WO2023061547A1

WO2023061547A1 - A novel process for the preparation of nano-natural hydroxyapatite by two different methods

Info

Publication number: WO2023061547A1
Application number: PCT/EG2021/000033
Authority: WO
Inventors: Yasser Mohamed ELKAMARY
Original assignee: Elkamary Yasser Mohamed
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2023-04-20

Abstract

Natural hydroxyapatite has a many unique properties that enable it to be candidate of multiple medical applications. The nano hydroxyapatite is a high performance substance due to high surface area to volume ratio that can produce rapid remodeling of bone defects, tissue engineering application & drug delivary system. The natural hydroxyapatite in a nano-scale is superior than micro &macro scale in all different application.

Description

A novel process for the preparation of nano-natural hydroxyapatite by two different methods

Technical Field:

Biomedical Application

Background Art:

Biological materials are commonly used medicinally in orthopedics , dentistry , jaws and face surgeries.

This biological material (natural or synthetic) could be introduced into living bodies for a certain period to treat or replace a tissue , an organ or a function in this body.

These biological materials should possess physical , chemical .biological and mechanical properties that might vary according to the intended use.

The principle property that should exist is that its biological acceptability which ensures its safety and avoidance of inflammatory or allergenic effects accompanying body rejection.

Hydroxyapatite has many unique properties that enables it to be a candidate of multiple medical applications.

It is a highly bioactive material capable of chemical binding with bony or cartilaginous tissues when implanted in living tissues where it remains fixed in place . These positive properties are mainly due to the chemical and structural similarity with biological apatite which forms the major hard tissue in human and animal bodies.

It constitutes about 98% of enamel , 85% of dentin and 65-70% of bones based on dry weight where the remaining percentage collagen ,non collagen proteins ,fats and sugars in minute quantities.

Publications about this issues point out two main approaches for its preparation ; Firstly; where the starting materials are not natural but of synthetic chemical compounds as phosphoric acid , calcium hydroxide, nitrate or carbonate, sodium phosphate or ammonium phosphate that produces a product named "synthetic hydroxyapatite" .Secondly, where the method is based on the use of primary natural material as coral reefs and animal teeth and bones to yield "The natural Hydroxyapatite".

Synthetic hydroxyapatite preparation process may include thermal solid state reactions, solution precipitation reactions , ultrasonic exposure, vapour condensation or transformation of colloidal into gel semisolid state (sol-gel).

Natural hydroxyapatite may be prepared through anionic exchangeable reaction between calcium carbonate and ammonium phosphate salt solution or by removal of organic components of animal teeth or bones to get natural hydroxyapatite. The removal of proteins , fats and sugars is performed by alkali treatment or by burning in ovens under ambient conditions at about 1000 °C.

Disclosure of Invention:-

This invention aims at:

1-Preperation of hydroxyapatite in high purity with high degree of fixed chemical , physical ,biological and mechanical properties.

2-Achieving the highest biological efficacy when introduced into human body by maintaining the chemical and structural characteristics .

3- Preparation from cows bones which allows the use of an oxidizing air current during inceniration of the material .

Powder size adjustment could be then controlled by milling and sieving processes.

4-Based on the mentioned above , variable sizes of HA granules in the ranges 250-1000 pm , 1000-2000 pm , and less than 250 pm are fractionated . Particles of size below 250 pm are solubilized followed by recrystallization into nanohydroxyapatite .

The obtained particle ranges could be used to satisfy variable orthopedic , maxillofacial surgeries , craniomaxillofacial surgeries and tissue engineering. 5-The starting material and the chemicals used and equipment are available in low cost that allows the availability of the product in an affordable price.

6-AII the industrial process takes place at temperature not exceeding 650°c that offers low energy consumption and reduces environmental hazards. Other by- products could be recycled into useful adhesive glues .

7-The yield in average is not less than 65% of the starting bone weight.

