?

Usually, a scratch is created with a sharp object such as a pipette tip or a needle. The best way would be to use a 20ul tip. Keep it straight without twisting, applying constant pressure.

3. Is your treatment aimed to hasten or slow down the healing process?

Regards,

Malcolm Nobre

The saline swelling method is often more representative of wound healing applications, as wounds typically have a moist environment with exudates. This method better simulates the conditions a wound dressing or drug delivery system will encounter in situ, leading to more accurate release profiles for such applications.

The Franz diffusion cell could also be used if the study’s goal is to understand permeation through intact skin. However, for direct wound applications, the saline method aligns more closely with physiological conditions and may provide a more reliable indication of drug release behavior in an actual wound setting.

I am not entirely clear how you wish to use your 'drug'.

Assuming that it is a small molecule 'drug' you can use it to supplement any serum-free media formulation. We published a protocol for this 15 years ago: see doi: 10.1089/ten.tec.2007.0428. Lonza purchased the rights to our formulation, but buried it when they introduced their Serum-replacement formulation. They have since improved this further with their BulletKit® formulations.

Recommend that you dig into the literature from the mid-late '00s for protocols. Accurate formulation concentrations and absence/presence of drug-binding species is going to be critical. Make sure that it does not alter the pH, osmolarity, and osmolality!

Are you asking about the specific data you can obtain about the biocomposite itself? Or are you asking about how cryo-EM can be used to support the hypothesis that your biocomposite promotes wound healing?

For the first question, the results depend significantly on the type of biocomposite you are examining. Essentially, it can provide data about the size of the material. Additionally, it can offer insights into the material's crystallinity, enabling you to calculate all necessary crystallographic parameters.

For the second question, I doubt cryo-TEM would be useful. Wound healing is a complex process that occurs on a much larger scale. However, you might be able to confirm the binding of a wound healing agent to the composite.

Aagam Bamb

Instead of using a plastic petri dish, glass Petri dishes are a better alternative. If plastic ones are to be used, it is often suggested that you can coat them before parafilm or wax paper, if that doesn't interfere with your desired experiment.

Ensure that the TPP is being added slowly and under constant stirring to prevent clumping, or pre-dissolve TPP in a small amount of water before adding it to the main solution. This can help in achieving a more uniform distribution. If the Tpp particles are clumped up you can sonicate them before.

The freezing temperature you have used during lyophilization can cause the problem of less strength of the hydrogel sample; you could refer to these papers for better understanding.

Try using freezing, primary drying and secondary drying methods, if that doesn't affect the stability of your hydrogel.

1. Ionically Crosslinked Chitosan Membranes Used as Drug Carriers for Cancer Therapy Application DOI: 10.3390/ma11102051.

2. Preparation and characterization of macroporous chitosan-gelatin/beta-tricalcium phosphate composite scaffolds for bone tissue engineering DOI: 10.1002/jbm.a.10153.

Making a hydrogel is all about trial and error, and often, solute, if not dissolved properly, can hamper the formation; simultaneously, sudden temperature change also may affect the formation.

I hope this helps.

Manal, I suggest you check out the following review article and even read some of the articles sited within this review to start finding some of the answers you seek -

I hope this is helpful.

Hello Lidia Maeso,

You may perform wound healing assay using either established cell lines or primary cells.

When you perform such assay, it is important that you maintain

conditions as consistent as possible for a successful assay. For established cell lines, culturing protocols which include information such as the recommended growth medium, sub-culturing and expected doubling time are provided to you. But for primary cells, the protocols are often developed by the individual and are often more difficult to standardize.

I prefer using cell lines, though primary cells closely mimic the physiological state of cells in vivo and are expected to maintain their in vivo functions under optimal conditions for a short period of time.

Among cell lines, you could either use fibroblasts or keratinocytes. Both are

the major cell types that respond to the inflammatory phase in the cutaneous repair/regeneration process. Inflammatory signals activate the proliferation and maturation of these two cells types, which is essential for wound healing. Among the fibroblasts, you could use 3T3L1 cells and for keratinocytes you could use HaCaT cell line.

Best.

Alireza Moradi Chou-Yi Hsu Thankyou for your answers. This helped!

1. Prepare the Wound: Create the wound on the mouse skin using surgical procedures like excisional wounds or punch biopsies.

2. Apply Silicone: Carefully apply a thin layer of silicone material over the wound site, ensuring complete coverage.

3. Secure the Silicone: Use medical adhesive tapes or bandages to secure the silicone in place, gently pressing the edges to ensure adhesion without causing discomfort.

