Preparation of Polycaprolactone Scaffolds using TIPS

Silva, I. M. Group 3, Class B bio11008@fe.up.pt FEUP Faculdade de Engenharia da Universidade do Porto, May 2012

I.

INTRODUCTION

III.

RESULTS

Tissue Engineering is a novel field that uses biomaterials, in particular porous scaffolds that allow cell adhesion, proliferation and differentiate and are applied to the body for repair or regenerate a damaged tissue. To obtain an ideal support for the proliferation of cells, the scaffold architecture must be well defined and properties like the pore-size should be optimal. [1] Polycaprolactone (PCL, (C6H10O2)n ) is a semicrystalline aliphatic polyester, biodegradable and non-toxic, with low melting point (60C) and glass transition temperature around -60C. This synthetic polymer used in many biomedical applications such as drug delivery and sutures, as good solubility in must solvents and low degradation rate (more than 24 months to lose total mass). The choice of PCL over other biomaterials like poly-lactic acid (PLA) is based on its more stable and flexible structure in ambient conditions, more available quantities and cheaper price. [2] The size of the PCL structure pores can be controlled by handling the freezing temperature as presented bellow (low temperature, correspond to smaller pores; higher temperature, bigger pores). But there are other important parameters such as the concentration of polymeric solution (the increasing of concentration leads to smaller pores), the solvent type and the template where the scaffolds are freezing (This process is gradual, leading to an incomplete freezing in the interior of the scaffolds in bigger templates, that result in different pore-size in the same sample). [1] The PCL scaffolds were prepared and analyzed using Energy-Dispersive X-Ray Spectroscopy (EDS) to see if there were contaminations, and Scanning Electron Microscope (SEM) in order to observe the influence of the freezing temperature in the size of the pores. II. MATERIALS AND METHODS

The EDS technique, like XPS, expresses the chemical composition of the material but in a deeper analysis. The results, showed in figure 1, reveal that there are no significant contaminations derived from the scaffold synthesis. The only peaks observed were carbon (C) and oxygen (O) respecting to the biomaterial and a group of gold (Au) and palladium (Pd) used to cover the samples in the SEM analysis, for better conductivity and to improve resolution. The results/pictures of the SEM technique are exposed in the figure 2. There are presented two pictures from each freezing temperature (4C, -20C and -80C). According to that, low freezing temperatures can be associated to smaller pores and the increasing of the temperature leads to bigger pores. Some pores at -80C can confuse because there are bigger than some presented at -20C but there are some much smaller pores at -80C. Its also observed the presence of interconnectivity between the pores, an essential characteristic for tissue engineering by allowing the migration of cells (macroporosity) and the transfer of nutrients and vascularization (microporosity). IV. DISCUSSION

The results observed match with the expected and represent a correct relation between the increasing of the pore size with the increasing of the freezing temperature. In fact, for lower temperatures, there is a faster freezing of the 1,4-dioxane, potentiating faster crystallization, obtaining smaller crystals and after sublimation, smaller pores. To obtain a better analysis of the pore-size could also be analyzed the influence of the concentration for example. The EDS analysis was essential to assure the truth of the results.
[1,2]

V.

CONCLUSIONS

The PCL porous scaffolds were obtained by the Thermally Induced Phase Separation method (TIPS). 1.0114 g of Polycaprolactone (Sigma-Aldrich; 440744) were added to 50,57 mL of 1,4 dioxane (organic solvent; >99%; Sigma-Aldrich; 360481) to obtain a 2% w/v solution. Sequentially, the solution was mixed 1 hour until complete dissolution. In each of 3 petri dishes were placed 15 mL of the solution and then stored overnight at temperatures of 4C, 20C and -80C. After this, the petri dishes were moved to the freeze dryer, occurring lyophilization (sublimation of the solvent and formation of pores). [3] To the SEM and EDS analysis were executed cross sections in the scaffolds, and then glued to a metallic support. The EDS was performed at 10keV.

This experience showed that the pore-size is a dependent variable of the freezing temperature. In the preparation of a scaffold, the pore-size is essential to its success when applied to the body. VI. BIBLIOGRAPHIC REFERENCES

[1] Sravanthi R., Preparation and characterization of poly (caprolactone) PCL - Scaffolds for tissue engineering applications, Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, May 2009. [2] Domingos M.,Dinucci D., Cometa S., Alderighi M., Brtolo P., Chiellini F., Polycaprolactone Scaffolds Fabricated via Bioextrusion for Tissue Engineering Applications, International Journal of Biomaterials, vol. 2009, Article ID 239643, June 2009. [3] Gonalves, I. Preparation of Polycaprolactone Scaffold Experimental Protocol, Biomateriais 2011/2012

VII. ANNEXES

Fig.1 Chemical composition of the PCL Scaffold using EDS.

4C

-20C

-80C

Fig.2 SEM pictures of the PCL Scaffolds. The images from left show scaffolds with freezing temperature of 4C, in the middle the temperature is -20C, and at right -80C