Technical Problem:

Inspite of the success achieved in the medical application of hydroxyapatite relative to other biomaterials ,th is success is limited due to many reasons:-

A-synthetic Hydroxyapatite :

1-Non-uni formity and instability of finished product properties due to the complexity of manufacturing process and variables included such as purity of the materials used, their strength and reaction temperature.

2-The multiple crystalline phases that could be obtained as a result of the reaction between calcium and phosphate ions as dicalcium phoshphale dehydrate, anhydrous calcium phosphate , alpha or Beta tricalcium phosphate, tetracalcium phosphate and calcium hydroxyapatite . 3-Based on that it is essential to get pure hydroxyapatite that ratio between calcium ions and phosphorus ions in the reaction medium to be exactly equal to their stoichiometric ratio in their molecular formula in the compound ( ie. 1.67).This ratio is different from natural hydroxyapatite in bone tissues (1.6- 1.65) which affects adversely the performance level of the synthetic one .

4-preparation procedure involves the application of not less than 1000 °c which causes thermal decomposition of the compound into p-tricalcium phosphate and tetracalcium phosphate according to the following equation :

Caio(PO₄)₆(OH)₂

Therefore; synthetic hydroxyapatite is usually mixed with the above mentioned compounds in variable quantities that lead to its instability with respect to the readability of interaction with living host tissues and degree of dissolution by the surrounding physiological environment.

5- The high cost of the process due to the need of highly professional personnel and expensive chemicals and equipment.

B-Natural hydroxyapatite

It has been proven that this type -specially that obtained from bones-gives better results if compared with the synthetic one. This was attributed to the ability of the compound to resist different treatments during its preparation and remains very close to the biological apatite, however, Ca/p ratio shows relatively variable degrees of non-fixed properties that could affect its function due to the following reasons:

1-The transformation of coral reefs to hydroxyapatite is usually incomplete resulting a product mixed with different ratios of calcium carbonate.

2-Dental hydroxyapatite is the hardest and strongest tissue in living organisms due to the high crystallinity and high calcium to phosphorous ratio than the theoretical value .This adversely affects its biological efficiency due to the reduced response with the surrounding physiological environment. This strong structure needs thermal treatment more than 1000c with the possibility of transformation of great part of the hydroxyapatite to p-tricalcium phosphate and tetracalcium phosphate.

3-Contrary to that mentioned above ,bony hydroxyapatite has low degree of crystallinity with less stability and low Ca/P ratio (<1 .67) that renders its crystals small with high specific surface and high chemical and biological activity .This could hinder its preparation during the process of organic material removal that could be obtained by: a-Alkali treatment is not sufficient for removal of organic materials but should be aided by incerination (while in powder form) .This limits the development of oxidizing atmosphere by the use of air current because this will scatter the existing fine powder therefore much higher temperature is used that results in the above mentioned undesired transformation . b-When only direct incerination is used without alkali treatment , much much higher temperature should be applied that would increase the undesired transformation and would increase the crystal size of the HA and consequently reduce its efficiency as a biological material.

Solution The Problem :

The method used is the result of a continuous research and trials over many years and aimed at keep in the chemical and structural features of animal bones biological HA and avoidance of any changes that could affect its biological activity : The process involves the use of the minimum temperature of incerination to get rid of organic materials remaining after alkali treatment. Therefore the process involved many steps based on three main added new approaches :

Firstly:

In addition to the alkali treatment , soaking in an ionic detergent solution step has been applied followed by hot water treatment under high vapour pressure(> 100) that reduces the water polarity and surface tension rendering it better solvent for organic materials and increases its penetrability.

Secondly;

Alternative repeatition of alkali and vapour treatment several times till almost complete removal of all proteins , fats .sugars and other organic materials .

Thirdly;

The use of nickel-chrome electric furnace of 55x60x90cm dimensions that accommodates about 7kg of bones and has openings in the front ,back and side walls with air-current control aids to allow strong compressed oxidizing air stream into the incerination chamber and act also as a duct for exhaust gases.

