1. INTRODUCTION:

Tissue engineering a new approach which integrates the principles and knowledge of life sciences to tissue or device to repair and reconstruct the building of the tissue and organs in order to preserve and improve their function. Scaffold plays an important role, in tissue engineering as it can maintain and help the growth of cells and tissue. Ideal scaffolds have the following characteristics: non-toxicity, mechanical strength, biocompatibility, it should biodegrade with a rate that can match the rate of tissue regeneration, and the biodegraded material should not have a negative effects on the surrounding tissues or organs. It should also have high porosity with interconnected porous structure to give enough space for the cells’ growth, seeding and proliferation. Methods to develop porous three-dimensional biocompatible scaffolds for tissue engineering, includes gas-forming foam, thermal-induced phase separation, three-dimensional printing, electrospinning and freeze-drying. Scaffolds of natural biomaterials are generally prepared by freeze-drying, because it is favorable for the ones dissolved in aqueous media [1].

The porogenic template is a different and flexible procedure for the preparation of porous materials. Water being cheap and benign to the biological systems, a number of porous scaffolds for tissue engineering is prepared by freeze drying, in which ice is used as a porogenic template. They disperse the inorganic particles in water to control the growth speed and orientation of ice crystals and get porous scaffolds after sublimation of ice crystals, which act as templates and left pores. The pore size of the materials is controlled by the size of the ice crystal, which can be adjusted by changing the concentration of the solution [2,3].

Gelatin is a natural polymer. It is usually used in production of drug carriers, wound dressing and scaffolds for tissue engineering due to their good biocompatibility, biodegradability and high water adsorbing ability in vivo [4,5,6]. Alginate is a naturally arising anionic and hydrophilic polysaccharide. It is one of the most common biosynthesized materials, and it is derived mainly from brown seaweed and bacteria. Alginate is blocks of (1–4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) monomers. Alginate is of specific interest for a broad range of uses such as a biomaterial and as a supporting matrix or carrier system for tissue regeneration and repair. Having so many outstanding properties in terms of biodegradability, biocompatibility, chelating ability and non-antigenicity, it has been extensively used in a diversity of biomedical applications such as tissue engineering, drug delivery, etc [5]. The biocompatibility of Gelatin and Alginate makes them ideal for the scaffolding and hence a combination of gelatin and alginate would have better properties for tissue engineering applications. The objective of our study is to prepare gelatin-alginate composite scaffold by freeze drying technique and examine their surface morphology by microscopy.

2. MATERIALS AND METHODS:

2.1. Materials:

Gelatin and Alginate were purchased from HiMedia, India. Other standard glasswares, magnetic stirrer and-20ºC freezer and lyophilizer were needed.

2.2. Preparation of gelatin solution:

About 15 g gelatin was added into 100 ml distilled water contained in a beaker and then it was stirred at a speed of 350 rpm to form a homogeneous 15 wt% gelatin solution.

2.3. Preparation of alginate solution

About 10 g alginate was added into 100 ml distilled water contained in a beaker and then it was stirred at a speed of 450 rpm to form a homogeneous 10 wt% alginate solution.

2.4. Scaffold Fabrication:

Gelatin and alginate solutions were mixed at three different ratios of 30:70, 50:50 and 70:30 in small beakers using spatula and then poured into moulds of 6 mm diameter and 20 mm height. Gelatin-alginate mixtures were frozen at -20 ºC for 24 hours. The solidified mixtures were then lyophilized at-40 ºC under a vacuum of ̴ 0.09 mm/Hg for 24 hours.

Table 1: Scaffold compositions

Sample code	Gelatin(%)	Alginate(%)
GA3070	50	50
GA5050	70	30
GA7030	30	70

2.5. Characterization:

The surface morphology of the scaffolds was initially observed with Optical Microscopy. The top and the bottom scaffold surfaces were viewed at 50x and 100x. The scaffold with best porosity and interconnection of pores was further examined under scanning electron microscopy.

3. RESULTS AND DISCUSSION:

3.1 Scaffold Texture and Size:

The scaffold surface was pale yellow in colour with visible porosity. The texture of the scaffold was brittle and shiny.

(a) GA5050

(b) GA3070

Figure.1: Photographic images of the scaffolds

3.2 Optical Microscopic Images:

The optical microscopic images illustrate the pore structure and interconnection of the pores formed by the crosslinking of gelatin and alginate in different ratios at 50X (Fig.2) and 100X (Fig.3).

(a)GA5050

(B) GA3070

Figure.2: Optical microscopic images of the scaffolds at 50X

(A) GA5050

(B) GA3070

(C)GA7030

Figure.3: Optical microscopic images of the scaffolds at 100X

3.3 Scanning Electron Microscopic Images

(a) GA7030

(b) GA7030

Figure.4: Scanning electron microscopic images of the scaffolds

The scaffold was brittle. From the images of microscopy we can infer that GA7030 was turned out have the best structure with an optimum pore size and a good interconnection between the pores is observed in the images. The other two ratios i.e. GA5050 and GA3070 did not turned out be as good as the third ratio this suggest us that the conditions we used to prepare the scaffold were optimum or best suited for the GA7030 ratio. Scaffold GA7030 was visualized at 330X magnification. The images showed clear pore topography and interconnections between the pores.

The gelatin- alginate scaffolds were successfully prepared using freeze-drying technique and the obtained scaffolds had high porosity and proper interconnection of pores. This was obtained as a result of proper crosslinking of gelatin and alginate in different ratio that was visualized in microscopic images.

4. ACKNOWLEDGEMENT:

We would like to express our special thanks of gratitude to our teacher, Dr. Manjubala I who gave me this golden opportunity to this wonderful project of Tissue Engineering as a part of our J-Component. We would also like to thank ‘VIT’ for giving us the desired infrastructure to complete this project.

5. REFERENCES:

1. Wu, X., Liu, Y., Li, X., Wen, P., Zhang, Y., Long, Y., and Gao, J. (2010). Preparation of aligned porous gelatin scaffolds by unidirectional freeze-drying method. Acta biomaterialia, 6 (3), 1167-1177.

2. Zhang, H., Hussain, I., Brust, M., Butler, M. F., Rannard, S. P., and Cooper, A. I. (2005). Aligned two-and three-dimensional structures by directional freezing of polymers and nanoparticles. Nature materials, 4 (10), 787.

3. Lv, Q., and Feng, Q. (2006). Preparation of 3-D regenerated fibroin scaffolds with freeze drying method and freeze drying/foaming technique. Journal of Materials Science: Materials in Medicine, 17 (12), 1349-1356.

4. Huang, Y., Onyeri, S., Siewe, M., Moshfeghian, A., and Madihally, S. V. (2005). In vitro characterization of chitosan–gelatin scaffolds for tissue engineering. Biomaterials, 26 (36), 7616-7627.

5. Sun, J., and Tan, H. (2013). Alginate-based biomaterials for regenerative medicine applications. Materials, 6 (4), 1285-1309.

6. Kang, H. W., Tabata, Y., and Ikada, Y. (1999). Fabrication of porous gelatin scaffolds for tissue engineering. Biomaterials, 20 (14), 1339-1344.

Received on 06.04.2018 Modified on 15.06.2018

Research J. Pharm. and Tech 2018; 11(9): 3816-3818.

DOI: 10.5958/0974-360X.2018.00699.6