Preparation and Characterization of Hydrogels of Several

Chapter 7

Downloaded by COLUMBIA UNIV on June 25, 2012 | http://pubs.acs.org Publication Date (Web): December 14, 2010 | doi: 10.1021/bk-2010-1061.ch007

Preparation and Characterization of Hydrogels of Several Polysaccarides for Biomaterials Applications Hydrogels for Biomaterials Applications Ayse Z. Aroguz,*,1 Kemal Baysal,2 and Bahattin M. Baysal3 1Department

of Chemistry, Engineering Faculty, İstanbul University, 34320 Avcılar-Istanbul, Turkey 2TUBITAK Research Institute for Genetic Engineering and Biotechnology, Marmara Research Center, P.O. Box 21, 41470 Gebze-Kocaeli, Turkey 3Department of Chemical Engineering, Bogazici University, 81815 Bebek-Istanbul, Turkey *[email protected]

The advantages of hydrogels as biomedical materials and their performance in applications depend on their molecular structures. Two hydrogels derived from biopolymers such as alginate and chitosan obtained from natural sources were prepared and characterized for biomaterials applications. Morphology of the crosslinked material was investigated by using SEM. Swelling behavior of chitosan/polycaprolactone and chitosan/alginate hydrogels in different compositions was studied. The biopolymer hydrogels thus prepared were used for cell growth experiments. To measure the degree of cell proliferation, the gels were seeded with L929 mouse fibroblasts. The cell growth properties were followed by SEM photographs. It was found that the population of cells in the cross-linked polymer gels increased.

© 2010 American Chemical Society In Contemporary Science of Polymeric Materials; Korugic-Karasz, L.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Introduction The structure and properties of polymeric gels are important for their applications in tissue engineering (1–4). Chitosan is linear polysaccaride commenly used in the tissue engineering applications for its biocompability (5–7). Chitosan is often crosslinked with other polymers such as polycaprolactone and alginate to modulate its general properties such as mechanical property for biomedical applications (8). Alginate is another natural polysaccaride obtained from brown seaweeds. It is commonly used in the food industry as additives and thickeners (9). It has also an excellent biocompatibility, potential bioactivity and non-toxicity and is used in tissue engineering drug systems (10, 11). Many review articles have been published on the use of natural polymeric materials in a broad range of biomedical applications (12–14) Polycaprolactone (PCL), an aliphatic polyester, is one of the most important synthetic materials for commercial applications as a biodegradable material. Recently, blending PCL with the other polymers has been studied to obtain a more economical version of the degradable polymer (15, 16). Blends of polycaprolactone with chitosan were studied for tissue engineering applications by Madihally and coworkers (17). Our laboratories have extensively studied preparation, characterization and various properties and applications of these biomaterials in drug release and cell growth potentials (18–22). In this study, chitosan/alginate and chitosan/polycaprolactone crosslinked gels were prepared in the presence of the cross-linking agent. To obtain a porous polymeric structure, solvent removal method by freeze-drying was used. The swelling behavior and morphological properties of the gels were studied. Adding acrylic acid (AA) to the chitosan/poly caprolactone crosslinked material during the preparation process increased the stability of the hydrogels.

Experimental Materials Chitosan powder with a medium molecular weight, alginic acid sodium salt and polycaprolactone with the average molecular weight (Mw) of 80,000 g/mol were supplied from Aldrich Chemical Company Inc, U.S.A. Acrylic acid was purchased from Fluka. The crosslinker, N,N′- dicyclohexylcarbodiimide (DCC) was also purchased from Aldrich Cartsan DBTDL, stannous dilaurate (T-12) used as catalyst was obtained from Cincinnati Milacron Chemicals Inc., U.S.A. All the polymers were used without further purification. Preperation of Hydrogels The gel preparation reactions were performed in acetic acid solutions to obtain products in high yields. First, stock solutions of the polymers were prepared: Chitosan was dissolved in 0.5 M acetic acid at 121 °C; Stock solutions 94 In Contemporary Science of Polymeric Materials; Korugic-Karasz, L.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


