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Interview with an expert

Because of the sensitivity of my blog topic about preclinical testing, I was only since today allowed to put the read out of my interview with an expert online. First, Prometheus had to give their approval.

For my interview I asked professor Marina Maréchal to tell us about her experiences with animal testing. I wrote out a text that can serve as a complete summarization of my posts and I think it will give you all a clear view on the system and organs behind preclinical and to a lesser extent clinical tests.

Interview with Marina Maréchal

Introduction

Marina Maréchal graduated in 1983 as a dentist and in 1987 she became a specialist in Periodontology. In this period she already did some clinical trials using students as a guinea pig. After her specialization she started a private practice in the periodontology for about 13 years. Her passion for scientific research let her apply for a PhD funded by an IDO project in cooperation with the departments of periodontology, rheumatology, MTM, Biomechanics and prosthodontics. In this collaboration Marina did in vivo experiments with rabbits as animal testing in preclinical trials. After her PhD she started working at Tigenix where she became head of Preclinical in cartilage repair. When the R&D department was shut down, she made a come-back to KU Leuven in preclinical research at Onderwijs&Navorsing, Gasthuisberg.

First steps towards animal testing

The main goals of performing preclinical trials (animal testing) is collecting data about feasibility, iterative testing and safety to define the ultimate safety profile of a new product before clinical trials start. To collect valuable data out of these preclinical experiments, it very important to design these animal experiments as if they are human clinical trials.   As logical as this statement is, the acceptation of doing an animal testing is already provided. An animal is to be treated as humanely as possible. Surgical procedures should be performed using sterile material and under aseptic conditions and using general anaesthesia. During postoperative check-ups the recovery of the animal will be followed checking the experience of pain, monitoring the body temperature, evaluation of the food intake or the occurrence of infections. This implies that the animal may not experience pain or suffer from a severe infection. If this is the case and some medical treatments (e.g. antibiotics, painkillers) do not solve the problem, humane endpoints determine if and when the animal will be sacrificed. Since sacrificing animals before the endpoint of the experiment decrease the number of data collected during the experiments, the animals will be treated as good as possible and also cage enrichment (some toys in the cage) will improve their well-being. Just like with humans, if an animal is feeling happy in general it will be healthier and the delivered data will be more useful.

Ethical commission

In vivo experiments cannot start before the ethics committee approves the experimental template of the study. An ethical commission form has to be prepared and submitted, and without any approval the study is not allowed and no test animals will be available for the study. The number of test animals and the type of animals that will be used in the study will be compared to the preliminary results and the expected results. Mostly in preclinical trials animal testing will involve two different species. One will start using some small animals such as mouse or rats, … followed by a larger animal based on which animal will give the best correlation with a human trial, such as a rabbit, pig, a goat, a sheep, a horse, … The choice of using mice, rabbits, horses or another animal for example is not only one of having the best correlation with a human setting, but safety, efficacy, ethics and money will also be important factors.

To the clinical trials

Before any treatment or product will be commercially available, three clinical test phases are necessary after the preclinical experiments. Phase I clinical trial is conducted in a small group of healthy volunteers (mostly male) to check mainly safety issues that could not yet be answered in the preclinical trials. Phase II clinical trial evaluates the efficacy and also safety issues in a selected population of patients (the target group) and includes a large patient group (around 100-500). Phase III clinical trial is performed in a real-life population (500-5000 patients) when efficacy and safety have already been established and therefor the goal will be to generate additional data on efficacy and safety in relatively large number of patients. Before clinical trials can be done, an enormous amount of preclinical data is needed to convince the ethics committee and the FAGG (in Belgium), put down in a clear document where all issues are mentioned. When one of these two authorities gives negative advice, the clinical trial is not approved. The importance of these extensive controls and the growing importance of preclinical data are demonstrated by some terrible incidents: http://en.wikipedia.org/wiki/Thalidomidehttp://en.wikipedia.org/wiki/TGN1412

Marina Maréchal

I asked her the question if she was still emotional at the moment she had to sacrifice an animal used in a preclinical trial and her reply was clear: “Doing preclinical experiments will include surgical procedures, postoperative care and sacrifice. If at a certain point  I would not feel any difficulty anymore to sacrifice an animal, I will not perform preclinical experiments anymore”.

