The Patents Rules, 2003

COMPLETE SPECIFICATION

(See section 10 and rule 13)

Bioactivity of fluorophosphate glasses and method of making thereof

Pandian Bio-Medical Research Centre

36, Sivagangai Road

Madurai-625 001

Tamil Nadu. INDIA.

The following specification particularly describes the invention and the manner in which it is to be performed DESCRIPTION

Field of invention

The present invention describes the composition of fluorine added phosphate based glasses by melt quenching technique for different bio-medical applications.

Background of invention with regard to the drawback of associated known art

A number of materials have been examined for their ability to regenerate new bone. Currently, autologous bone remains the preferred material in bone graft and regenerative procedures. This bone, taken from a secondary site within the body is often excised and reimplanted. It contains both the inorganic mineral hydroxyapatite as well as the cell characteristics of bone. Even this is not a living material, but can remodel into new and functional bone. Autografts may be combined with supplementary agents such as growth factors or synthetic bone replacement materials. The disadvantage of autologous bone usage is the creation of a secondary trauma site that has to heal. Further, its usage is limited by availability. The materials taken from cadavers (allograft) are not constrained by supply. The above material is often demineralized, leaving behind a collagenous scaffold for the growth of new bone. Further, it fails to function as an osteoinductive but only as osteoinductive material. These materials always carry the risk of disease transmission with them.

The third generation of materials used for the regeneration of new bone is bioactive glass. These materials have the unique ability to form a chemical bond with the host tissue when implanted. Thus, it allows to retain the structural integrity and to resist movement at the implantation site. The chemical bond that occurs through the formation of hydroxyapatite on the glass surface converts the glass into bone rich material. The beneficial effects of bioactive glasses on cellular growth also substantiate to their use.

Attempts were made to synthesis artificial bone graft material which is suitable for different biomedical applications. L.L. Hench in Imperial college, London succeeded in developing silica based material, named Bioglass, in the year of 1960. But, it was hard to get good compatibility with biological tissues because of their relative insoluble nature. Silica-free phosphate glasses, namely bioactive glasses, were made in the year 1996. These glasses show better bioactivity due to their chemical composition, which is compatible with that of natural bone. But still the slow rate of biological conversion of bioactive glasses hampers its clinical application. To circumvent these pitfalls a better composition of bioactive glass with higher porosity, elastic moduli, mechanical strength, bioconversion and controlled rate of dissolution near that of natural bone is continuously searched. Object of invention

Fluorine molecule is used to promote bone formation and this fact is well known by years of clinical experience and hence is used in medical practice. As per the guidelines from environmental protection agency (EPA), India, 4 ppm of fluorine is the permitted level in water. Consuming excess amount of fluorine above that for a long period of time is known to cause fluorosis, a disease of abnormal bone formation even in soft tissues in the body. This observation is taken into consideration to induct fluorine in an optimal nontoxic dose as a component for higher rate of bone growth to produce a specific type of glass, namely fiuorophosphate glass, which is having higher rate of apatite forming ability than the available bioglasses and bioactive glasses.

Fiuorophosphate glasses so far made have been put into several industrial applications. US patents such as 20130135714, 20130134362, 20120258848, 20120090358, 20090325774, 20040023786 etc. are few examples for the use of fiuorophosphate glasses in laser amplification, optical fiber, battery electrode applications. To the best of our knowledge, no study has been done on fiuorophosphate glasses for bone tissue engineering. To develop a better composition of fiuorophosphate glasses which is having higher biological conversion and less toxic to biological tissues for different biomedical application is the objective of the present invention.

Statement of invention

1. The protocol for melt quenching method to produce fiuorophosphate glass which will incorporate fluorine in it has been identified.

