Polymer Scaffold Structure for Tissue Engineering Lau Yao Loong Calvin1, Tong Yen Wah2 Abstract Biodegradable

polymers are very important in tissue engineering because these structures provide a substrate on which liver cells can grow on. The aim of this paper is to investigate various methods of making scaffolds from these biodegradable polymers and to find out the best method. For all experiments used in this research, the polymer used is PLGA [poly(lactic-coglycolic acid]. The methods used are solvent casting-particulate leaching as well as porogen leaching and freeze-drying techniques. The polymer scaffolds produced are examined using the BET (Branuer Emmit Teller) to evaluate the suitability of each method by measuring the pore sizes, total pore surface area and average specific pore surface area. Knowledge of these properties of a biodegradable polymer reveals the suitability of a particular method. The experiments were repeated several times to ensure repeatability and that the results were not affected by other unexamined factors. Introduction Every year, thousands of people across the globe die of cancer or other organ failures. Many patients who are seeking organ transplant to replace their diseased organs die waiting for donor organs. The deaths of people with curable diseases are due to an acute lack of organ donors. In United States alone, 15% of potential candidates for organ transplant died while on the waiting list1. The solution to this problem is the use of tissue engineering to grow organs in a controlled environment to replace diseased ones. In this relatively new and promising scientific field, the patients own cells are grown from a small biopsy before culturing them on a temporary biodegradable polymer scaffold in a sterile environment. The tissue engineered organ is then transplanted into the patient without the slightest risk of tissue rejection of foreign cells by the body6. In this innovative approach, polymer scaffold structures are extremely important. Besides providing growth factors for cells and determining the shape of the eventual organ, these structures must have a highly porous structure with extensive interconnectedness and a high surface area so that cells have adequate space to grow in3. In addition, the polymer structure must not exhibit immunogenicity or cytotoxicity. Several tissue engineering methods have been carried out which fit these criteria. They include fiber bonding(unwoven meshes), solvent casting or particulate leaching, gas foaming, porogen leaching and phase separation or emulsification. In this research project, porogen leaching, solvent casting and particulate leaching were carried out. In each method, several factors were varied to investigate the effect on the polymer scaffold formed. The bigger the pore size and pore surface area, the more suitable the method used to make the polymer because cells can grow in adequate space.

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1 2

SRP student, Raffles Junior College Department of Chemical Engineering, National University of Singapore

Table 1: Properties of polymer scaffolds produced by different methods2 Methods Fiber Bonding (Unwoven mesh) Porosity (%) 81 Advantages Highly porous scaffolds with interconnected pores Disadvantages Use solvents which are poisonous to cells immersed in them for a long time Organic solvents used contaminate polymer Very long time required. Organic solvents used contaminate polymer Solvents used are poisonous. Pore size (m) 500

Solvent casting/Particulate leaching

87

Structure has high strength or electrical conductivity

100

Gas foaming

93

biocompatible

100

Phase separation/emulsification

95

Pore size and porosity easily changed

13 - 35

Materials and Methods The instruments used are freeze-drier, BET (Branuer Emmit Teller), test tubes, mixer, pipette, weighing balance, fume cupboard and aluminium moulds. The chemicals used are PLGA [poly(lactic-co-glycolic acid], chloroform, liquid nitrogen, distilled water and sodium chloride particles (200m). In the solvent casting-particulate leaching method, a constant mass of PLGA (0.0745g) was placed in labeled test tubes. Sodium chloride particles (200m) were then placed in each of the test tubes. Excess volumes of chloroform were then pipetted into each of the test tubes. The volume of chloroform should be sufficient enough to dissolve the PLGA. The exact volume of chloroform is not important because the PLGA just needs to be dissolved and chloroform will not form part of the resultant polymer structure. The test tubes were sealed up tightly to prevent chloroform from evaporating because it is a very volatile liquid with a low boiling point of 60oC. Sealing was also done to prevent chloroform from spilling out during mixing. The test tubes were then placed on the mixer for at least 30 minutes so that the PLGA was dissolved in chloroform completely. Next, the seals for the test tubes were taken off and all the test tubes were placed in a freeze drier. The chloroform was quickly evaporated, leaving a polymer scaffold. After three days, the test tubes were removed from the freeze-drier. Each of the resultant polymer scaffolds in each of the test tubes were extracted from the test tubes. The polymers were totally immersed in distilled water in a petri-dish to leach out salt and any other contaminants, resulting in a complete polymer with hollow pores. Pore morphology was examined using the BET. Pore size, average pore surface area and total surface area were recorded down for every polymers. As the pressure at which the mercury is absorbed into the mercury increases, the volume of the mercury increases. Thus, the surface area of pore and pore volume can be calculated from mercury volume and pressure.4

In porogen leaching and freeze-drying technique, liquid nitrogen was used5. The samples were prepared first by using a method similar to the initial steps of the solvent casting-particulate leaching technique. In this case, 0.2036g of PLGA was placed into each of 2 test tubes and chloroform is used to dissolve the PLGA with the aid of a mixer. Liquid nitrogen was then placed in a plastic dish. Next, a pipette was used to add droplets of distilled water to the liquid nitrogen. The distilled water solidifies into spherical solid ice immediately in liquid nitrogen because liquid nitrogen is at an extremely low temperature. The spherical solid ice particles are put into each of the two test tubes immediately. Next, the test tubes are sealed tightly and put into a freeze drier. After 3 days, two complete polymers with hollow pores are obtained. Pore morphology was examined using BET again.

