Extraction of Hydroxyapatite from Caprine Bones and its Anti-Bacterial Study

Bones are mineralized connecting tissues consisting of 70% of Hydroxyapatite (HAP); it is the major component of the bone. Hydroxyapatite (HAP) is most important biomaterial possessing all distinguish features such as antibacterial activity, bioactive and non-inflammatory behavior. Hydroxyapatite is mainly synthesized by two routes i.e. i) using inorganic substances and ii) by exploiting natural sources. The aim of this study is to extract hydroxyapatite using solid waste (animal bones) and to investigate its antibacterial activity. The extraction consisted of alkaline technique and calcination methods. During alkaline process bones became very soft and were easily converted into powder using mortar and pestle without utilizing any milling process and anti-bacterial response was higher when compared to previous studies. The characterization techniques that were utilized are; X-Ray Diffraction (XRD), Fourier Transformed Infrared Spectroscopy (FT-IR) and SEM (Scanning Electron Microscopy (SEM) in order to reveal phase composition, functional groups and surface morphology of HAP. Antibacterial activity was checked by zone of inhibition


INTRODUCTION
ones are mineralized connecting tissues consisting of 20-30 Wt% organic phase, 60-70 % is inorganic phase and around 5% consist of water [1]. The organic phase mostly consists of collagen, in addition to proteins, carbohydrates, lipids etc. that are also present in small concentration. A combination of both hydroxyapatite and collagen provides stiffness and mechanical properties to the bone. A model component present in bone is hydroxyapatite [2]. Hydroxyapatite having formula Ca10 (PO4)6 (OH)2 is bioactive ceramic material that belongs to calcium phosphate family [3]. The composition and biological structure of Hydroxyapatite resembles to natural bone mineral component. Natural HAP is one the most important biomaterial possessing all distinguish features, such as bioactive, non-inflammatory, non-toxic, osteoconductive, non-immunogenic and anti-bacterial property [2]. Due to these distinctive features, it appeals to the researcher to use it variety of biomedical applications as well as water treatment to disinfect bacteria [4].
Pathogenic microorganisms are harmful for human health and can be cause of diseases. Micro-organism can be transferred in human body by various sources. Polluted water causes many diseases and infection [20], on the other hand dental implant and bone substitute implants can also lead bacterial diseases [21]. It is found in literature that hydroxyapatite can be used to disinfect bacteria [22,23]. It is reported that mostly hydroxyapatite are obtained by chemical reactions are used in water treatment [24]. Chemically synthesized methods are often complex and costly [11]. In previous studies the chemically synthesized hydroxyapatite was used to observed antibacterial activity, but the procedures of chemically synthesized HAP are complex and not economical. In this research HAP was obtained by natural source (Solid waste, animal Bones), simple and cost effective procedure was followed by using alkaline technique and calcination methods. The antibacterial performance of naturally extracted HAP was investigated through zone of inhibition.

Materials
Caprine bones were obtained from the local slaughter house of Hyderabad, Sindh. Acetone (CH3COCH3, 99.5%), Sodium hydroxide (NaOH, 99.0 %), hydrogen peroxide (H2O2, 30.0%), Eosin methylene blue agar (EMB) were purchased from local market and all were of analytical grade. Deionization water (DI) were used throughout the research work. Table 1 shows all the chemicals and materials which were used in this research. Fig. 1 shows the flow chart of the process and Fig. 2 shows the pictorial representation of the process.

Cleaning
First of all, bones were washed thoroughly with tap water for several times and boiled for 3-5 hours for removing of muscular attachments. Then bones were treated with solution of acetone and sodium hydroxide for further removing of any attached impurities.

Alkaline method
A solution containing 10% of NaOH was prepared for the immersion of bones. Bones were immersed in a solution for one day. After that bones were washed several times with distilled water to maintain its pH as a neutral before calcination. After chemical treatment bones had become very soft and these were easily converted into powder form without utilizing any milling process.

Calcination
The bone powder was then calcined in electric muffle furnace at 1200°C and holding time was 5 hours to obtain hydroxyapatite.

Fourier Transform Infrared (FTIR) technique analysis
Fourier transform infrared was used to find the presence of functional groups. Potassium bromide (KBr) was utilized for the analyzing. Bone sample and KBr was mixed with the ratio of 1:10 and grinded thoroughly with the help of mortar and pestle.
Hydraulic press was used to form transparent disk of mixed material and it was scanned for analyzing the functional groups. Fig. 3 shows the FTIR graph. In FTIR analysis, the bands at 1000-1100 cm -1 and 560-609 cm -1 were identified as phosphate group. The strongest ranges of phosphate group are 1043 and 609 cm −1 , found by Barakat [25]. The bands at 1460-1530 and 870-880 were identified as carbonate group. Hosseinzadeh [26] also found that carbonate group have intensive peak at 1417 and 873 cm −1 . The absorption bands within 3456-3775 cm -1 are due to O-H ion. In FTIR spectra, in the region within 1500 to 3000 cm -1 , there is no intensive peak which indicates that there is complete removal of organic components during chemical immersion of bones.

Scanning Electron Microscopy (SEM)
Scanning Electron Microscopy (SEM) JEOL JSM 6380_L model was used to study the surface morphology and crystal size of the HAP samples. The samples were coated with gold to give conductivity and protection from the beam damage.

Fig. 5 reveals the Scanning Electron Microscopy
(SEM) results of prepared hydroxyapatite. SEM determines the surface morphology and particle size of HAP extracted from caprine bone. SEM graph shown that particles had non uniform shapes including small spheres and agglomerated in some areas. It was observed that the particles were swelled up because of molar concentration of NaOH.

Anti-bacterial study
Anti-bacterial activity of naturally extracted HAP disk was studied by using disk diffusion method. Following standard procedure, for the cultivation of microorganism, EMB (Eosin methylene blue) agar was poured and evenly distributed into Petri plates. E. coli (Escherichia Coli, KCCM 40880, gram negative) that was incubated in tryptic soy broth for 24 hours at 30 °C was spread into nutrient containing plates by spread plate method. 2 disc samples of 10 mm diameter were prepared by using hydraulic press. The produced HAP discs was placed into the Petri plate and incubated at 30 °C for 24 hours. After that clear area around the disc was studied and measured by ruler.

Anti bacterial response (Zone of inhibition)
The result of antibacterial bacterial activity is shown in Fig. 6. The clear area around the HAP disc shows the antibacterial activity. The zone of clear area was measured by ruler [27]. The diameter of clear zone around the disc was 25mm.

CONCLUSIONS
An experimental study was carried out to prepare the HAP using natural method. On the basis of this study, it can be concluded that hydroxyapatite was successfully extracted from caprine bone using alkaline method. It is found that the process for the extraction of HAP is simple and cost effective. Antibacterial activity was determined by zone of inhibition; the diameter of clear zone was 25mm thereby indicating the antibacterial activity of HAP. An improved response of anti-bacterial activity was observed than that of previous study.