2. The proportion of fluorine which will yield the best bioactivity has been optimized.

3. Physical properties of fiuorophosphate glass have been accessed.

4. The byconversion of fiuorophosphate glass has been ascertained.

5. Nontoxic nature of fiuorophosphate glasses up to the addition of 10 mol% of fluorine content has been confirmed.

A summary of invention

Fluoride added phosphate based glasses and methods of making and the use thereof are disclosed. A rich layer of hydroxyapatite (HAp) is formed on the surface of fluorophosphate glasses during in vitro studies. The ex vivo cell culture studies confirmed that the prepared glasses are non-toxic and exhibit better cell viability with the addition of fluoride molecule up to 10 mol% in glass composition. The physical properties of the fluoride added phosphate based glasses were assessed using ultrasonic velocity measurements, x-ray diffraction (XRD) patterns, Fourier transform infrared spectra (FTIR), pH variation of the sample glasses soaked 21 days in the laboratory prepared simulated body fluid (SBF) solution, scanning electron microscope (SEM) images, energy dispersive x-ray spectra (EDS) and x-ray photo electron spectrographs (XPS).

The bioconversion was confirmed using in vivo animal studies by implanting the glass into femoral condyle of matured rabbits for 10 weeks followed by SEM images, EDS spectra, confocal laser scanning microscopy (CLSM) images.

Brief Description about Drawings

The experiment details and evidences such as density variations, elastic moduli, SEM images, EDS spectra, XRD pattern, FTIR spectra, CLSM image, pH variations during 21 days of in vitro studies, cytotoxicity studies, etc. of the prepared glass samples for the proposed invention are shown in the Figs. Fig. 1 shows the flow chart for synthesis protocol. Figs. 2, 3a and 3b show the density variations and elastic moduli of the prepared glass samples as a function of added CaF ₂ . Fig. 4 shows the XPS spectrograph of the glass sample FP3. Fig. 5 and 6 show respectively the SEM image and EDS spectrum of the prepared glass sample FP3; Fig. 7 shows the pH variations of the glass samples FP1, FP2, FP3, FP4 and FP5 during 21 days of in vitro studies. Fig. 8 and Fig. 9 show respectively the FTIR spectrograph XRD pattern of all the glass samples after 21 days immersion in SBF solution. Figs. 10 and 11 show the SEM image, EDS spectrum of the glass sample FP3 after in vitro studies; Fig. 12 shows the optical microscope image of cell viability test of the sample FP3. Figs. 13a, 13b and 13c show the SEM images of un-decalcified section of the femoral condyle of the rabbit bone after 10 weeks implanting. Figs. 14a and 14b show the CLSM images of the un- decalcified section of the implant FP3. Description of invention

Fluorophosphate glass composition P2O5— CaO— Na ₂ 0— CaF ₂ , methods of preparation and use thereof are disclosed. The glasses can be used for different bio-medical applications such as bone substitute, prosthetic implants, stents, screws, plates, tubes, controlled drug delivery etc. Fluorine was added in incremental pattern in various compositions, keeping the P/Ca ratio as constant. The fluorophosphate glasses were prepared in the mentioned compositions include various salts in mol% within the following ranges:

S. No. Salt Ratio in mol%

1. Phosphorus pentoxide (P2O5) 35-65

2. Calcium oxide (CaO) 22-36

3. Sodium oxide (Na ₂ 0) 11-32

4. Calcium fluoride (CaF ₂ ) 0.01-20

To access the variation if any, the following chemicals were alternatively used as per its proportionate molecular weight:

S.No. Required salt Alternatively used

1. p ₂ o ₅ Ammonium di hydrogen phosphate, Phosphorous chloride, ammonium phosphate, calcium phosphate, sodium phosphate, silver phosphate etc.

2. CaO Calcium sulphate, calcium carbonate, calcium fluoride, calcium fluorophosphate, calcium chloride, calcium caseinate, calcium bicarbonate etc.