 

I want to thank Marina for giving me this inside view on the whole mechanism around animal testing and especially by giving me her own perspective on this subject.

 

I hope you now all have a view on why animal testing is done and also how that it has to be performed. I want to thank you all for blogging together those months and for giving me your personal contribution to my topic.

 

Thank you,

Jeroen

Testing on non-human primates

On the occasion of the protest that occurred in front of the royal palace yesterday, I wanted to know what you people think about animal testing on non-human primates. The link to the article at ‘de redactie’ about the demonstration you can find here. Yesterday there was a protest by some members of the anti animal testing organization with the slogan: “Monkeys are no utensils!”. Testing on primates is already been forbidden also in Belgium, but non-human primates are still used for scientific animal testing. The organization wants our government and even our king to stop this testing and to stop the discrimination between different apes.

monkey_1958030c

Associations against animal testing and in particular against testing on (non-human) primates are numerous. I found a website where they are suggesting alternative ways for this scientific tests. Some of these are:

– Embryonic stem cell test: using mouse-derived cells to assess potential toxicity to developing embryos, has been validated as a partial replacement for birth-defect testing in rats and rabbits.

– Human skin model tests: such as the validated EpiDerm™ test, which has been accepted almost universally as a total replacement for skin corrosion studies in rabbits.

– Use of human skin leftover from surgical procedures: or donated cadavers can be used to measure the rate at which a chemical is able to penetrate the skin.

Of coarse if testing is possible without the use of primates and with the use of such alternatives, I would think nobody would understand why it is still done. End even more when you use such pictures like I used in this post. But that is just the reason why these experiments are still done: it is not possible yet. In vivo experiments, especially experiments with drug release and drug absorption involve the totality of the complexity of a living being. No in vitro tests can already cope with this, but the amount of data of these tests is increasing at rapid speed.

Maybe, in future an almost completely digital version of life will be available. What do you think? And should we already stop with testing on non-human primates?

Another interesting article that appeared in the ‘daily telegraph’ in 2011 can be found here. It is about the reasoning, efficiency and the justification of scientific testing on monkeys.

Tissue renewal

Because of the post with the bionic eye, I looked up some recent articles to explain what I mean with regenerating tissue.

In this simple article in De Standaard there is explained how damaged cardiac tissue because of an myocardial infarction can be replaced or regenerated with autogenous cardiac stem cells. Because it was really promising it also appeared in the Cardiovascular Research magazine. First tests were performed with test animals and now the first trials on human are done.

nursing diagnosis for myocardial infarctoin

Myocardial infarction

Another article in De Standaard tells us that a trachea was transplanted that was completely made in vitro already in 2011. A conductive scaffold was used as a matrix for seeding the stem cells onto. This construct was cultured in vitro and the stem cells successfully proliferated and differentiated into a trachea that was accepted by the patient.

clpt2011270f3

Tissue engineering concept

transplant

Cultured trachea

Sorry for the english people but I hope you understand my short summaries and with the help of the pictures you hopefully can imagine something like it.

My thesis

To come back on my thesis, the last post to tell you how far the situation is.

So there were three main goals of my thesis:

1. Investigate the dissolution characteristics of different CaP scaffolds

2. Investigate the cell seeding efficiency of the different scaffold types

3. Investigate the gene expression of cultured cells on the different scaffolds

To start, the in vivo used scaffolds were analyzed with nanoCT first to investigate the structural characteristics of the scaffolds. Together with the material characteristics we, in the end, try to correlate this to the results of the different parts.