**The steps of the new process includes

1-5 to 7 kg of adult cancellus cow bones are carefully collected after primary screening and removal of fragile porous parts surrounding the joints .

2-Mechanical removal of fats , flesh residues,. . .etc by knives

3-Washing bones with running water 4-Cutting the bones with electric saw into cylindrical pieces of about 4-7 cm length.

5-Soaking (12-24 hours) in aqueous (2-25%) ionic detergent in bone/solution weight ratio of 1-3 tol-7

6- A steel brush is used to scrap off the the external bone surface

7-After washing ,the bones are soaked in distilled water in a press boiler in the ratio 1-3 to 1-7 bone to water weight ratio .The boiler is operated for 2-5 hours under 5-8 bars

8- After washing , bone pieces are soaked in aqueous sodium carbonate solution (20-35%), boiled for 3-6 hours with compensation of vaporized water.

9-Steps 7&8 are repeated alternatively for 3-5 times

10-After washing with running water , bones are dried at 130-1 70°c for 12-24 hours

11- To ensure quality , the porosity is measured according to Arshcmedis method till fitting the required value (25-55%). When the test is within limit , next step is started, if not , step 7&8 are repeated till achieving the required value .

12- cylindrical bone pieces are piled and arranged after drying in horizontal position on perforaed refractory base inside the oven to allow the flow of compressed air stream from frontal openings in the rate 2-20 L/min with increasing . the temperature in the rate 0.5-2 °c/min till reaching 500-600 °c where the conditions are maintained for 2-5 hours .

13- After fixing of maximum temperature , air direction is reversed but in the same rate from back openings for the same period of time.

14-Stop air current , close all oven openings and switch off electricity and let the oven cool spontaneously .

15-A sample is analyzed for structural and chemical composition by XRD, XRF & FTIR

16-Clean the collected bones after cooling with a stream of compressed air, crush and grind the bones using ball mill . Fractionate the collected granules into portions using a set of sieves to get different size ranges 1000-2000 pm , 250-1000 pm and less than 250 pm .

17) particle fraction < 250 pm was further used to prepare nanohydroxyapatite by either of the following methods :

A-Top-down approach :

Ball mill was used where particles < 250 pm were loaded into the mill on wet state using a non solvent liquid . The mill was allowed to rotate at a speed of 200-400 rpm for 30-50 hours . The product was filtered , allowed to dry in an oven at 50-70 °c for 12-24 hours .

B- Buttom up approach : a- A weight of 50-150 grams of HA particles fraction < 250 pm was dissolved in 1 - li\₂ liters of 0.2-1 MHCL with stirring till obtaining a clear solution and complete dissolution of HA . This takes almost 6-12 hours . b- Add dilute ammonium hydroxide (NH₄OH) to the above solution and leave maintaining a pH in the range 6.5 - 8.5 overnight . c- Separate the formed nanohydroxyapatite by centrifugation for up to 20 hours . d- Wash several times using deionized water followed by drying in an oven at 50-70 °c for 12-24 hours

18) The obtained powder fractions are packed in pharmaceutical glass vials away from moisture or under nitrogen in weights of 1,2, 4,6 and 10 gms.

19) After fixing the rubber cap and the aluminum closure on the vial neck , the bottles are sterilized by exposure to gamma radiation (30kGy=3mega rad)using Co60 . The sterilized bottles should be used once after first opening.

Claims

1-AII the manufacturing process according to the sequence mentioned above

2-The use of cancellus cow bone parts in the process of preparation

3-lntroduction of treatment with anionic synthetic detergent solution.

4-lntroduction of aqueous high pressure steam treatment and its repeated alternative use with alkali treatment for the complete removal of organic materials accompanying natural apatite .

5-Testing the porosity as a routine test for evaluating the efficiency of organic matter removal process .

6-The furnace design and the way of generating oxidizing environment and the mode of controlling the entering air current.

7- The process of dissolution and reprecipitation of HA to obtain nanohydroxyapatite as well as its separation .

8- Particle size ranges selected .