Results Solvent casting-Particulate Leaching Experiment 1 2 3 4 5 6 7 8 Calculations Average values Standard deviation Mass of NaCl (g) 0.0895 0.1025 0.0935 0.0915 0.0955 0.0975 0.0995 0.1015 Diameter of Pores () 33.586 58.926 199.362 610.034 829.334 1003.945 342.783 214.682 Surface Area (m2) 0.1064 0.9583 2.9837 4.9290 9.3045 18.3945 12.982 5.148 Specific Surface Area (m2/g) 1.7161 8.953 24.943 56.6557 100.893 153.889 91.994 27.012 Specific Surface Area (m2/g) 58.2570 53.2083

Diameter of Pores () 411.5815 362.6450

Surface Area (m2) 6.8508 6.3090

Porogen leaching and freeze-drying techniques Experiment 1 2 Mass of PLGA (g) 0.2036 0.2036 Diameter of Pores () 117.965 119.842 Surface Area (m2) 0.0061 0.0075 Specific Surface Area (m2/g) 0.0599 0.0651

Some Results from the BET (Branuer Emmit Teller) showing graphs and pore diameters.

Discussion Several deductions can be made from the method of solvent casting-particulate leaching. The general trend shown in the table indicates that the greater the mass of sodium chloride added to the solution of PLGA and chloroform, the greater the diameter of pores, surface area and specific surface area of a pore. It is observed that the diameter of pores increases from 33.586 to 1003.945 as the mass of NaCl increases from 0.0895g to 0.0975g. These results can be attributed to the fact that the greater mass of salt occupies more spaces in the solution so that the resulting polymer will have more pores. This has major implications for tissue engineering which requires substrate surfaces to have a large surface area so that tissues can grow rapidly and unobstructively in a short period of time. However, as the mass of NaCl increases from 0.0995g to 0.1015g, the diameter of pores, surface area and specific surface area of the pores register an unexpected decrease. In view of this, a possible postulate can be put forward to explain this phenomenon. The relatively high mass of NaCl added clump together and actually hinder the formation of more pores on a microscopic level. The excessively high amount of NaCl can also prevent the polymer scaffolds from linking with each other strongly, thus resulting in less pores. This has adverse effects on tissue engineering because it decreases the surface area on which the cells can grow. Results also show that the diameter of pores, pore surface area and specific surface area are generally lower when using the porogen leaching and freeze drying techniques as compared to the method of solvent casting and particulate leaching. Thus, solvent casting and particulate leaching is a more favourable method of making polymer scaffolds.

Acknowledgement I would like to thank Dr Tong Yen Wah, my mentor, Ms Lim Siew Eng, the teacher adviser, and the science mentorship programme committee in national university of Singapore for giving me this opportunity to conduct this enriching and insightful research into tissue engineering. I would also like to thank Ivan, Chaw Su and Choon Ying, students at the research laboratories, for giving me some advice when I conduct the experiments. Lastly, I would like to thank anyone who helped me in one way or another. References 1) Ulrich A.Stock and Joseph P.Vacanti. 2001. Tissue Engineering: Current State and Prospects. Annual Reviews 2) http://www.ejb.org/content/vol3/issue2/full/5/ 3) Shoufeng Yang, Ph.D., Kah-fai Leong, M.S.E., m.s.m.e., ZhaoHui Du, Ph.D., and Cheekai Chua, Ph.D., The Design of Scaffolds for use in tissue Engineering PartI. Traditional Factors. Tissue Engineering. Volume 7, Number 6, 2001 4) Sari Westermarck, 2000, University of Helsinki(FIN), Use of mercury porosimetry and nitrogen absorption in characterization of the pore structure of mannitol and microcrystalline cellulose powders, granules and tablets. 5) Guoping Chen, Takashi Ushida, Tetsuya Tateishi, Development of Biodegradable porous scaffolds for tissue engineering. http://80isi2.isiknowledge.com.libproxy1.nus.edu.sg/portal.cgi?DestApp=WOS&Func=F 6) David J. Mooney and Antonios G.Mikos, Growing New Organs April 1999 issue