3. Na ₂ 0 Sodium carbonate, sodium citrate.

4. CaF ₂ Calcium di fluoride, calcium tri fluoride, calcium fluorophosphate, sodium fluoride etc.

The influence of fluorine on phosphate glass system is studied in terms of density measurement, ultrasonic velocity measurement, SEM image, EDS spectra, XRD pattern and pH variations during in vitro studies and by in vivo studies using animal model. The structural role of fluorine is noticed in the form of loose packing of glass network. The hydroxyapatite (HAp) forming ability of prepared glasses is carried through in vitro studies in simulated body fluid (SBF). The scanning electron microscopy (SEM) images before and after in vitro studies show the formation of HAp in all glass surfaces, while a higher rate of formation of HAp is evidenced on fluorine added glasses rather than fluorine free glass. Further, FTIR spectra and XRD patterns that are observed support its higher bioactivity. In vivo animal studies confirmed the bioconversion of glass to bone. Hence fluorophosphate glasses are found to be more suitable for synthetic bone graft material. Materials and method

Fluorophosphate glasses were prepared by melting the homogeneous mixture of phosphate, calcium, sodium and fluoride salts followed by sudden quenching of the melt. The derivatives of phosphate, calcium, sodium and fluoride salts were weighed accurately and ground using mortar and pestle/ planetary ball mill . The mixture was fed into alumina crucible and then preheated at a temperature ranging from 140 °C to 190 °C for 1 h in a closed furnace and cooled to room temperature at a rate of 1 °C per minute. The preheated mixture was ground using mortar and pestle/ planetary ball mill. The mixture was taken in a platinum crucible and melted at the temperature ranging from 1050 °C to 1400 °C for 1 h. The temperature in the furnace was kept constant throughout the process. The melt was poured in a preheated graphite/ steel mould and cooled to room temperature. In this method, the entire synthesis protocol is successfully done without the usage of any toxic chemicals.

The method for preparing fluorophosphate glasses comprises the following steps:

a) The calculated quantities of the chemicals were weighed accurately using an electronic balance.

b) The weighed chemicals were ground using mortar and pestle/ planetary ball mill to obtain the homogeneous mixture.

c) The mixture was preheated at a temperature ranging from 140 °C to 190 °C for 1 h in a closed furnace and cooled to room temperature at a rate of 1 °C per minute.

d) The preheated mixture was ground using mortar and pestle/ planetary ball mill to obtain the homogeneous powder.

e) The obtained homogeneous mixture was taken in a platinum (10% Rhodium doped) crucible and melted at the temperature ranging from 1050 °C to 1400 °C for 1 h in electric furnace.

f) The melt was suddenly quenched in a preheated steel/ graphite mould at temperature of 300 °C - 600 °C and then cooled to room temperature.

g) The obtained solid glass sample is annealed at 300 °C - 500 °C for 1 h and cooled at the rate of 0.5 °C - 2 °C per minute.

h) The prepared glass sample is cut into required shape and size using a diamond cutter. Example 1:

21.5549 g of P ₂ 0 ₅ , 5.6770 g of CaO, 2.7681 g of Na ₂ 0 pure chemicals were taken in agate mortar/ planetary ball mill. Ethanol was added with the mixture and ground for 1 h to obtain a homogeneous mixture. The mixture was dried at 100 °C -200 °C for 1 h and ground using agate mortar/ planetary ball mill to obtain a fine powder. The powdered mixture was melted in platinum doped with 10% Rhodium crucible and heated at the temperature range of 1050 °C - 1400 °C for 1-4 h then quenched in a preheated stainless steel/ graphite mould of temperature 300 °C - 600 °C and then cooled. The prepared glass sample was annealed at 300 °C- 500 °C for 1 h and then cooled at the rate of 0.5 °C - 2 °C per minute to release the stress in the glass sample. The prepared glass sample was cut into required size and shape using a diamond cutter. The code for the present sample is called as FP1.

