Technique: The Simplest Way to Make a Collagen Scaffold

Collagen scaffolds are very common within the field of tissue engineering. They are varied in composition as different types of collagen can be used, and virtually anything added s a supplement. In this post I’m going to discuss the simplest method of creating a collagen scaffold, a method that I employed for two out of the three materials I made for my PhD project. The first material was a plain fibril bovine collagen type I scaffold, and the second was the same except with an added hydroxyapatite component. I actually made the hydroxyapatite myself, through the standard precipitation method (topic for a future technique post) in order to eliminate any byproducts present in commercially available product.

Basically, all materials in the final scaffold are mixed together to form a homogenous aqueous solution, which is then centrifuged and vacuumed in order to remove any air bubbles. The solution is then placed in a mold (we used PVC) and frozen at the desired temperature (in my experiments I used -20 degrees Celsius, though in past experiments I have also used -80 degrees Celsius freezers). The samples are then lyophilized (a.k.a. freeze-dried) for a few hours, pushed out of their molds, and then lyophilized some more. Note: beware of removing the samples too early; they dry from the outside, so if moisture is still present in the center they can collapse later. Finally, before use, samples are trimmed and cut as desired. I found that some trimming was necessary as sem micrographs showed walls of collagen on the outer edges, hindering cell and nutrient penetration.

Like any other polymer scaffold, the size and shape of the pores can be easily modified for different desired pore sizes etc. Given a set amount of collagen in an area, pore size is directly controllable by modifying the freezing temperature. At lower temperatures, ice crystals nucleate faster, and therefore the crystals formed will be more numerous and smaller. Since final pore size is directly related to aqueous crystal size, this means that the lower the freezing temperature, the smaller the pores. If the pores are more numerous, this means that the number of walls between pores also rises; but, since the total amount of collagen remains the same, this means each individual wall is thinner. One of the advantages to collagen and this fabrication method is the variation of the scaffold in its micro-porosity under 100 micrometers. It has actually been shown that variation on this scale is more conducive to biological activity, a long as the larger pore structures average the same as an otherwise similar scaffold.

Advertisements

Growing more enamel in our sleep?

The following is a draft of a short piece written for the National Institute of Dental and Craniofacial Research.

The concept of circadian rhythms, that is, the regulation and fluctuation of biological processes over a 24-hour period, has always been a popular idea. Are blind animals still attuned to what time of day it is? Can humans kept in special chambers on 28 hour days function normally? How do long-distance airline pilots cope with multiple time zone, and the accelerated/decelerated daylight exposure?

Although long based on empirical observation, modern science has allowed researchers to find the biochemical evidence of a circadian clock, and to study this phenomenon all the way down to the genetic level, and in organisms as far back as a simple fungus. A group of scientists funded by the NIDCR set out to answer the question:

“Do we grow more enamel in our sleep?”

In order to answer this question we have to discuss previous work that has been done in the field of enamel biochemistry. Previous scientists had done staining experiments that showed bursts of enamel being deposited on a daily basis, as contrasted to a slow, steady, build-up. Because this was on a 24-hour rhythm, speculation arose that the process might be circadian.

Enamel growth has been shown to be two-step process: the production of a protein matrix made of amelogenin and other enamel-specific proteins followed by the deposition and growth of bicarbonate crystals as directed by this matrix and concomitant removal of matrix. Snead et al. set out to show that these two steps were directly related to the circadian clock, with protein matrix being grown during light hours and crystal growth occurring during dark hours.

The group of researchers attacked the problem through three different analysis methods. Gene sequence analysis was carried out to see if promoter sequences known to be circadian-clock-regulated (E-box sequences) were associated with enamel-specific protein and crystal production genes. Through the culturing of circadian-synchronized cells and the dissection of molars from pups kept in a strictly circadian environment, RNA was extracted and analysed at 4 hour intervals to see if enamel genes showed fluctuation in a circadian-specific manner. Additionally, to demonstrate the presence of circadian-regulating proteins in the mice, molars were extracted and stained for these proteins. Thus, through DNA analysis, gene and protein expression analysis, the scientists were able to conclude that the two steps of enamel production did indeed take place sequentially and on a 24-hour cycle.

The circadian clock modulates enamel development.

The NIH-OxCam Program

I thought I would give a brief shout-out for the NIH-OxCam program that I’m on, for any people considering graduate programs who are reading this blog. Applications are now open until January 2nd, and the official website and application information can be found here:

NIH OxCam Webpage

Just over a decade ago, as graduate students were first starting to make their way onto the National Institutes of Health (NIH) campus, it was decided that the NIH would form what is now known as the Graduate Partnerships Program (GPP). It was decided that the NIH was not going to be degree-granting institution. However, they wished to provide funding for students to do research on campus and to work towards degrees at partnering Universities.  One of the first partnerships was with Oxford and Cambridge.

Each graduate partnership at the NIH works differently, but the OxCam program, as it is known, gives American citizens the opportunity to study a biomedical –related field at either Oxford or Cambridge as well as at the NIH. (There is a similar program called the NIH-Wellcome program for non-US citizens.) While the NIH funds the degree, the university in England is in charge of the academic side of affairs, including examination and degree conferral. The goal is for students to evenly split their time between the two locations, working in collaborating labs and pulling together a cohesive project.

As with any course of graduate study the success of the project is dependent on many variables, the three main ones being mentorship from supervisors, tenacity of the student, and topic choice. The great thing about the program is that it does in many respects offer a middle-ground between British and American attitudes towards PhD projects. One of the main aspects of this is the fact that funding is given for five years, which is longer than the British timeframe, but shorter than the American time-frame. This seems to work out just right for the students in the program who generally seem to graduate after about 4.5 years.

The program is certainly not for everyone in that in addition to the normal criteria for a PhD candidate, you also need to add a good set of communications skills, self-motivation, and extra tenacity. But it can, and does, work out, and I would definitely encourage anyone interested to apply. And please do contact me if you have any questions.

Holidays Can Be Dangerous

In searching for relevant Hannukkah-related articles on Pubmed, an interesting article jumped out at me.

Child Injury in Israel

In this article, the authors studied trends in injuries admitted to the emergency room at the Petach Tikvah Children’s Medical Center in Israel. Interestingly, they found that injury rates in children rose in specific ways related to certain Jewish holidays. In particular, they found that there was a significant rise in burn injuries surrounding the holidays of Hannukkah, Lag BaOmer, and Passover. Hannukkah involves the nightly lighting of candles, and Lag BaOmer traditionally involves bonfires. While Passover does not explicitly involve any fire-related activity, the authors speculate that this is due to an overall increase in cooking in the house around this festive period. What is clear, however, is that there is a sudden jump in accidental poisonings around Passover, due to cleaning products used during holiday-mandated spring cleaning. Around the days surrounding, and including Passover, there is a spate of bicycle and skateboard related injuries. Cars are not driven during Passover, so this means that children have free-rein of the roads, and use the opportunity to promptly injure themselves.

So be careful when lighting those Hannukkah candles.

Oh, and if you’re Christian, this is advance warning to beware pokey objects near eyeballs and other Christmas-related injuries:

Christmas-related eye injuries: a prospective study.

Propeller and jet-ski injuries during Christmas and New Year in Western Australia