Since bone formation needs osteoconduction, -induction and -genesis, we investigate the dissolution of the scaffolds for the decomposition of Calcium and Phosphate ions as an inductive path for the cells to stimulate differentiation to osteoblasts. The conductive way of stimulating the cells is investigated through material and structural information on the scaffold. Osteogenesis or bone formation by cells can be assessed in vitro by doing RNA extraction of the cultured cells and to quantify certain gene markers. If certain genes like RUNX2, OCN, Col1A1 are overrepresented (compared to housekeeping genes) we can gently predict that the scaffold in vivo will stimulate bone. This can then be correlated with the dissolution characteristics, cell and scaffold properties to make up an overall discussion, based on other research results found in literature.

To tell you how far I am. The lab work of steps 1 and 2 are finished but the results are being investigated. Step 3 will-be started this thursday and the lab work will be done after 5 weeks. Then still the results will have to be investigated.

I hope you have a clear picture of what my thesis is. The results I cannot show but when my thesis is finished you can always ask me for the results if you would like to know.

Thank you for your time 🙂

The animal discussion will be continued for a few more weeks.

Animal testing: Cosmetics and Fur?

Again we continue with the ethical aspect of animal testing. A few days ago I saw the posters of GAIA in the train station of Leuven. Maybe you saw them too? If not you can check them here. I also recommend you to look at the video.

200-IssuesClothingFurAnimalsRaccoon

Now, everyone knows what is going on and I do not think I have to tell you. But what can be really interesting is to know how we (all members of this blog group) think about this next statements. I made the statements, but it does not imply that it is true for me.

1) Cosmetics should never be tested on animals. (link)

2) Someone who buys bond is an animal hater and/or an asshole.

3) Someone who wears bond is an animal hater and/or an asshole.

Please comment short but clear and separately (just number 1,2,3) for each statement.

Fur_Carcasses_Sergey_Maximishin2

Video

Thank you and I am looking forward to the discussion!

Scaffold analysis

Back to my thesis…

As I told you before, a scaffold is a carrier wherein cells can attach themselves and this cellularized scaffold can then be implanted to regenerate more efficiently new tissue. The scaffolds we are using are biodegradable, made from CaP and some have a collagen matrix distributed between the CaP grains.

Bio-Oss-Collagen.6692106_std

An important issue is the search after the optimal morphology of a scaffold (apart from the material type). This includes some parameters as average pore size, total void volume, porosity, specific surface area, etc. The difficulty here is that a scaffold is small and cored out of a larger block, delivered by the manufacturer. Structural properties between different blocks are quite homogenous, but between different scaffolds there are quite some variations between these structural parameters. Of course analyzing these scaffolds is the solution, but this takes an enormous amount of time (scanning and computing thousands of pictures).

A possible way to calculate these structural parameters is micro-CT analysis. It takes around 2000 pictures. These pictures are made from the scatter that x-ray beams of different positions (angle, height, …) experience through the sample. Computer software then can calculate 3D parameters. The outcome of the first 8 samples of each scaffold type resulted in next graphs:

1: Total scaffold volume 2: Total void volume

1: Total scaffold volume
2: Total void volume

1: Porosity 2: SSA

1: Porosity
2: SSA

As you can see it is very difficult to cut every scaffold manually with the same size. This is a necessity since it will be important to have scaffolds of the same size in further experiments. The intrinsic parameters are independent of size and though very only between the different scaffold types.

So now you guys are also up to date! 🙂

Animal testing part 2

Since I saw that there was well responding on the animal testing post, I come up with a second part of this subject.

First of all to summarize your reactions:

The most of you do recognize it as an acceptable approach since it can be useful for us, but it has to be ‘controlled’.

Well, I can assure you that all tests first have to be recognized by the institutional guidelines, for example KU leuven’s animal agreements. One of this agreements includes the minimum use of a test animal. An other important rule is the hierarchy of test animals. First experiments on mice are done, later on dogs, later on primates and finally humans. Between these there are more groups of animals.

Types_of_vertebrates_v2en

Animal testing for cosmetics are still conducted in the US, but in Belgium and most other European countries this is strictly forbidden.

My next question to you:

Do animals feel pain? Answer with yes or no for mice, rabbits, cat, dogs, sheep and primates