Example 2:

20.6220 g of P ₂ 0 ₅ , 5.4313 g of CaO, 3.0015 g of Na ₂ 0, 0.9459 g of CaF ₂ pure chemicals were taken in agate mortar/ planetary ball mill. Ethanol was added with the mixture and ground for 1 h to obtain a homogeneous mixture. The mixture was dried at 100 °C -200 °C for 1 h and ground using agate mortar/ planetary ball mill to obtain a fine powder. The powdered mixture was melted in platinum doped with 10% Rhodium crucible and heated at the temperature range of 1050 °C - 1400 °C for 1-4 h then quenched in a preheated stainless steel/ graphite mould of temperature 300 °C - 600 °C and then cooled. The prepared glass sample was annealed at 300 °C- 500 °C for 1 h then cooled at the rate of 0.5 °C - 2 °C per minute to release the stress in the glass sample. The prepared glass sample was cut into required size and shape using a diamond cutter. The code for the present sample is called as FP2.

Example 3:

19.1441 g of P ₂ 0 ₅ , 5.0421 g of CaO, 4.6439 g of Na ₂ 0, 1.1699 g of CaF ₂ , pure chemicals were taken in agate mortar/ planetary ball mill. Ethanol was added with the mixture and ground for 1 h to obtain a homogeneous mixture. The mixture was dried at 100 °C -200 °C for 1 h and ground using agate mortar/ planetary ball mill to obtain a fine powder. The powdered mixture was melted in platinum doped with 10% Rhodium crucible and heated at the temperature range of 1050 °C - 1400 °C for 1-4 h then quenched in a preheated stainless steel/ graphite mould of temperature 300 °C - 600 °C and then cooled. The prepared glass sample was annealed at 300 °C- 500 °C for 1 h and then cooled at the rate of 0.5 °C - 2 °C per minute to release the stress in the glass sample. The prepared glass sample was cut into required size and shape using a diamond cutter. The code for the present sample is called as FP3. Example 4:

18.8827 g of P ₂ 0 ₅ , 4.9732 g of CaO, 4.9077 g of Na ₂ 0, 1.2364 g of CaF ₂ , pure chemicals were taken in agate mortar/ planetary ball mill. Ethanol was added with the mixture and ground for 1 h to obtain a homogeneous mixture. The mixture was dried at 100 °C -200 °C for 1 h and ground using agate mortar/ planetary ball mill to obtain a fine powder. The powdered mixture was melted in platinum doped with 10% Rhodium crucible and heated at the temperature range of 1050 °C - 1400 °C for 1-4 h then quenched in a preheated stainless steel/ graphite mould of temperature 300 °C - 600 °C and then cooled. The prepared glass sample was annealed at 300 °C- 500 °C for 1 h and then cooled at the rate of 0.5 °C - 2 °C per minute to release the stress in the glass sample. The prepared glass sample was cut into required size and shape using a diamond cutter. The code for the present sample is called as FP4.

Example 5:

17.0042 g of P ₂ 0 ₅ , 4.4785 g of CaO, 7.2185 g of Na ₂ 0, 1.2989 g of CaF ₂ , pure chemicals were taken in agate mortar/ planetary ball mill. Ethanol was added with the mixture and ground for 1 h to obtain a homogeneous mixture. The mixture was dried at 100 °C -200 °C for 1 h and ground using agate mortar/ planetary ball mill to obtain a fine powder. The powdered mixture was melted in platinum doped with 10% Rhodium crucible and heated at the temperature range of 1050 °C - 1400 °C for 1-4 h then quenched in a preheated stainless steel/ graphite mould of temperature 300 °C - 600 °C and then cooled. The prepared glass sample was annealed at 300 °C- 500 °C for 1 h then cooled at the rate of 0.5 °C - 2 °C per minute to release the stress in the glass sample. The prepared glass sample was cut into required size and shape using a diamond cutter. The code for the present sample is called as FP5.

Physico-chemical and in vitro studies

1. Density Measurements

The density of the prepared glass samples was measured using Archimedes' principle

W _a

with water as a buoyant and the relation, P ~ ^~ — 7^ ^~ P _b , where W _a is weight in air,

^W a ~ ^W b

W _b is the weight in water, and p _b is the density of water. A digital balance (model

BSA224S-CW; Sartorius, Gottingen, Germany) with an accuracy of ±0.0001 g was used for weight measurements. The measurements were repeated five times to find an average and an accurate value. The overall accuracy in density measurement is ±0.5 kg/m ³ . The percentage error in the measurement of density is ±0.05.

