Macroporous Keratin Scaffold – a Novel Biomaterial for Biomedical Applications

Tissue engineering has recommended the development of biomaterials for implantable porous scaffolds at the injured site, which temporarily acted as the supporting materials for ingrowths of the cells and degraded along with extra cellular matrix production, vascularization and tissue regeneration. Usually, three dimensional scaffolds play the most important role in the design of Tissue Engineering Construct that affects the cell fate process. Natural Polymer biomaterial such as Collagen, Keratin mimics the extra cellular matrix at the site of injury for regeneration of tissue. Collagen is one of the protein biomaterials and already prominently placed in the development of tissue engineering contructs.But due to poor environmental conditions, Collagen denatures into gelatin which lost triple helix conformation and also affects the cell fate process. Keratin is one of the fibrous proteins found in the extra cellular matrix, providing outer covering such as wool, hair feathers and nail and alternative to collagen biomaterials. The presence of high cysteine content in the keratin helps to immobilize the bioactive molecules for drug delivery application. The extracted keratin from horn meal is made into the macro porous scaffold by freeze drying method. The molecular weight of extracted keratin was determined by SDS.Poly acryl amide gel electrophoresis and was characterized by CD spectroscopy, FTIR. The scanning electron microscopy studies of macro porous Keratin scaffold confirms that the scaffold has interconnectivity heterogeneous pores that will help to bind with drugs or cells for design of drug delivery systems and tissue engineering constructs. INTRODUCTION The regeneration of tissues at the site of damage is really a challenging task in the field of Tissue Engineering. The need of suitable biomaterials, which influence the formation of in vivo extra cellular matrix and support cellular proliferation for tissue regeneration, have motivated the word of sciences towards tissue engineering which in turn recommended the development of biomaterials for implantable macroporous scaffolds at the damaged site which temporarily act as the supporting materials for ingrowths of the cells and degrade along with in vivo extra cellular matrix production, vascularization and tissue regeneration. Biocompatibility and biomaterial surfaces to favour cell attachment and enhance cell fate process are the fundamental requirements for development of biomaterial scaffold. The scaffold is a three dimensional substrate and it acts as a template for tissue regeneration. The ideal scaffolds should have an appropriate surface chemistry and microstructures to facilitate cellular attachment, proliferation and differentiation. In addition to that, the scaffolds should possess adequate mechanical strength and degradation rate without any undesirable by-products that should not cause any immunological effects. Generally, Biomaterials made from fibrous protein mimics the extra cellular matrix at the site of injury for regeneration of tissues. Collagen is one of the most prominent protein based biomaterials and already ruled in the development of tissue engineering constructs with few limitations. Due to extreme conditions, Collagen denatures into gelatin which lost triple helix conformation. So, the collagen biomaterials needs cross linking which INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES ISSN: 22775005 Vol. 1 (4) Oct-Dec 2012 www.ijpcsonline.com 1448 normally cause cytotoxity in the cell culture system. Keratin, which is less thermolabile, is one of the fibrous proteins found in the extra cellular matrix, providing outer covering in the fibrous tissue such as wool, hair feathers and nail and alternative to collagen biomaterials. At a molecular level, the most distinctive feature of keratins is the high concentration of half –cysteine residue (7  20 number % of the total amino acid residues).Most of these half – cysteine residues are localized at the terminal regions of the proteins. Keratin biomaterials in the form of sponge, film were already developed from wool and human hair for various biomedical applications such as wound dressings and neural tissue engineering application. . Keratin contain cellular-binding motifs which mimic the sites of cellular attachment found in the native extra cellular matrix which could make the keratin to be used for the development of tissue engineering constructs. Keratin extracted from wool, silk, and human hair contained cell adhesion sequence, RGD and LDV which are found in the extra cellular matrix proteins such as fibronectin. The protein based porous scaffolds have several advantages to enhance the growth of cells at the site of damage and with respect to the microstrurues of the porous scaffolds, high porosity (greater than 90%) and interconnected pore network are desirable for the development of scaffold. Moreover, the preferred pore size in the scaffold is generally needed in the range of 50 -500 microns to permit the ingrowths of cells, vascularization and regeneration of tissues. The reduced extracted keratin from horn meal, is formulated into the keratin porous scaffold and then characterized for various tissue engineering applications and drug delivery devices. MATERIALS AND METHODS Preparation of Horn Meal Raw horns of slaughtered cattle and buffaloes collected from the local slaughterhouse at Perambur Chennai were washed and subjected to high steam pressure (40psi) in a wet rendering plant (FMC, Australia) for three hours (raw horn water ratio 100:30 w/v). The resulting