of alginate and polycaprolactone were prepared in water and in glacial acid at room temperature, respectively. The polycaprolactone solution was slowly added to the chitosan solution under continuous stirring. To obtain crosslinked chitosan/PCL gel, catalyst T-12 and DCC crosslinker were added into the mixture. The crosslinked product was poured into Teflon petri dishes of 2 cm diameter, and subsequently evaporated in a hood at room temperature until a concentrated gel was obtained. All samples were prepared by using the same procedure, but at different mixing ratios of the original materials. The same procedure was also used to prepare chitosan/alginate crosslinked gels. The alginate solution was slowly added to the chitosan solution, then T12 and DCC were added to this mixture to obtain a crosslinked chitosan/alginate hydrogel. Chitosan/PCL/AA gels were prepared using the same procedure as the one for the chitosan/PCL hydrogel. AA was added to the mixture before adding the catalyst and crosslinker. All the samples were evaporated in the Teflon petri dishes to obtain concentrated hydrogels. Lyophilization Lyphlock 6, a Labconco Model Freeze Drier system, was used for lyophlisation of the crosslinked materials and obtaining porous structures. The concentrated gel products were first frozen for 2 hours at −80°C. Then the frozen gels were lyophilized within the freeze drier for 6 hours at –50°C to obtain the porous scaffold. The lyophilized freeze-dried samples were used in the cell growth and drug release experiments. Swelling of Gels Dynamic swelling experiments of the hydrogels were performed by immersing dried disc samples in distilled water (pH=5.6) and in phosphate buffered saline solution (PBS) (pH=7.4) in a temperature-controlled bath at 298 ± 0.1 K. The mass of the gel sample was messured as a function of time. The equilibrium mass swelling is expressed as the amount of water in the hydrogel:

Where, m0 is the mass of the dry gel and m∞ the mass of the swollen gel at equilibrium. A sample of the chitosan/PCL gel was dried in vacuum oven at 50 °C until a constant weight of the gel was reached. Then certain amount of chitosan/PCL dry gel film was immersed in distilled water and in PBS separately, and stored at room temperature for several hours. The gels were removed from the solution and wiped gently with tissue paper and weighted quickly. Thus, the hdyrogel was weighed in the swollen state, and the equilibrium mass swelling was calculated using Equation (1). The equilibrium mass swelling values were compared for various compositions of the gel and different amounts of the crosslinker used in the gel. 95 In Contemporary Science of Polymeric Materials; Korugic-Karasz, L.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Morphological Analysis


The morphology of the crosslinked gels was analyzed by a JOEL-JSM T330 scanning electron microscope (SEM). Ambios Universal Scanning Probe Microscope AFM was also used to compare the morphologies of the samples. For SEM observation, the samples were coated with gold before analysis. The coated samples were placed in the vacuum chamber of the instrument and observed at the voltage of 5 kV. The results are shown in Figs. 1 for a chitosan / PCL gel and Fig. 2-3 for a Chitosan/alginate gel. Figs. 4-5 show the AFM results of the chitosan/alginate hydrogel.

Results and Discussion Table 1. The composition and swelling properties of the Chitosan/PCL hydrogel Chitosan/PCL (w/w)

67/33

Swelling medium

water

Crosslinker (wt%) in the original mixture 0.83

40

0.67

91

0.41

180

water 50/50

0.70

PBS

95 48 17

25/75 50/50

Equilibrium mass swelling ratio

water

1.5

53

75/25

58

25/75

16

50/50

PBS

1.5

42

75/25 Chitosan/PCL/AA (w/w/w) 50/25/25

32

water

14 0.37

PBS

6

96 In Contemporary Science of Polymeric Materials; Korugic-Karasz, L.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 1. SEM micrograph of the surface morphology of chitosan/poly caprolactone (1/1) (x500). Swelling Ratio The equilibrium mass swelling ratios of the chitosan/PCl and chitosan/ alginate hydrogels are listed in Table 1 and Table 2, respectively. In Table 1 the mass swelling ratio increases with a decreasing crosslinker content in the gel. The ratio also increases with an increasing amount of chitosan in the gel. Similar results are observed for the chitosan/alginate hydrogels (Table 2). An increasing amount of chitosan in the chitosan/alginate gel increases the swelling ratio. The mass swelling in PBS solution is less than it is in water for the two hydrogels. Adding AA decreases the swelling of the two gels. Morphology Figure 1 shows the morphological structure of the chitosan/alginate hydrogel. A porous and fibrous sructure is evident. The crystalline structure between the holes comes from PCL. The arrows in Figure 1 represent the holes. Figure 2 shows the surface of the chitosan /alginate hydrogel. The surface of the chitosan /alginate hydrogel after being seeded with cells is seen in Figure 3. The arrows in Figure 3 show the growing seeded cell. AFM results of chitosan/alginate hydrogel are seen 97 In Contemporary Science of Polymeric Materials; Korugic-Karasz, L.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

in Figs. 4-5. AFM images support the porous structures of the gels. These images reveal a homogenous distrubution of holes in the gels. The porous structure is suitable for use as a scaffold for cell growth experiments.