4. Monitor and Replace: Regularly monitor the wound healing progress and replace the silicone material as needed to maintain effectiveness.

5. Handle with Care: Handle the mice carefully to avoid dislodging or damaging the silicone barrier, minimizing stress or handling that could interfere with wound healing.

6. Ethical Considerations: Ensure compliance with ethical guidelines and regulations for animal research, obtaining necessary approvals and following institutional protocols.

Altought i didn't know about this until i read your question, i think that yes there are tetraspanin like markers in plants (alias TET), but not the CD markers we known in humans.

Some of these have structural similarity with the human ones, and that is why the antibody work. In the paper, probably dont known the right TET maker that is detected and they use the common term.

Could you please specify what type of tegaderm you used?

Flora Lise Nicholls Thank you for your advice!

Check out the attached articles.

Thank you a lot prof dr Sundus Hantoosh and for prof dr Stephan C for your recommendations of the question

But Dr Sundus is there is any study about the relation between debridement technique steps which include bizarre removal of dead burned tissues on subsequent cosmetic appearance ?

Hi

Try zinc oxide tape on shaved body.

In lab work or research studies focusing on wound healing tests, resveratrol is typically used in its purified form. The most common forms of resveratrol used for laboratory experiments are:

Resveratrol can be administered to cells or animals in various ways, including as part of a topical cream or ointment for local application to wounds, or through oral administration to study its systemic effects on wound healing.

When conducting lab experiments with resveratrol for wound healing tests, researchers should consider the appropriate dosage, treatment duration, and appropriate controls to ensure the validity of their results. It is also crucial to follow ethical guidelines and obtain necessary approvals when using animal models for wound healing research.

Gouranga Dutta

The color change you are observing in your hydrogel loaded with CuO (copper oxide) nanoparticles can be attributed to a phenomenon known as "surface plasmon resonance" (SPR). Surface plasmon resonance is the collective oscillation of electrons on the surface of a material in response to incident light.

In the case of copper oxide nanoparticles, their color is primarily determined by their size and shape, which influences the SPR phenomenon. Generally, smaller nanoparticles absorb light in the blue or ultraviolet (UV) range, resulting in blue color. In comparison, larger nanoparticles can absorb longer wavelength light, such as green or red, resulting in a corresponding color change.

Here's a possible explanation for the color shift you observed:

1. Ash Black: Initially, when the hydrogel is loaded with CuO nanoparticles, the color appears as ash black. This could be due to the agglomeration or clustering of the nanoparticles, which often occurs during the preparation or storage of nanoparticle dispersions. In this state, the particles may not exhibit a pronounced SPR effect.

2. Light Green: After a few days of storage, the nanoparticles may undergo some oxidation or transformation. This transformation can lead to changes in the size and shape of the nanoparticles, resulting in a shift of the SPR wavelength towards the green region of the spectrum. As a result, the hydrogel may appear light green.

3. Light Blue: As the storage time increases, further oxidation or transformation of the nanoparticles can occur, changing their size and shape. This change in size and shape may cause a shift in the SPR wavelength towards the blue region of the spectrum, resulting in a light blue coloration of the hydrogel.

Factors that could contribute to these color changes include exposure to air, moisture, temperature variations, and other storage conditions. The specific mechanisms responsible for the transformation and oxidation of the nanoparticles would require more detailed analysis and characterization.

To minimize these color changes, optimizing the synthesis and storage conditions of the CuO nanoparticles is importantaccumulationaggregationessential This might involve controlling the size and shape of the nanoparticles during synthesis, ensuring proper dispersion and stabilization of the nanoparticles in the hydrogel matrix, and implementing appropriate storage conditions to minimize oxidation and agglomeration.

It is also worth noting that while color changes may not necessarily impact the wound healing properties of the hydrogel, it is important to investigate and monitor any potential changes in the physicochemical properties and biological activity of the hydrogel over time to ensure its efficacy for wound healing applications.

Pls refer to the following link it has a detailed explanation:

It is by trial and error, if direct method with proper controls, like dmso 5-10%, dosent give reliable results, you use the pther metod

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

Hello Kunal Madhav

Why don't you contact

National Institute of Biologicals

Government of India Plot No. A-32, Sector-62 Institutional Area, NOIDA-201 309 (U.P.), INDIA.

They have a centralized facility for bioassays (CFB) to cater for the cell based bioassays for various recombinant products.

