Technical Field:

Biomedical Application Background Art:

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

This biological material (natural or synthetic) could be introduced into l iv ing 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 acceptabi lity which ensures its safety and avoidance of inflammatory or allergenic effects

accompanying body rejection.

I iydroxyapatite 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 simi larity with biological apatite which forms the major hard tissue in human and animal boodles. lt constitutes about 98% of enamel , 85% of dentin and 65-70% of bones based on dry weight where the remaining percentage collagen ,ηοη 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 hydroxylapatite' ^, .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 col loidal 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 1 000 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- Preperation as medi um size lumps (not as fine powder )from cows bones which allows the use of an oxidizing air current during inceniration of the material . Powder size adjustment could be then control led by mi l ling and sieving processes.

4- Based on the mentioned above variable sizes of HA granules in the ranges 1 000- 2000, 600- 1 000, 250-600 and less than 250u could be obtained to satisfy variable orthopedic and maxillofacial surgeries.

5- The starting material and the chemicals used and equipment are avai lable i n low cost that allows the availability of the product in an affordable price. 6- A11 the industrial process takes place at temperature not exceeding 650c that offers low energy consumption and reduces environmental hazards. Other byproducts could be recycled into useful adhesive glues

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

I nspite of the success achieved in the medical application of hydroxyapatite relative to other biomaterials .this success is limited due to many reasons:-

A-synthetie 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 calci um phosphate , alpha or Beta tricalcium phosphate, tetracalcium phosphate and calcium hydroxyapatite

3- Based on that it is essential to get pure hydroxyl appatite that the 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 1 000c wh ich causes thermal decomposition of the compound into β -tricalcium phosphate and tetracalcium phosphate according to the following equation :

Therefore; synthetic hydroxyl appatite is usually mixed with the above mentioned compounds in variable quantities that lead to its instability with respect to the readabi lity of interaction with l iving 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 i f compared with the synthetic one. This was attributed to the ability of the compound to resist di fferent treatments during its preparation and remains very close to the biological appatite,however, Ca/p ratio shows relatively variable degrees of non-fixed properties that could affect its function due to the fol low ing reasons:

1 -The transformation of coral reefs to hydroxyl appatite is usually incomplete resulting a product mixed with di fferent ratios of calcium carbonate.

2- Dental hydroxyapatite is the hardest aiid strongest tissue in l iving organisms due to the h igh crystal l inity 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 environ.ment.This strong structure needs thermal treatment more than 1 000c with the possibility of transformation of great part of the hydroxyapatite to β-tricalcium phosphate and tetracalcium phosphate.

3- Contrary to that mentionrd above,bony hydroxyl appatite has low degree of crystal l inity with less stability and low Ca/P ratio (< 1 .67) that renders its crystals smal l w ith high speci fic 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-The infinitismal building level of tiny appatite granules integrated with organic tissues necessitates the alkal i or the thermal treatment of l ittle quantities (few gms) of fine powder (<500 urn) which limits the use of the finished product where higher particle size is recommended(500-2000um).

b-Alkal i treatment is not su fficient for removal of organic materials but should be aided by incerination (while in powder form) .This l imits the development of oxidizing atmosphere by the use of air current because this will scatter the ex isting fine powder therefore much higher temperature is used that results in the above mentioned undesired transformation . c-When only direct incerination is used without alkali treatment , much much h igher temperature should be applied that would increase the undesired

transformation and would increase the crysral size of the H A 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 keeping 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. There fore the process involved many steps based on three main added new approaches :

Firstly:

In addition to the alkal i treatment , soaking in an ionic detergent solution step has been applied followed by hot water treatment under high vapour pressure(> 1 00) 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 til l almost complete removal of al l 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 wal ls with ai r-current control aids to allow strong compressed ox idizing 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 cortical 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 ( 1 2-24 hours) in aqueous (2-25%)ionic detergent in bone/solution weight ratio of 1 -3 to I -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 udcr 5-8 bars

8- A fter washing ,bone pieces are soaked in aqueous sodium carbonate sol ution (20-35%), boi led for 3-6 hours with compensation of vaporized water.

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

1 0- After washing with running water ,bones are dried at 1 30- 1 70c for 1 2-24 hours

1 1 - To ensure qual ity ,the porosity is measured according to Arshcmedis method til l fitting the required value (25-55%). When the test is within l imit ,next step is started, if not ,step 7&8 are repeated till achieving the required value

1 2- cyl indrical 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/mi n with increasi ng the temperature in the rate 0.5-2 c/min till reaching 500-600c where the conditions are maintained for 2-5 hours

1 3- After fixing of maximum temperature ,air direction is reversed but in the same rate from back openings for the same period of time. 1 4- Stop air current , close all oven openings and switch off electricity and let the oven cool spontaneously .

1 5- A sample is analyzed for structural and chemical composition by XRD,XRI ^; &FT-1R.

1 6- Clean the collected bones after cooling with a stream of compressed air ,crush and grind the bones using ball mill and pass through a 2000u sieve. Frationate the collected granules into portions using a set of sieves to get di fferent size ranges 1 000 -2000 u,600- 1 000,250-600 and <600u

1 7- The obtained powder fractions are packed in pharmaceutical glass vials away from moisture or under nitrogen in weights of 1 ,2,4,6& 1 Ogms

1 8-After fixing the rubber cap and the aluminum closure on the vial neck ,the bottles are steril ized by exposure to gamma radiation (30kGy=3mega rad)using Co60.The sterilized bottles should be used once after first opening.