material was dried in a dryer (BHL, Ahmedabad) and pulverized in pulverizer (FMC, Australia) to get horn meal. 100 Kg raw horns give 60 Kg of Horn meal (average yield). Horn meal was collected from the pilot plant of Central Leather Research Institute and washed twice with distilled water to remove the rough dirts and soils from the material and dried at 60°C overnight. It was kept at room temperature and used as raw material. Extraction of Keratin from Horn meal The horn meal was washed with water, dried and defatted by soxhlet extraction using a 1:1v/v mixture of hexane and dichloromethane. The cleaned horn meal (10g) was mixed with 7M urea (180ml), SDS (6g), and 2-mercaptoethanol (15ml) in a 300ml round –bottom flask and shaken at 50C for 12 hr. Because keratins were not extracted at acidic pH less than 5 and underwent decomposition at alkaline pH greater than 9, the aqueous phase was maintained in a neutral pH range during the process. The resulting mixture was filtered through a stainless steel mesh. Subsequently, the filtrate was dialyzed against degassed water for 5 days. The solution was stable in a bottle at room temperature for at least 1 year. SDS PAGE of Keratin The aqueous solution of the reduced keratins were subjected to one dimensional slab SDS–PAGE at 15C using a 10 -15% gradient gel at 250V and 10mA.The protein bands in the developed gels were stained by Coomassie brilliant blue R-250 . CD spectra of Reduced Keratin CD spectra were taken on a JASCO J-20 spectropolarimeter in a thermostatically controlled 1mm jacketed cell. CD of extracted was examined in Jasco J-500A spectropolarimeter. Liquid nitrogen was circulated through the instrument for about 30 minutes to have a constant temperature that will not affect the nativity of the protein during the period of the experiment. A cell with 1mm path length was employed. Baseline adjustment was INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES ISSN: 22775005 Vol. 1 (4) Oct-Dec 2012 www.ijpcsonline.com 1449 carried out by taking the solvent 0.025 M acetic acid. CD measurement of each of the samples was made from 195 to 250 nm. The CD data were expressed in terms of the mean residue ellipticity, in deg cm2 dmol-1. FTIR spectra of Reduced Keratin FTIR spectra of the samples of lyophilized reduced keratin were obtained by using the ATR technique. The keratin powder crushed with potassium bromide for ATR technique in FTIR spectroscopy studies.Infrared spectra of keratin material were taken. Infrared spectra of the films of the materials under study were recorded from 400 to 2000 Cm, using a Nicolet 20 DXB FT-IR spectrophotometer. Preparation of Porous keratin scaffold The 10 ml of reduced keratin solution (100l containing 4.2mg of protein) was added to Petri dish (3cm in dia) frozen at 80 C for 2 days and lyophilized to form sponge (5 cm in dia,0.8 cm thickness) .The keratin sponges treated with 10 ml of 0.1M idoacetic acid in 0.5M Tris HCl buffer pH 8.5 at room temperature for the protection of a large amount of active SH group on the keratin proteins. The blocked scaffold was washed with Phosphate buffer saline solution at 60 C for 1 hr to remove any iodacetic acid and SDS. Scanning electron microscopy (SEM) for Porous Keratin Scaffold The surface and the cross-section of pure porous keratin scaffold were observed with scanning electron microscopy (SEM). The porous keratin scaffold was coated with gold using an Edwards E306 sputter coater .The stubs were introduced into the specimen chamber of a FEI-Quanta 200 Scanning electron microscope. The stubs mounted on the stage could be tilted, rotated and moved to the desired position and orientation. RESULTS SDS PAGE of keratin The electrophoresis analysis of reduced keratin from horn meal (fig 1) shows two high molecular mass bands (225–150 Kda) and the lower molecular mass band ( 20 Kda) attributed respectively. INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES ISSN: 22775005 Vol. 1 (4) Oct-Dec 2012 www.ijpcsonline.com 1450 The SDS page analysis of keratin and Silver staining of Keratin confirms that the molecular weight of keratin is 225 -150 Kda and contains cysteine amino acids. CD spectra of Reduced Keratin The CD spectrum of aqueous solution of keratin confirms anti parallel beta sheet structure with negative minimum absorption band at 225nm and a weak positive maximum absorption band at 195nm. FTIR spectra of Reduced Keratin The FTIR spectra of reduced keratin shown in fig show characteristic absorption band assigned mainly to the peptide bonds(-CONH-).The vibrations in the peptide bonds originate bands known as amide A, amide I, II, III. The amide A band,which falls at 3292.12 1/cm ,is connected with the stretching vibration of N-H bonds .The amide I band is connected mainly with the C=O stretching vibration and it occurs in the range of 1700 -1600 1/cm ,sharp peak comes at 1650.8 cmThe amide II, which falls at 1541.47 cm is related to N-H bending and C-H stretching vibration. The amide III band occurs in the range of 1220 -1300 1/cm sharp peak at 1231.18 cm and it results from in phase combination of C-N stretching and in N-H in plane bending, with some contribution from C-C stretching and C=O bending vibrations. INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES ISSN: 22775005 Vol. 1 (4) Oct-Dec 2012 www.ijpcsonline.com 1451 Preparation of porous keratin sponge The porous keratin sponge was prepared by chemical method and it was analyzed by scanning electron microcopy. The scaffold contains heterogenic pores and pores present in the scaffold evenly distributed in the scaffold. The diameter of pores in the scaffold varies from 40 microns to 200 microns.