2. Ultrasonic Measurements

Ultrasonic velocities (U _L , longitudinal and U _s , shear) and attenuations (U|_, longitudinal and U _s , shear) measurements were carried out using pulse echo method and cross-correlation technique. The measurement system consists of an ultrasonic process control system (model FUII050; Fallon Ultrasonics Inc. Ltd., ON, Canada), a 100-MHz digital storage oscilloscope (model 54600B; Hewlett Packard, Palo Alto, CA), and a computer. The measurements were carried out by generating longitudinal and shear waves using X- and Y- cut transducers operated at a fundamental frequency of 5 MHz.

3. X-ray Photoelectron Spectroscopy

Elemental analysis of the prepared glass sample was done using X-ray photo electron spectroscopy (model AXIS Ultra DLD; Kratos, Kyoto, Japan) with Al Ka source operating at 210 W. The glass sample was ground using planetary ball mill (model PM 100; Retsch, Haan, Germany) and the powdered glass sample was used for XPS analysis. A survey spectrum (0-1200 eV) was recorded and high-resolution spectra for Cls and Nls band were obtained. X-ray as the excitation radiation was used for the XPS measurements. The spectra were collected in a fixed retarding ratio mode with a bandpass energy of about 10 eV.

4. Fourier Transform Infrared Analysis

Infrared absorption of powdered glass samples after in vitro studies were analyzed from FTIR spectra. The FTIR absorption spectra were recorded at room temperature using an FTIR from 4000 to 400 cm ^"1 , (model 8700; Shimatzu, Tokyo, Japan) spectrometer. A 2.0-mg sample was mixed with 200 mg KBr in an agate mortar and then pressed under a pressure of 100 kg/cm. It gave a pellet of 13 mm diameter. For each sample, FTIR spectrum was normalized with blank KBr pellet. 5. X-ray Diffraction Analysis

To confirm the amorphous nature of prepared glasses and the presence of HAp layer on the surface of glass samples, XRD studies were carried out on each glass sample. An X-ray diffractometer (model PW 1700; Philips, Eindhoven, The Netherlands) was used with CuKa as a radiation source to obtain the XRD pattern in the range of a scanning angle between 20° and 80°. The glass samples were removed from SBF solution after 21 days in vitro studies and then washed gently with double distilled water. The washed glass samples were dried at room temperature. The dried glass was subjected to obtain the XRD pattern as discussed above.

6. pH Measurements

The prepared glass samples were soaked for 21 days in laboratory prepared SBF solution and kept in C0 ₂ incubator at the temperature of 37 °C in 6% C0 ₂ . The variation in pH values of simulated body fluid (SBF) solutions was measured on all the 21 days using a pH meter (model 3-star; Thermo Orion, Beverly, MA) for all glasses under identical conditions. The pH electrode was calibrated using the standard buffer solution with a pH value of 4.01, 7.00, and 10.01 before taking the measurements. The percentage error in the measurement of pH is ±0.005.

7. Scanning Electron Microscopy

The surface morphology of the prepared glass sample was explored using SEM studies. The glass samples were gently washed with double-distilled water and dried at room temperature. A thin layer of a gold film was coated on the surface of glass sample using sputtering technique. The SEM (model Ultra 55; Zeiss, Oberkochen, Germany) was used to obtain a surface image of all glass samples before and after in vitro studies to explore their surface morphology.

8. Energy Dispersive X-ray spectroscopy

Energy dispersive X-ray spectrograph (EDS) was taken for all the prepared glass samples before and after in vitro studies using EDS (model X-max 50 mm ² ; Oxford, Abingdon, England) for obtaining semi quantitative elemental information of the surface of samples. The percentage of error associated with the elemental composition analysis is ±0.1.