Table 2. The composition and swelling properties of the Chitosan/alginate hydrogel Chitosan/alginate (w/w)

Crosslinker (wt%) in the original mixture


1.5 33/67 3.1

1.5 25/75 3.1

Swelling medium

Equilibrium mass swelling

water

66

PBS

43

water

51

PBS

37

water

41

PBS

39

water

36

PBS

33

Figure 2. SEM micrograph of the surface and cross section morphology of chitosan/alginate (1/2). 98 In Contemporary Science of Polymeric Materials; Korugic-Karasz, L.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 3. SEM micrograph of the surface morphology of chitosan/alginate (1/2) after being seeded with L929 cells (x500).

Figure 4. AFM micrograph of chitosan/alginate (1/2) hydrogel.



Figure 5. Figure 4. AFM micrograph of chitosan/alginate (1/2) hydrogel.

Conclusions We studied two hydrogels, chitosan-polycaprolactone and chitosan-alginate. Lyophilizied hydrogel samples were used in-vitro cell culture experiments with L929 mouse fibroblasts to determine the effects of these polymeric gels on cell attachement and growth. Cell growth experiments were carried out using chitosan/PCL gels, but it was not succesfull because the crosslinked gels used as scaffolds were not stable. The acrylate containig chitosan/PCL/acrylate gels were used for this purpose. On the other hand chitosan/alginate gels were used succesfulluy for cell growth purposes. The details of cell-growth experiments will be published elsewhere. Drug loading and release experiments were carried out in phosphate buffer at pH 7.4 at room temperature using chitosan/PCL and chitosan/PCL/acrylate gels. Details of drug loading and release experiments using Lidocaine as the model drug will also be published elsewhere.


References 1. 2. 3. 4. 5.


6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Ma, L.; Yu, W.; Ma, X. J. Appl. Polym. Sci. 2007, 106, 394–399. She, H.; Xiao, X.; Liu, R. J. Matter Sci. 2007, 42, 8113–8119. Grodzinski, J. J. Polym. Adv. Technol. 2006, 17, 395–418. Vieira, E. F. S; Cestari, A. R.; Airoldi, A. R. Biomacromolecules 2008, 9, 1195–1199. Cui, Y. L; Qi, A. D; Liu, W. G.; Wang, X. H.; Wang, H.; Ma, D. M. Biomaterials 2003, 24, 3859. Hsu, S. H.; Whu, S. W.; Tsai, C. L.; Wu, Y. H.; Chen, W. H.; Hsieh, K. H. J. Polym. Res. 2004, 11, 141. Guibal, E. Sep. Purif. Technol. 2004, 38, 43–74. Muzzarelli, R A. A. Carbohydr. Polym. 2009, 77 (1), 1–9. Julian, T. N.; Radebaugh, G. W.; Wisniewski, S. J. J. Controlled Release 1988, 7, 165–169. Li, Z.; Ramay, H. R.; Hauch, K. D.; Xiao, D.; Zhang, M. Biomaterials 2005, 26, 3919. Li, Z.; Zhang, M. J. Biomed. Mater. Res. 2005, 75A, 485. Rinaudo, M. Rewiew. Polym. Int. 2008, 57, 397. Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Adv. Mater. 2006, 18, 1345. Drury, J. L.; Money, D. J. Biomaterials 2003, 24, 4337. Rosa, D. S.; Guedes, C. G. F.; Pedroso, A. G. ; Calil, M. R. Mater. Sci. Eng. 2004, 24, 663–670. Chun, Y. S.; Kyung, Y. J.; Jung, H. C.; Kim, W. N. Polymer 2000, 41, 8729. Sarasam, A.; Madihally, S. V. Biomaterials 2005, 26, 5500–5508. Kayaman–Apohan, N.; Karal–Yilmaz, O.; Baysal, K.; Baysal, B. M. Polym. 2001, 42, 4109–4116. Kayaman−Apohan, N.; Baysal, B. M. Macromol. Chem. Phys. 2001, 202, 1182–1188. Porjazoska, A.; Karal - Yilmaz, O.; Cvetkowska, M.; Baysal, K.; Baysal, B. M. J. Biomater. Sci., Polym. Ed. 2002, 13, 1119–1134. Tasdelen, B.; Kayaman−Apohan, N.; Güven, O.; Baysal, B. M. Int. J. Pharm. 2004, 278, 343. Tasdelen, B.; Kayaman−Apohan, N.; Güven, O.; Baysal, B. M. Phys. Chem. 2005, 73, 340–345.


Preparation and Characterization of Hydrogels of Several

Recommend Documents