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Typical requirements of a hybrid composite are; the composite should be able to be used in all areas of tooth with high superior and initialÂ polish retention with sound mechanical properties . It has been a trend in classification of resin based composites that as the filler size decreases the cosmetic properties, polishability, translucency and flowability increases providing large surface area. Although with the decrease of size of fillers the mechanical properties tend to decrease but heterogeneous combination of fillers has shown improvements to the mechanical characteristics .
To acquire these characteristics in resins based composites nano sized fillers were developed. Nano-hybrid resin based composites contain a hybrid mixture of colloidal silica particles of 20 to 60 nm and other inorganic fillers in the size range of 0.1-2.5Î¼m, such as borosilicate in conventional methacrylate based resin matrix .
Summita et al. compared the properties of cosmetic dental nano-composite, Filtek Supreme Universal RestorativeÂ (3M ESPE Dental Products, St. Paul, Minn) with conventional resins. They incorporated the resins with nano - fillers and investigated wear, fracture toughness, flexural strength, polish retention and surface morphology after toothbrush abrasion. They reported that nano sized hybrid composite resins have excellent translucency, high polishablility and polish retention (similar to microfills), ability to maintaining physical properties and comparative wear resistance to those of hybrid composites. . Venus Diamond (Heraeus Kulzer, Armonk, NY) is also recently reported as a new nanohybrid universal composite containing novel monomer TCD-DI-HEA, Bis-(acryloyloxymethyl) tricyclodecane. It is claimed to be a material with low polymerisation shrinkage with low viscosity .
The study carried out by Mitra et al. it is concluded that nanofill fillers improve flexural and compressive strength in comparison to those of packable and flowable resins but flexural strength decreases slightly. Nano - hybrid resins have high packing density, better flexure strengths, excellent surface properties and sound mechanical properties [107, 108 and 109].
Polymerisation methods for resin based composites
Resin based composites are can be self-cured (autopolymerisation) or light cured, both by production of free radicals which initiate polymerisation. Polymerisation of composites used to be done by mixing two pastes of monomers. One of the paste contained activator (for example tertiary amine), while the other contained initiator such as benzyl peroxide .
Later in 1970, UV light was started to be used to initiate free radical polymerisation by breaking ether linkage. For this type of polymers there was no need of two pastes as polymerisation would not initiate till exposure to UV light. The disadvantages of UV light included burns to soft tissue, damage to eyes, expensive mercury lamp and limited depth of cure (especially with the older lamp) [10. 95].
To overcome those problems visible light activated composite were marketed. In these composites camphorquinine has been used as photo - initiator and tertiary amine2 - ethylmethcrylate is used as activator in of matrix . They cure in the range of 400 - 500 nm and curing time is 40s . Excitation of these initiators is in visible, blue colour range. Advantages of this curing methods include use of cheap quartz halogen lamp, less energy of excitation so less damaging, better depth of cure and safe, as visible blue light makes it possible for the dentist to use the light at the damaged tissue only to avoid other tissue to be affected. Munksgaard et al. reported that halogen cure units do not have efficient depth of cure which allows leaching out of the monomer causing reduced biocompatibility, structural stability and wear rate and decrease in mechanical properties . Other than quartz halogen lamps following lamps have also been used to initiate polymerisation;
Blue light emitted diode (blue - LED)
Blue - LED is advantageous that it is energy efficient as it emits light at narrow range of 460 to 480 nm and mobile as it can be operated by small rechargeable batteries [33, 95].
Argon laser uses high intensity light source which can be optimised to initiate polymerisation to produce high degree and depth of cure in comparison to halogen lamps. It takes less time for the polymerisation during setting than halogen lamp which reduces the stress relaxation during polymerisation .
Plasma (xenon) arc lamps
They cure nearly at the same intensity as argon laser. The advantage of using plasma xenon lamps is less shrinkage stress due to slower polymerisation of resin and less cost. They have less curing time than halogen lamp cure and more output light energy .
Advantages and disadvantages of conventional methacrylate based resin composites
Composite's properties and bioactivity is affected by many factors, such as: size, shape and distribution of the filler particles . Resin based composites are strong, aesthetic and durable. Due to being multiphasic crack propagation is decelerated. They can then be polished to achieve better aesthetic properties. Generally, composite fillings are used to fill a carious abrasion involving highly visible areas (such as the central incisors or any other teeth that can be seen when smiling) or when conservation of tooth structure.
