EXTRACTION OF HYDROXYAPATITE (HA) AND PREPARATION OF
HA-CHITOSAN COMPOSITE
Abida*
1, Ayatullah Qureshi*
2, Ali Dad Chandio*
3, Khurshid Ali Mirza*
4*
1Department of Biomedical Engineering and Technology, Mehran University of Engineering and
Technology, Jamshoro, Sindh, Pakistan.
*
2Department of Metallurgy and Materials, Mehran University of Engineering and Technology,
Jamshoro, Sindh, Pakistan.
*
3Department of Metallurgical Engineering, NED University of Engineering & Technology, Karachi,
Sindh, Pakistan.
*
4Institute of Biomedical Engineering and Technology Liaquat University of medical and Health
Sciences Jamshoro, Sindh, Pakistan.
ABSTRACT
Hydroxyapatite is a biocompatible material composed of calcium and phosphate. It has widely been developed in biomedical applications due to its excellent biocompatibility, bioactivity, and osteoconduction characteristics. This material can be prepared from synthetic as well as natural resources. In comparison with synthetic, natural resources are more biocompatible and osteoconductive. The use of bones as a natural resource is efficient because of the high percentage of Cellulose acetate availability, economic and environmental factors. This study is based on the preparation of HA-chitosan composite by using solid-state mixing. The HA was obtained from Bovine, Caprine, and Fishbone. The main objective of this study was to extract Hydroxyapatite from the solid waste i.e., bones of Bovine, Caprine, and Fishbone by chemical solution method and thermal treatment. This was followed by preparation of subsequent composite with chitosan. Moreover, Scanning electron microscopy (SEM), X-RAY diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Energy-dispersive X-ray spectroscopy (EDX) performed. Results suggest that the HA obtained from Caprine bone is more preferable for biomedical applications than that of bovine or fishbone. This is because it offers more yield than that of HA obtained from fishbone and also offers optimum particle sizes. Results show the surface morphology of HA-composite, fusion of HA and chitosan, chemical composition of composite, and biocompatibility and osteoconductivity that prove an excellent repair and implant material for bone defects.
Keywords: Caprine bones, Extraction of Hydroxyapatite, Biocompatibility, HA-Chitosan Composite, Hydro-Support Implant, and Osteoconductivity.
I.
INTRODUCTION
Being rich in livestock growth, Pakistan has a sound name not only in delivering the meat but in the usage of wastage as the by-products. Hydroxyapatite is the calcium phosphate (Ca10 (PO4)6(OH) 2) which has a hexagonal layout and that have a resemblance to human bone apatite found in mammalian hard tissues. Comparing to other calcium phosphates,HA is stable in terms of thermodynamics and under the physiological condition such as body fluid configuration, pH, and temperature. On the premise of stoichiometry the temperature at which HA decomposed ranges from 800°C to 1200°C. A fusion of hydroxyapatite and chitosan creates a strong bone implant that can endure ordinary stresses experienced by the body. For increasing its strength HA can be entrapped with bio polymeric material so that it can be used as a replacement of bone and also can be used as a bone filler material. In the previous time, HA is naturally extracted from bovine and pig bone but the manufacturing cost is high. So here we extract HA from the caprine bone due to good biocompatibility property and its cost-effectiveness and is environmentally good.
Chitosan is derived from chitin. Chitosan is not only a polymer but also a polysaccharide that explains the biodegradable nature, which contains glycosides breakable bonds. Chitosan is biocompatible and nontoxic so that it can be used in medical applications as Antimicrobial and wound healing biomaterials. However, it is soluble in dilute aqueous acidic medium (pH < 6.5). The very first article which explains about the structure
and composition of bones, teeth and the different types of tissues that were classified has been appeared in the late seventeenth century. Fourcroy in the end of 18th century and current times it is called as (MCPA), (MCPM), (DCPA), and (DCPD) Organic and in-organic complex nature of bone has been familiar since 1788. Different methods were developed for the formation of calcium- deficient HA in 1807. In 1809 the production phenomena of HA, structure, properties and composition of bones were described in details. Chemical constitution of apatite was presented by the German mineralogist Gustav Rose in 1827. In 1832 β-TCP and α-TCP are introduced having the chemical name “tribasic phosphate of lime”. In 1846 Calcium phosphate existence in corals has been discovered and the solubility tests of first calcium phosphate have been reported. In the 19th century the very first well-recorded studies on allografts and auto grafts was published. After
trepanation, the surgically tear out parts of skull were replaced with auto graft of bone by German surgeon and ophthalmologist Philipp Franz von Walther. The American surgeon Fred Houdlette Albee in 1920, made the first trial for placing a laboratory-made calcium phosphate (TCP) as an unnatural material to mend surgically in rabbit bones generated fractures. In early 1930s, structure of apatite was described. This was demonstrated 1948 that only Particular kinds of calcium phosphates affect the process of bone healing. The distinct calcium Phosphate perimeter were strained between hydroxyapatite (HA) and OCP in 1950.The hydroxyapatite powder into dense and useful forms, also hot pressed was reported in 1969 .In 1975, the modern applications of calcium phosphate were started. For immediate tooth- root substitution, HA cylinders (in dense form) were used in 1979. In 1976, the background of calcium phosphate films, coatings and layers started although calcium phosphate based hybrid biomaterials and bio- composites started in 1981. The dental community in early 1980s started the use of hydroxyapatite blocks and coatings in restorative dental procedures to improve bone to peruse fixation; the tremendous biocompatibility and chemical stability of hydroxyapatite made this a choice for fascinating material. Therefore, the community of orthopedics started utilization of hydroxyapatite for an implant coating and in bone defect augmentation. In the 1980s, a rapid-commercialization of calcium phosphate bio ceramic in dental markets and orthopedics occurred and it was led by de Groot in Europe, Aoki in Japan and Jarcho in the USA. In 1985, Furlong implanted first HA by coating of main hip prosthesis in humans.