9. Cell culture and cytotoxicity assay

Nontoxic nature of the selected glass sample was assessed using cytotoxicity study in cell culture lines. Human gastric adenocarcinoma (AGS) cell line (ATCC-1739) was obtained from the National Centre for Cell Science, Pune, India. The cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture F-12 HAM (1 : 1) with 2mM L ^"1 glutamine supplemented with 10% fetal bovine serum, 45 IU ml ^"1 penicillin and 45 IU ml ^"1 streptomycin. Growth ingredients were also added and incubated in a humidified atmosphere at 37 °C in 5% C0 ₂ . After a number of passaging, the pure confluent AGS cell lines were obtained and cells at a density of 10 ³ were used to evaluate the cytotoxicity at a concentration of 100 mg ml ¹ for the selected bioactive glass samples. The morphology of AGS cell lines was observed regularly under binocular inverted microscope. After 48 h of incubation, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was performed to evaluate the viability of the bioactive glass treated AGS cells. The percentage of cell viability from triplicates of the bioactive glass treated and non-treated cells was calculated using optical density (OD 590 nm) as follows:

OD of the glass particles treated cells

Cell viability % = x lOO

OD of the cells

In vivo studies

Animal studies were done after getting the approval from animal ethical committee (Approval number IAEC-LDC/7/13/1 dated 4 ^th July 2013). Young rabbits of weight about

I.600 Kg were purchased from King Institute, Chennai as per CPCSEA guidelines. Under ketamine anaesthesia, one centimetre incision was made over the medical epicondyle of femur under image control. Under microscopic magnification, Periosteum was opened and a 2 mm drill hole was made. The selected sample of fluorophosphate glass rod of diameter 2 mm was pegged into the hole. After saline wash, wound was closed using single layer 3-0 ethilon sutures and then 250 mg of ceftriaxone was given intra-muscularly.

Rabbit was allowed to move freely immediately after recovery from anesthesia. Fluorescent calcein (Sigma Aldrich, Japan) (10 mg/kg) was administrated intramuscularly on the day of surgery and then every week until 2 days before euthanising to label newly formed bone continuously. After 10 weeks, the animals were euthanised using a lethal dose of ketamine and the lower end of femur harvested and preserved in 10% formalin and used for SEM, EDS, CLSM studies. Un-decalcified sectioning was done on the harvested femur perpendicular to the implant rod using microtome (Model SP1600; Leica, Nussloch, Germany) and getting slices of 1 mm thickness.

10. Histophysio!ogical analysis

Calcein fluorescence was used to examine the newly formed bone using a confocal laser scanning microscope (model Fluoview-IX70; Olympus, Tokyo, Japan). The excitation wavelength was set at 488nm (Ar laser). Calcein fluorescence was detected through a BP 515 -565 nm bandpass filter.

II. Histomorphological studies

The un-decalcified section of the implant was dried at room temperature using desiccator. A thin layer of a gold film was coated on the surface of glass sample using sputtering technique. SEM and EDS studies were done on the dried slice to find the formation of HAp at the glass-bone interface. Details obtained from the Figures:

Fig. 1 illustrates the protocol used for the synthesis of phosphate based glass samples with different contents of fluorine.

The density variation of the prepared glass samples as a function of added CaF2 content is shown in Fig. 2. A sudden decrease in the density from 2707 kgm ^"3 to 2630.5 kgm ³ due to the initial addition of calcium fluoride is noted. Further addition of calcium fluoride leads to increase in the density up to 2636.6 kgm ³ for 2.5 mol% of CaF ₂ . The observed behaviour explains the alteration of glass network by fluorine atom. Fig. 3a and 3b shows the variation of elastic moduli of all _. the prepared glass samples as a function of CaF ₂ content. Elastic moduli such as longitudinal, shear, young's and bulk modulus shows the same trend with the initial addition of CaF ₂ . Further addition of CaF ₂ increase all the elastic constants. The glass containing 2.5 mol% of CaF ₂ showed the maximum moduli value than the other CaF ₂ added glasses.