The CFB is equipped with workstations Class II type B2 Biosafety cabinets, centrifuges, CO2 incubators, refrigerators/freezers, microscopes, cell counters, cryopreservation equipment and multi-mode plate reader for detection mode of absorbance, luminescence, fluorescence, and time-resolved fluorescence (TRF) for cell-based immunogenicity assays and bioassays including Anti-Proliferation assays, Complement dependent cytotoxicity (CDC), Antibody dependent cell mediated cytotoxicity (ADCC), Apoptosis assays, Reporter gene assays and Neutralization assays.

They may be able to help you.

Please refer to the link provided below.

This is the closest place from Delhi that you could approach.

There is another lab at Bangalore, Bioneeds India Private Limited. Please refer to the link below for more information.

I can add another lab, Zelle Biotechnology Private Limited located at the following address:

A/7 MIDC, Mira Industrial Area, Western Express Highway, Mira Road,

Thane – 401104. Maharashtra.

Please refer to the link blow for more information.

I hope these labs will be helpful!

Good luck!

Low cost technique which can replace open ICG probes

it is possible to dissolve up to 20% (w/v) silk fibroin in Ajisawa's reagent, although the optimal concentration may vary depending on the specific application.

I appreciate your help !

(Human) dermal fibroblasts (HDF) originate from mesenchymal cells and are the main cell type present in skin connective tissue (dermis). They are located in particular, in the dermis and are the main actors of extracellular matrix (ECM) production and homeostasis. Fibroblasts interact with epidermal cells during hair development and in interfollicular skin. Moreover, they play an essential role during cutaneous wound healing and in bioengineering of skin.

In researches, Human Dermal Fibroblasts (NHDF) are isolated form the dermis of juvenile foreskin or adult skin from different locations like the face, the breasts, the abdomen, and the thighs. Those whoe are isolated from the foreskin are specifically labeled Human Foreskin Fibroblasts (HFF).

Such cells then undergo cryopreservation and can be used for wound healing studies and dermatological research to investigate diseases like scleroderma, fibrosarcoma, fibrosis, xeroderma pigmentosum, and histiocytoma. Moreover, fibroblasts are important for cancer research, tissue regeneration, and tissue engineering studies.

Hi Marjan,

To give you a little more context, we plate the cells and wait until they're confluent, then we do a scratch with a scraping tool. This is a 24 well plate that is then imaged under a microscope/incubator combo until the wound heals 100%, with images taken every 20 minutes.

What is abnormal is that sometimes the wound is very large and takes maybe 20 hours to heal and other times (on other days) the wound is small and takes maybe 12 hours to heal. Our treatment seems to be producing an effect in both scenarios when I plot the data but I don't know how to graph this data together or combine them as the timeframes are different.

You can use Dialysis membrane or bag for the drug release study.

I think you mean PRP for platelet rich plasma is this correct?

Hello Eunji Han

The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity. This colorimetric assay is based on the reduction of MTT to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduces the MTT to formazan. The insoluble formazan crystals are dissolved using a solubilization solution and the resulting-colored solution is quantified spectrophotometrically. The darker the solution, the greater the number of viable, metabolically active cells.

On the other hand, Wound Healing assay is a standard in vitro technique for probing collective cell migration. In this assay, a cell-free area is created in a confluent monolayer by physical exclusion or by removing the cells from the area through mechanical, thermal, or chemical damage. The exposure to the cell-free area induces the cells to migrate into the gap. In this assay it is essential to inhibit proliferation of cells because closure of the wound should happen only due to cell migration and not by cell proliferation. For this reason, the cells need to be starved for at least 16 hours before the scratch (wound) is created. You may provide the cells with 0.5% serum containing media which may help prevent cell death but at the same time inhibit cell proliferation.

So, MTT assay measures cell viability and is mostly used to check the cytotoxic effects of compounds under study. Wound healing assay measures the basic cell migration parameters such as speed, persistence, and polarity. Cells at the edge of the created wound polarize and migrate into the wound space. This assay does not require the use of specific chemo attractants or gradient chambers, and it generates a strong directional migratory response. It is most reliably analyzed when performed using time-lapse imaging, which can also yield valuable cell morphology/protein localization information.

In short, MTT assay checks cell viability and Wound Healing assay checks cell migration in terms of speed, persistence and polarity.

Best.

Dear Jun,

yes, there are methods.Just read the attached article.

regards

Horst Liebl

ahmed how are you?

Do you happen to have a picture to clarify better? there is a classification regarding the wound and the evolution of healing, but it would help me to answer you. last year i studied something very similar.(I'm not very good at english)

Acidic pH is helpful.