In 1859, C. Rouget published his findings that “modified chitin” could be prepared by treating chitin with boiling, concentration solutions of potassium hydroxide in water and he observed that chitin can be manipulated via chemical- and temperature-dependent treatment. In 1930s chitosan derived from chitin source such as shrimp shells, crabs, lobster, krill, and mushroom had been confirmed by Rammelberg. In 1960s, chitosan was studied as a hemostatic agent for its ability to bind with red blood cells, for water treatments, and detoxification. The period of application started from 1970. Although this historical view is not comprehensive, it just highlighted the work done by researchers and those who have contributed to enhance our knowledge for chitin throughout the 220 years of its history.
II.
MATERIALS AND METHODOLOGY
Materials
Raw Materials for HA Preparation
For the extraction of hydroxyapatite, the bovine, caprine, and fishbone were utilized as the raw materials. In addition, sodium hydroxide (NaOH) and hydrogen peroxide were used for synthesis as shown in Figure 1.
Figure 1: Shows the raw materials used to prepare the HA
.
Materials for Preparation of HA-Chitosan CompositeFor the preparation of HA-chitosan composite, the HA from the bones of Caprine, Maleic acid, Zinc oxide, Calcium oxide and all necessary apparatuses such as Beakers, China Dishes, spatula, electronic weight machine and gloves etc. were required as shown in Table 1.
Table 1: Shows the raw materials required for HA-Chitosan Composite
Methods
The following processes and treatments were carried out in this research as shown in subsequent sections. Methods for HA Preparation
Cleaning
First of all, 250gm of raw goat bones (Caprine) were collected. The bones were washed with tap water and then boiled to remove the muscular attachments. The remaining tissue parts were detached with the help of dagger. To remove further impurities, the bones were treated with solution of acetone and deionized water for 4-5 hours. The cleaning process is shown in Figure 2.
Figure 2 Shows the cleaning bath along with bovine, caprine, and fish samples. Chemical treatment
Cleaned bones were divided into nine equal set of samples of 250gm each. These samples were immersed in three different concentrations of different solutions. Solution A consisted of 5% sodium hydroxide (NAOH), 2.5 % hydrogen peroxide (H2O2) and 92.5% deionized water while solution B contained 10 % NAOH, 5 % H2O2 and
85% deionized water. Whereas, solution C contains 15% NAOH, 10% H2O2, 95% deionized water for 100ml of
solution. In order to analyze the effect of different temperature and concentration of NaOH, 3 samples were treated at room temperature for different hours i.e., 24, 48, 96 and 192 hours while other 3 samples were
Materials Weights Extracted Hap 63.10g Chitosan 23.1g Calcium oxide 7.0g Zinc oxide 4.8g Malic acid 2.0g
treated in water bath for 3 days. Moreover, rests of three samples were thermally treated. Process flow diagram is shown in Figure 3 which exhibits the sequences and other details.
Furthermore, after every 24 hours, the solutions were renewed. The beakers were enclosed with sample dishes. As time passed the pH reduction was observed in all samples. This was due to the breaking of bonds between inorganic and organic molecules of bones. Finally, after chemical treatment, the samples were dried by using a dryer.
Thermal Treatment
All the chemically treated samples were placed in Evaporated dishes (china dishes). The dishes were placed in programmed automatic furnace for 5-6 hours at 1000℃ and 1200℃. After thermal treatment samples were ground to reduce the particle sizes. The physical view of the sample is shown in Figure 4.