Fig. 4 shows the elemental composition of the glass sample FP3 using XPS. The observed intensity at the binding energies at 437.6 eV, 348 eV, 135.2 eV, 1072.5 eV, 534.8 eV, 684.8 eV shows respectively the presence of CaO, CaF ₂ , P205, Na20, O and F in the glass sample FP3. SEM image of the prepared glass sample FP3 is showed in Fig. 5. The smooth surface of the glass sample FP3 obtained from SEM image exhibits the amorphous nature of the sample. Fig.6 shows EDS spectrograph of the glass sample FP3. A close observation is noted between experimental and nominal composition of the glass sample FP3. Fig. 5 and Fig. 6 confirm the presence of fluorine atom in the glass network.

21 days of in vitro studies were made on all the prepared glass samples. The observed pH variations during 21 days in vitro studies help to assess the bioactivity of the prepared glasses. The pH variations of all the prepared glasses during in vitro studies are given in Fig. 7. The initial release of phosphate ions lead to form phosphoric acid resulting in sudden decrease in pH value of all the glass samples. At the end of second day of immersion, sodium ions are released and hence the increase in pH value. At the end of 21 days in vitro studies, the sample FP3 shows higher pH value than all the other samples.

FTIR spectrograph of all the prepared glass samples after in vitro studies is shown in the Fig. 8. The FTIR absorption assignment band at 470 cm ^"1 , 530 cm ^"1 , 720 cm ^"1 , 893 cm ^"1 , 1000 cm ^"1 , 1116 cm ^"1 , 1275 cm ^"1 , 1630 cm ^"1 , 3640 cm ^"1 are respectively of vibration bands of P0 ^3" , HAp, P-O-P (asymmetric mode), P-O-P (asymmetric mode), P0 ₃ ^2" , P-0 (stretching mode), hydrogen bending mode vibration of water and water associated in HAp. The presence of HAp confirms the bone bonding ability of all the glass samples. XRD patterns of all the glass after in vitro studies are shown in Fig. 9. In the obtained XRD shoulder peak at 31.912° and a weak one at 31.792° in the sample FP3 shows respectively the rich concentration of HAp and Weak concentration of FAp. Fig. 10 shows the SEM image of the glass sample FP3 after in vitro studies. The SEM image confirms the rich deposit of Ca-P layer on FP3 glass surface. The size of the deposited particles is in the order of 20-100 nm. Fig. 11 shows the elemental composition of the deposited precipitate using EDS. The Ca/P ratio of the deposit is about 1.6 indicating the deposit is HAp. But the presence of fluorine molecules in the surface precipitate confirms the existence of fluoroapatite. The EDS spectrograph clearly indicates the presence of hydroxyapatite and fluoroapatite on the surface of the glass sample FP3.

Fig. 12 shows the optical microscope image of cell viability test for the sample FP3. The image clearly shows there is no cytotoxicity observed in the glass sample FP3. Fig. 13a shows the SEM image at 100X magnification of un-decalcified section of the femoral condyle of the rabbit after 10 weeks of glass implanting. The micrograph shows bone conversion morphology of the glass sample FP3 in femoral condyle of the rabbit. A uniform change in glass size and structure of the implanted glass is well observed on the entire circumference. Fig 13b at 200X magnification the differential layering between the glass and bone in the form of an interface, binding the glass to bone is clearly shown. Fig. 13c shows the EDS spectrum of the glass-bone interface. The presence of minerals such as P, Na, Ca, O and F at the bone-glass interface confirms the transformation process of glass into bone. 14a. is the CLSM image showing the morphology of the bone-glass interface. Fig 14b shows the fluorescence at the bone-glass interface. The brilliant wide fluorescence indicates high calcium turnover at the interface over a period of 10 weeks. This activity confirms the transformation process of glass to bone.