Kindly extend the professional expertise if you have any idea of laboratory evaluation for wound healing activity of a drug, drug product, plant extract, herbal formulation etc, without using Laboratory animals?
Drug development research on wound repair is challenging and healing depends on the complex nature of wound and scarring processes and many biological models are available in vitro or in vivo are used for preclinical drug testing (including cited by Dr.Rob Streeper ).Based on type and nature of wound healing effect aimed ,you can try the effects of compounds on their efficacy primarily as anti-infective/ antimicrobial agents, their anti-inflammatory effect, anti -histaminic effect (anti-bradykinin effect for burn wounds)also.
Yes, there are several in vitro models and i want to refer you to some publications which can help
Mensah, A. Y., Sampson, J., Houghton, P. J., Hylands, P. J., Westbrook, J., Dunn, M., Hughes, M. A. and Cherry, G. W. (2001). Effects of Buddleja globosa leaf and its constituents relevant to wound healing. Journal of Ethnopharmacology 77, 219-226
Wound healing potential of methanol extract of Spathodea campanulata stem bark forumlated into a topical preparation
K Ofori-Kwakye, AA Kwapong, MT Bayor - African Journal of Traditional, …, 2011 - ajol.in
Chick Choreoalantoic membrane model of angiogenesis is used as in vitro model for wound healing...
Scrape wound models of cultured epithelial cells (cell line or primary) are used in respiratory research as a model of wound healing. I guess it depends what organ you are interested in as to the appropriate model of epithelial / keratinocyte wounding.
yes, agree with John Holloway . Make lines with a pippete tip in a full confluent monolayer cells. and evaluate the gap distance under microscope. Easy
Using time lapse microcopy allows you to quantify rate of wound healing e.g. doi: 10.1152/ajplung.00255.2002
Since Angiogenesis plays the most prominent role, there are a few assays named - Matri Gel Assay (very simple, quick and easy to analyze), Aortic explants assay (ex-vivo model) but the most robust one.
Different in vitro models, based on co-culturing techniques, can be used to investigate
the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists
to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions.
In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human
dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells).
Dear Nikhil,
Jill Trendel is right, scratch assays are the simplest and less expensive to run. Please consult the detailed method published in 'Nature Protocols' (you can obtain it from http://web.mnstate.edu/provost/Scratch%20assay%20from%20Nature.pdf) For wound healing you would like to use keratinocytes (see attached paper from PLOS) . Moreover, it is regarded as a quite good approximation to cancer as the molecular mechanisms governing the bidimensional migration of cancer cells lines in this setup are quite dependent in a relevant set of genes (see http://www.biofocus.com/_downloads/publications/2009/biotech-international.pdf) However, scratching the plate with a sterile pipette tip, would lead to quite a variable "wound" area, and the resulting shape is quite difficult to measure (time consuming!!!).
If you want to automatise it follow these instructions: Invest some money in an ORIS PLATYPUS kit (look at the video http://www.platypustech.com/discoverAssay.html). It consists on a series of inserts that do not allow the cells to grow in the middle of the microwell, and the areas are very consistent accross the plate. We recycle the inserts by Washing/sterilizing them in the laminar flow cabinet with sterile PBS and EtOH 70%, in the latest solvent we store them between assays. They fit quite well in other standard microplates so we use them again and again.(rinse well with sterile PBS before each assay!)
We stain the cells with trypan blue (inexpensive) and scan the microplates in a Fujitsu SnapScan (any other high quality scan would do but NEED TO BE ABLE TO SCAN USING TRANSPARENCY MODE (LIGHT coming FROM TOP TO BOTTOM) not the usual reflective scan.
Then we process the images with the aid of Photoshop. Use the Magic Wand tool to click on the empty central area, the software will select the whole cell free area and give its measure in pixels. Afterwards it is a matter of statistical work.
We published recently some data with this technique in Natural product communications. 01/2012; 7(6):757-760.
I hope these directions gives you a nice starting point for your experiments.
Feel free to ask me for more details if needed. Yours. Jose
We are using a scratch test assay on cells which is widely described in many research paper. However, you need to make sure that the scratch is standardised. I suppose that would be the tricky bit
Skin cell formation and proliferation, and burn wounds using elecron microscopy
As mentioned above, scratch assays sound like the best starting experiment. Practice on monolayers until you produce a consistent scratch first. Put marks on the plate to make sure you're taking the photo in the same place over time. Avoid inserts as the cells are not 'wounded' but rather didn't grow in the area of interest - in our hands the results you obtain can differ when the cells are wounded vs. not wounded.
You're right Ashley: wounded vs. not wounded will certainly differ, very good point. For my cancer studies I do not need the wound, but to open a new space to the cells.
Yes, you can use cell culture model for wound healing activity of a drug, drug product, plant extract, herbal formulation etc, without using Laboratory animals.
Hi Dr. Nikhil,
I have attached a publication and hope it will be helpful.
regards
Amarnath Pisipati
Highly technical question. the effectiveness of wound healing property of any drug varies from individual to individual. Cell Culture test may help us to understand the effect of the drug, but the measurement of wound healing rate is a tough task. We have to seek the opinion from the expert. Please go through the reference papers quoted by the contributor.
This is not impossible for Wound healing activity to using without laboratory
Thanks for the paper provided by Dr Amarnath Pisipati, and Dr. Jose Prieto.
Dr. Matadeen bharti, the question is - Is it possible to do wound healing activity by invitro without using animal models. Without laboratory we cannot do any research....... My kind suggestion to the questionaire is to go through cell culture studies. It is most useful, but the optimal conditions is required to perform this studies
Laboratory experimental models of wound healing (in-vitro and in-vivo) depends on type of wound nature we select (the model selected should reflect the closeness to actual wounds which are diverse and complicated & needs different types of treatments(.....burn wounds,ulcers,foot sores, surgical wounds etc. )
The following article shall provide some guidelines for elite researchers of the topic
1. Guidance for Industry Chronic Cutaneous Ulcer and Burn Wounds ...
www.fda.gov/downloads/Drugs/.../Guidances/ucm071324.
Center for Drug Evaluation and Research (CDER). Center for ... Wounds — Developing Products for ..... rationale and clinical data support clinical activity of a product in more than one wound type, it ... healing and wound care) for drugs and biological products. .... Measurement of laboratory markers for diabetes mellitus ...
In vitro 3D reconstructed human epidermis from normal human keratinocytes cultured on an inert polycarbonate filter at the air-liquid interface, in a chemically defined medium. This model exists at different stages of maturity. The Creative Bioarray Reconstructed Human Epidermis Model (RHE) is very similar to human skin in terms of morphology, of differentiation markers and of functional characteristics.
Learn more at: https://www.creative-bioarray.com/in-vitro-reconstructed-human-epidermis-rhe.htm
Published by De Gruyter April 19, 2016
In vitro wound healing assays – state of the art
Anne Stamm, Kerstin Reimers, Sarah Strauß, Peter Vogt, Thomas Scheper and Iliyana Pepelanova
From the journal BioNanoMaterials
https://doi.org/10.1515/bnm-2016-0002
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