Figure 3 Shows the schematics of thermal treatment carried forward. Methods for HA-Chitosan Composite
Weighing Samples
The prepared hydroxyapatite material appeared chalky having is 2.78µm average in diameter. The samples were measured by using the weight balance machine. In total, one set of material consisted of 100 g of composite as shown in Table 1.
Chemical Treatment
23.1g of chitosan was soaked in water thoroughly for about 30_40minutes then 83.1 g of hydroxyapatite is taken and is properly mixed with 7 g of CaO and 4.8 g of ZnO upon a magnetic stirrer about 1 hour. The temperature is kept 50° C for 30minutes to achieve the uniformity in the mixture and then it reduces to zero the soaked chitosan were added into the hydroxyapatite blended mixture at the time when temperature has been dropped. After a while when mixture turns into a gel like appearance malic acid was put in to it to form a paste. Composite Formation
The composite has achieved a desired consistency and it can be formed into cylindrical balled shaped as that of the human bone. Small amount of composite paste is taken as a sample for the characterization.
Figure 5 Shows the prepared HA-chitosan composite. Characterization Techniques
In this study, the following characterization techniques were utilized for the both HA and HA-Chitosan composite.
FTIR (Fourier Transform Infrared Spectroscopy)
FTIR is a high-strung method specially for identifying the organic chemicals in entire range of applications; however, FTIR can also use to characterize some inorganics. For instance, resins, polymers, paints, coating, drugs and adhesives. It is an essential tool for separating and characterizing organic components.
SEM (Scanning Electron Microscopy)
The Scanning electron microscopy (SEM) is a versatile and powerful tool for characterization of a material. SEM is used to determine the crystal size and surface morphology of a material.
XRD (X-ray Diffraction)
X-ray powder diffraction (XRD) is a fast analytical method basically used for phase identification of a crystal structured material that provides the information on unit cell dimensions. The material that has been analyzed is finely ground, homogenized, and average bulk composition is examined.
EDX (Energy Dispersive X-ray)
EDX is a x-ray procedure used to recognize the natural composition of materials. Applications incorporate materials and investigating, deformulation, product research, and more.
III.
RESULTS AND DISCUSSIONS
FTIR
Table: 2 Shows the functional Groups and Their Characteristic Wavelengths
S No: Functional Groups Characteristic Wave length(cm-1)
1 OH 2500 -3300
2600 -3600
2 PO4 560-609
3 CO3 870 -880
1460 -1530 (intensive peak) 4 Absorbed water 2600 -3600 Cm-1
Figure 6 Shows the FTIR spectrum of HA Bovine Sample
Figure 6 showing the FTIR spectrum of HA extracted from bovine Sample. The first demonstration of hydroxyapatite is the presence of phosphate group in the calcined sample at about609 and 1040 cm−1. The strongest ranges of phosphate group are 1043 and 609 cm−1, found by Perklin spectrum. Two peaks at 1068 and 1452 cm−1.
In FTIR spectrum there is not any intensive or minor peak between the region of 1500 to 3000, which is showing complete removal of organic components or deproteinization. Fig 6 FTIR spectrum of HA Bovine sample (24, 48, 96,192h, 3days (WB)), showing that when we treat the samples for more hours the removal of organic substances increases. At 192h is showing more removal of organic substances indicating more deproteinization.
Figure 7 Shows the FTIR spectrum of HA Caprine Sample
The figure 7 demonstrating FTIR spectrum of HA extracted from Caprine Sample. Likewise, bovine, Caprine 192h spectrum is better than 24, 48 and 96h due to increase in time. While the peaks of sample WB is suitable than all other samples. Similarly, the removal of organic components is greater in the samples which were treated for more hours. Hence, Caprine sample spectrum showing best result. As samples were treated with greater concentration of NaOH, the deproteinization effect is better.
Figure 8 Shows the FTIR spectrum of HA Fish Sample
Figure 8 demonstrating the FTIR spectrum of HA extracted from Fish sample .At Water bath, sample were treated on 24h,48h,96h,192h temperature, concentration same interval of time .Graph is showing the greater removal of OH and deproteinization of organic substances. But Caprine bones are softer and their internal structure is more porous that favors more degradation.
Figure 9 Shows the illustration of different samples of FTIR spectrum of HA
The ranges of transmittance spectrum of HA powder is 4000‐450 cm−1 . The typical FTIR spectra of the samples had Wavelength bands corresponding to PO4 -2, OH− and CO3 -2. Through comparison between spectra, this can be observed that only transmission peaks were found in all treated samples of HA.