Dear Shaunak Raole

In addition to chemical signals, other factors act in the healing process. Cell types and cytokines regulate each stage of the wound healing response. ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblast growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HA, hyaluronic acid; IL, interleukin; MMP, matrix metalloproteinases; PDGF, platelet-derived growth factor; TGF, transforming growth factor; tPA, tissue plasminogen activator; uPA, urokinase-type plasminogen activator; VEGF, vascular endothelial growth factor.

I hope this helped you.

Regards,

As you suggested, pH would be my first thought. For glutamine, there will be significant degradation in 24hr at 37°C which would be accelerated in the presence of 10% FBS, but I don't think the effect would be that drastic. Anyway, the cells are cultured in the same medium at 37°C for days. You can run an experiment with control medium warmed at 37°C for 24hr outside of the incubator (without CO2) and compare with the medium pre-incubated inside the incubator (assuming it is not in an air-tight vessel/container).

Dear Noor,

Typically, the as-prepared hydrogels are dried before characterization (as you did). After the freeze-thaw process, it is also important to purify the hydrogel to remove non-crosslinked chains or other chemicals. The presence of liquid within the swollen hydrogel can lead you to the wrong interpretation of results collected from the experiments mentioned by you (swelling, water retention, etc.). Taking in mind that the drying process used by you (oven, vacuum, lyophilization, etc.) can also affect your results.

This paper can be useful: https://www.sciencedirect.com/science/article/abs/pii/S1226086X19303600

Regards, André

Sam Augustine Kandathil NO sir, I didn't used coated plates

Dear all, I think the use of plants and plants extracts in wound healing are not a matter of discussion nowdays. It is an old science, art, and tradition. Please tell if you are working on a special system plant/polymer for electrospinning. My Regards

10.5772/intechopen.80215

What does the outcome variable look like; what scale is it?

Since you only have 2 groups (normal healing, delayed healing), t-test makes more sense.

You could indeed use a paired test to compare the difference between the two wounds in each pig.

The data from day 15 and 20 are only useful to test a hypothesis if you have something to test it against. For instance, you could test if day 15 with delayed healing is still 'less healed' than normal day 10.

You could also find good reviews in PubMed etc.

For wound healing, can we use THP-1 cell lines.

For a new molecule, it is interesting to see first, a mathematical modeling for example with the equation of Pott and Guy (there are others but which do not provide more information) All the mathematical models are absolutely false, but they first allow us to verify whether the molecule has the potential to cross the skin barrier or if it is very limited. If your drug has a log P between 1 and 4 and a MW of less than 500, the amount absorbed may be sufficient. A peptide of high molecular weight will not cross the skin barrier, or so weakly that it will be of no benefit. You already have an idea if the molecule will be able to cross the barrier (the melting point, if it is a salified form, the formulation are other factors to take into consideration) If your molecule has a potential absorption, you can test in 2nd on "franz cells" human or porcine skin, otherwise it is a waste of time.

Greetings Shreshtha Gaur

There are good papers about the desired aim. I suggest you some below:

I hope have helped you.

Best Regards,

No.

We do this simply by using GraphPad Prism.

I think will be a cause for complications, as greater opening in anastomosis, sepsis.

I agree 100% with Dr. Laborde especially with the first of 14 responses.

Your question can be answered best by Prof Pierre Basmaji, a good friend of mine in Brazil

Kind regards

Horst Liebl

hl@el-cit. com

Hi there,

yes, you're in the right track...

As usual, it depends on what you want to measure. However, measuring the whole image would be quite meaningless.

In this case, what you say is that you want to measure the whole area, and to do so you should first define your whole area (of the sample). One of your channels (usually the one that is a counter-staining) should be the right one to do so.

So, assuming the blue one is this one for DAPI (maybe I am wrong but still ok for the workflow):

-Over your RGB image (btw, when I opened your .png is a kind of 8-bit color image so I converted it to RGB)

1-Image>Color>Split Channels

-Take the image that better represents the whole area of the image (I took blue, however since the conversion 8-bit color to RGB it may contain artifacts but yours will not if you have the three separated channels to start with)

2-Image>Duplicate

3-Image>Adjust>Threshold

Adjust it manually to cover the whole area of the sample

4-Process>Binary>Close

5-Process>Binary>Fill holes

Here you get a binary image representing the whole area of your sample

Then:

6-Analyze>Analyze Particles...

Here you can try to filter by size and circularity (I used 1000-Inf for size, to get rid of the small particles). Check the Add to manager option as indicated in the workflow picture.

