Construction of Collagen Scaffolds That Mimic the Three-Dimensional Architecture of Specific Tissues
KAEUIS A. FARAJ, Ph.D., TOIN H. VAN KUPPEVELT, Ph.D., and WILLEKE F. DAAMEN, Ph.D.
Introduction
The structure of ECM greatly influences alignment and therefore development of cells in the bioscaffold. The team concentrated on mimicking the ECM architecture of tissues, specifically the lung, tendon, and skin through controlling freezing rates, type of suspension medium and additives. Scaffolds with a specific 3D structural design resembling the actual ECM of a particular tissue may have great potential in tissue engineering.
Materials and Methods
The preparation of insoluble type I collagen consisted of defatting Bovine Achillies tendons and freezing in liquid nitrogen (-196°C). These pieces were then pulverized with a cutting mill and sieved through a 0.5nm sieve. This allowed isolation of type I collagen via salt solutions (0.1M and 1.0M sodium chloride in demineralized water), diluted acetic acid and acetone. The collagen was then lyophilized in a Sublimator 500 II freeze dryer.
The scaffold resembling the lung was prepared by slowly freezing collagen suspension in 0.25M acetic acid at -20°C before lyophilization. For the tendon scaffold, the collagen suspension in 0.25M acetic acid was injected directly into liquid nitrogen-cooled nitrogen for faster freezing. To obtain a matrix resembling skin, collagen suspension in water containing 2.8% ethanol and frozen at -80°C.
Transmission electron microscopy was performed on the collagen fibrils with a JEOL 1010 electron microscope. Scanning electron microscopy was performed on collagen fibrils with a Philips XL30 ESEM FEG apparatus at 10kV. Scanning electron microscopy was performed on lyophilized collagen scaffolds with a JEOL JSM-6310 SEM at 15kV.
Results
The scaffold’s pores were formed during freezing due to ice crystal formation, which were removed during lyophilization. Thus, freezing temperature and rate greatly affect pore size. The relationship can be seen below, in which lower temperatures and faster freeze rates resulted in smaller pore sizes, while slow freezing at higher temperatures resulted in much larger pores.
Figure 1: The pore sizes of the three cross sections of the pan used for scaffolding at three varying temperatures.
Figure 2: Scanning electron micrographs of collagen suspension in 0.25 M acetic acid. A, D, G were frozen at -20°C; B, E, H were frozen at -80°C; C, F, I were frozen at -196°C.
Besides freezing conditions, varying the amount of water used in suspension also affected scaffold morphology. Collagen suspensions in water developed more thread-like structures than suspensions in acetic acid. The structural walls were also smoother in the acetic acid suspension.
Figure 3: The influence of acetic acid on scaffold morphology is apparent in the scanning electron micrographs above. Image A was a collagen suspension prepared in 0.25 M acetic acid, and B was prepared in water. Both are frozen at -20°C.
Discussion
The structure of the scaffold can be controlled by varying freezing conditions as well as by changing suspension composition. Fast cooling rates at low temperatures, such as by liquid nitrogen, forms ice crystals simultaneously and quickly, resulting in many small ice crystals, and therefore pores. Slower freeze rates allows large ice crystals to form, leaving large pores. Collagen suspensions in water form thin fibered scaffolds. Adding ethanol to the collagen suspension in water formed a closed surface, but with porous scaffold underneath and no thread-like structures. The scaffolds are mechanically weaker in comparison to their respective tissue type. For instance, the tensile strength of tendon is 50-100Mpa while the scaffold is approximately at 100kPa, with chance of improvement via chemical cross-linking to approximately 700 kPa. The goal of the experiment was to influence scaffold structure with simple laboratory techniques, such as freezing and with simple materials, and this has been successfully demonstrated.
Critique
The paper demonstrated several important relationships with scaffold morphology; the ECM morphology can be controlled by both freezing conditions as well as suspension composition. The results of the experiment have a broad impact to tissue engineering, as it provides a base for further exploration into constructing scaffolds that ultimately allow for development of specific tissues. Unfortunately, the developments are still insufficient to be clinically used because the scaffolds are mechanically too weak. The authors should focus their work on lung scaffolding, because their scaffold tensile strength is much closer to that of the lung (1-2 Mpa), and thus has more potential in the near future to be a viable solution for lung tissue engineering.
6 comments:
It’s really impressive that the authors are able to control pore size as well as they are. I am wondering why the authors chose to explore this technique rather than using a decellularized native matrix though. It seems to me that a native matrix would have more complexity and be better equipped for the tissue’s ultimate final purpose. I suppose in the event of a shortage of donors or for the sake of advanced preparation, this is a useful technique. Also, as time goes on and we are able to introduce greater and greater complexity to scaffolds, this technique will probably prove much more useful.
The careful control of cryopreservation and the tuning of freezing temperature is also fascinating. Normally the holes created by ice during freezing are seen as a menace to cells, but puts a positive twist on that phenomena.
I would also be interested to see if any techniques other than TEM were used to characterize the scaffold. It seems to me that mechanical tests would be of particular importance in a scaffold construct.
The authors of this article presented a fairly straightforward procedure for the production of relatively well controlled microstructure scaffolding using equipment likely to be found in a lab, albeit a well equipped one. The consistency in pore size per sample is quite impressive, as specific pore sizes do indeed dictate cellular and wound healing responses.
As Ashley had also mentioned, some of the procedure seemed pretty harsh, exposing the scaffold material to extremely low temperatures. Typically ice crystal formation would puncture cells, but considering that this is an acellular matrix, and considering the results, this is not a problem.
Personally, I was hoping the authors had mentioned future projects to involve testing these scaffolds with live cells and implantation. However, this was not the original purpose of the study, and instead, served as a suitable proof of concept.
I find the method of the controlling the pore size of ECM matrix using varying freezing temperature, rate, and amount of water quite interesting and unique. However, it's unclear how well the synthetic scaffold actually works for the different cell types. In addition, how did they pick -20 and -80 degrees? Also, can they obtain consistent results of the microstructure?
The author's methods are very unique, but, as you did mention, do not address the mechanical loads that may be applied to that porous material.
Also, I wonder if this technique, or one similar to this, would be useful in the construction of other organs that depend on a specific porosity?
The method of varying to the freezing temperature to create a collagen matrix with a controlled pore size is quite interesting. Could this method be used to create a scaffold of collagen mixed with some other solution (gelatin, etc.)?
Once mechanical strength is addressed, how would the cells interact with the scaffold (in vitro and in vivo)?
For the selection of freezing temperatures, the specific choice of -20C and -80C is unclear, but I assume it is from trial/error.
For the cell-scaffold interaction question: the basis of the paper is that the structure of ECM directly affects development of the cells (in vitro or in vivo) into specific cells types as needed
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