SEM
The surface morphology and particle size of extracted HA were determined by SEM (scanning electron microscope) analysis. The SEM (scanning electron microscope) micrographs of the samples obtained from Bovine, Caprine and fish bones are shown below.
Figure 10. Shows SEM analysis of bovine
These SEM micrographs are indicating that the particles have non-uniform and asymmetrical in shape. Moreover, it can be observed that Bovine a) particles are swelled up more as compare to bovine b), because greater concentration of NaOH in b) but the c) is indicated with higher concentration. Moreover, the samples treated on water bath bulged more as compare to those samples which were treated at room temperature because with increase in temperature the removal of organic substances also increases that lead to more degradation of structure.
Figure 11. Shows SEM analysis of caprine
SEM analysis of Caprine a, b and c samples on Water bath. The SEM micrograph shows that HA particles are non-uniform in shape. It can be observed that Caprine b) is more deteriorated than Caprine a) due to higher concentrations of NaOH in b) samples. While comparing bovine and Caprine bones the structure of bovine bone is denser than Caprine bone so it take more time to deform, hence Caprine c) WB favors’ more degradation and allow greater removal of organic substances.
Figure 12 SEM analysis of fish
SEM analysis of Fish a), b) and c) samples on water bath. The SEM micrograph shows that HA particles are non-uniform in shape. It can be observed that Fish S1 is more deteriorated than Fish S2 due to higher concentrations of NaOH in a) samples.
While comparing bovine and Caprine bones the structure of fish bone is lighter than Caprine and bovine bone so it took less time to deform, hence fish c) WB favors’ more degradation and allow greater removal of organic substances.
XRD
The XRD sharp peak of bovine sample. It indicates the better crystallinity structure at 31.8 A°.
Figure14. Shows the XRD of caprine The XRD sharp peak of caprine sample. The characteristic peak of caprine bone at 31.7
.
The XRD sharp peak of fish sample. Figure shows the good characteristic structure of fish bone XRD peak at 31.8.
Figure 16 Shows the illustration of different samples of XRD
The XRD sharp peaks of fish, caprine, and bovine sample. Figure shows the good characteristic structure of sample XRD peaks at 32.
SEMOFHA-CHITOSANCOMPOSITE
Figure 17 Shows the SEM of ha-composite
These SEM micrographs are indicating that the particles have uniform and symmetrical in shape. Moreover, it can be observed that particles are little swelled up, because greater concentration of NaOH in sample but the f) is indicated with higher concentration.
Figure 18 Shows the XRD of HA-composite
The XRD sharp peaks of HA-composite sample indicate the better crystallinity structure at 32.8 and 45 A°.
FTIROFHACOMPOSITE
Figure 19 Shows the FTIR of HA-composite
Figure 19 showing the FTIR spectrum of HA composite Sample. The first demonstration of hydroxyapatite is the presence of phosphate group in the calcined sample at about 509 and 1040 cm−1. The strongest ranges of phosphate group are 1043 and 809 cm−1, found by Perklin spectrum. Two peaks at 1068 and 1452 cm−1. In FTIR spectrum there is not any intensive or minor peak between the region of 1500 to 3000, which is showing complete removal of organic components or deproteinization.
Figure 20 Shows the EDX of HA-composite
Figure 20 shows x-ray procedure used to recognize the natural composition of materials. EDX of HA-composite showing the presence of Ca at 0.3 and 3.8, Zn at 1 and 5.7, P at 2, Al at 1.5, C at 0.2 and Mg at 1.3.
IV.
CONCLUSION
This comparative study was carried out to extract HA from Bovine, Caprine and fishbone. This was followed by the formation of composite using HA and chitosan together with varying compositions. The aim of this study was to understand the preparation methods and to understand the performance of HA-chitosan composite. This was followed by the characterization using FTIR analysis confirmed the presence of carbonate, phosphate and hydroxyl groups along with complete removal of organic substances in HA. SEM analysis determined that the HA particles have irregular shapes including small spheres and accumulated together in some areas, and XRD analysis of HA was also performed. Results suggest that HA extracted from the caprine bone yields better results and the pattern of crystalline structure. Moreover, composites prepared using mixture of HA and chitosan showed excellent biocompatibility.
ACKNOWLEDGEMENT
I would like to express my sincere gratitude to all those who provided me the necessary support, motivation, and guidance to complete the research work. Very special thanks to my Supervisors Dr. Ali Dad Chandio (Chairman and Associate Professor, NED University Karachi, Sindh) and Engr. Ayatullah Qureshi (Lecturer at Metallurgy and Materials, MUET Jamshoro, Sindh) for being a constant source of guidance and inspiration. I also extend my wholehearted thanks to my family for never ending support.
V.
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