7-In the ROI manager: More>Save

This will save as a .zip file the ROIs corresponding to your binary mask derived from the image corresponding to the whole area of your image.

Then click over ("activate") your channel of interest (red channel in this case)

8-Analyze>Set measurements (check what you want to measure)

9-ROI manager>More>Multi measure

And you will get a list of the measurements in the different ROIs.

That's to measure the whole-area immunoreactivity of you red channel (picture attached). You can try to write a macro for all these steps to save some time for further analysis.

There are also plugins to measure vascularity (I haven't used them though), to do it manually you should define what's a vessel in your red image and then count the number of vessels in the whole area of your sample. To do so, I'd basically operate the red image as it was done with the blue channel in the example until you have particles that are vessels. You will have to optimize the thresholding and other binary operations may not be required. Then, again, you can use the ROI manager to filter by size and circularity (and play with the options) to get a count of the number of vessels and express it as number of vessels per area unit in each one of your conditions,

Cheers,

J

Before doing the expressions studies, you will have to think,

Which genes?

Why these genes not others?

How many genes you will want?

Is there any relationship between these genes?

Are those genes part of the pathway?

Why that pathway?

Have those genes reported earlier?

If yes, collect references?

Have the primers for those genes reported earlier?

If no, design primers by yourself?

Record primers as much as possible?

Check these primers are for qPCR or simple PCR?

Select a reference gene?

Then perform expression analysis on the selected genes?

EVERYONE!! Or, at least, antiseptics.

You may be right. If you consider that healing is permanent and thus obviates the risk of subsequent infection, then his one month healing rate pertains. However, healing is not binary, because most wounds heal with a scar that is inferior in quality to native tissue. As a result, many wounds recur after appearing to be healed due to recurrent trauma or illness. Consider the fact that venous stasis ulcers of the ankle have a high relapse rate while an injury to the scalp heals rapidly after injury with minimal incidence of infection or recurrence. The main differentiating factor is the quality of blood flow and delivery of oxygen. Hypoxia is characteristic of the ankle due to its dependent position when we stand or sit.

A quick intuitive response to the question on whether PMA-differentiated THP-1 can migrate: No, as far as I know from imaging experiments. The cells become very large, spread out and don't look very active or motile. The differentiation primarily turns them to the gene expression profile needed to study macrophages, but that does not necessarily reconstitute the phenotype in vivo.

Questions about how to culture and differentiate THP-1 have been asked many times in this RG forum.

Briefly, the optimized dose is 5 ng/mL PMA. The exactly procedure can be found in this highly cited paper:

Previous Q&A: https://www.researchgate.net/post/I_have_got_new_cell_line_THP-1_can_anyone_tell_me_what_will_be_suitable_media_for_their_culture

Good luck!

Thank you Fauzi Mb sir for the kind information

Great option!

Nimal Raveendran Thank you Nimal sir for helping me with your wisdom

antioxidant

Thank you Shaymaa Fadhel for the kind information

Shaymaa Fadhel even with several measurements at different time points?

I think garlic (Allium sativum) is the best plant.

Sure lipofection may induce cell death -among other things - due to membrane stress.

Dear sir

Contact any pharmaceutical company manufacturing topical products.They formulation chemist can help and provide guidance. Try to contact GSK veterinary division.

I think yes every formulation should be tested because excipients will be different in each formulation and may lead to increased or decreased bioavailability.

Regards

please look at this link , it will help you (rat model not rabbit)

Please go through the following RG links and PDF attachment.

Anyone has recommendation for human Rage WT antibody?

Dear Sunilkumar Shukla

Check this

https://www.frontiersin.org/articles/10.3389/fmed.2019.00159/full

On the medical use of mummies, especially the powder called "Mumia vera" you can find a lot of information by googling "Mumia vera" o "Mummia vera" for instance: https://www.smithsonianmag.com/history/the-gruesome-history-of-eating-corpses-as-medicine-82360284/

Thank you so much.. it massively helps and supports our research study as well. Thank you Sir Vineet Sharma

Please see the following RG links.

I guess you need to do a pilot study.

W.

Sounds like a bad idea. Phenytoin is a small molecule, and would presumably get into the bloodstream or lymph when added to an open wound. As such, it could cause fibrosis at distant sites.

Let the body do it's natural healing, or use a wound-healing treatment that's local.

Also, by what mechanism does phenytoin cause fibrosis?

After documentation of the therapeutic effect on wound healing moddels

perfformed:

LD50

Local toxicological effects on the skin

effects on liver and renal functions

carcinogenic effect