Main disadvantage of methacrylate based composite resins included polymerisation shrinkage . During polymerisation polymers have to approach their neighbour molecules to form chemical bonds which lead to loss of volume causing polymerisation shrinkage. The bond of composite resin to tooth can be affected by contamination due to moisture and cleanliness of the prepared surface. Other than polymerisation shrinkage they were reported with the problems with marginal adaptation, inappropriate proximal contact, leakage and appearance of secondary carries . Unreacted monomers may leach out of the polymer system and cause damage and irritation to surrounding tissue .
First bioactive composite hydroxyapatite / polyethylene composite was prepared and investigated in 1980 to study the bone resorption . Bioactive composites act as alternatives to autogenous grafts . Bioactive fillers such as hydroxyapatite, sintered b-tricalcium phosphate, apatite/b-tricalcium phosphate biphasic ceramics, glass-ceramic A-W containing crystalline apatite and wollastonite have been used in history to produce bioactive composites .
In 1995 HydroxyapatiteÂ reinforced high densityÂ polyethyleneÂ (HA/HDPE) composite HAPEXâ„¢ was introduced as first bioactive ceramic-polymer composite. It used PE matrix which is biocompatible and ductile which allows incorporation of large amount of ceramic filler . Use of biodegradable matrix can be used as advantage to allow natural growth of the tissue but some successful studies for bioactive composites take non degradable polymers into account .
Wang et al. used Tricalcium Phosphate/ Polyhydroxybutyrate composite as biodegradable material for clinical applications and found to be a promising advancement to enhance tissue growth by bioactive composites .
Kishimoto et al. chose Polyamide (PA) as biocompatible with high polarity with inorganic bioactive polar fillers (NA). NA is similar to tissue in morphology, structure, composition and crystallinity. They claimed NA to be better in bioactive response for bone growth . Jie et al used NA slurry and polyamide solution to prepare nano - composite and then they fabricated it with highly bioactive NA content. The NA provided the excellent bioactivity for the composite by forming bone-bonding with the natural tissue. PAÂ enhanced its toughness and mechanical strength. The composite developed direct connection with the host bone tissue .
The composite under investigation in this research project is silorane resin based composite used in operative dentistry as direct restorative material. Its unique ring opening polymerisation mechanism provides low shrinkage to the matrix ; which makes it distinct from conventional resin matrix such as TEGMA, UDMA or Bis - GMA. Silorane is known for their low shrinkage and outstanding stability in physical and chemo-physical forces and influences. 
Composition of Silorane
It is formed by reaction between siloxanes and oxiranes representing hybrid monomer systems. Siloxane monomer provides it with distinct hydrophobicity, while oxirane makes it able to face high forces and challenging physical environment. Oxirane has been used in manufacture of sports equipment.
Fig 6: Silorane chemistry
Properties of Silorane
Silorane is biocompatible , hydrophobic  posterior restorative composite with low mutagenic potential . Weinmann et al. absorbed that nutritional dyes to silorane based restorative materials and found it to have less absorption due to silorane backbone having hydrophobic nature.  Hydrophobic nature of silorane also leads to less adhesion of bacteria to resins which was confirmed by Ralf et al. .
Polymerisation of silorane
Conventional methacrylates follow reduction oxidation reaction to ingenerate free radicals through double bonds. [90, 95] They face higher contraction at polymerisation. During polymerisation molecules have to approach their neighbour molecules to form chemical bonds. This results in loss of volume resulting in polymerisation shrinkage . Silorane has ring opening cationic polymerisation which fevers compensation of significant amount of polymerisation shrinkage occurs at ring opening step.
Silorane - Volumetric Shrinkage <1%
Methacrylate - Volumetric Shrinkage
Fig 7: Product information: Filtekâ„¢ Silorane Low Shrink Posterior Restorative System. 3M ESPE UK & Ireland.
Fig 8: Product information: Filtekâ„¢ Silorane Low Shrink Posterior Restorative System. 3M ESPE UK & Ireland.
Silorane (3,4 - epoxycyclohexylethylcyclopolymethylsiloxanebis- 3,4 -epoxycyclohexylethylphenylmethylsilane
Filler (silicon dioxide, ytterbium trifluoride)
Table 1: Product information: Filtekâ„¢ Silorane Low Shrink Posterior Restorative System. 3M ESPE UK & Ireland.
Silorane based Restorative materials have advantages over conventional methacrylate based resins
Conventional methacrylates use free radical polymerisation by initiating hydrogen radicals to form crosslinking and branches and hence growth at those sites. In the process of gelation the viscosity increases decreasing the volume % (polymerisation shrinkage . This polymerisation shrinkage affects the physio - mechanical properties of those polymers . The polymerization shrinkage of resin based composites depends upon cavity configuration factor (C-factor). "The C-factor is ratio of bonded to non-bonded surfaces, whereby a high ratio of bonded surfaces limits the materials ability to flow; which induces higher interfacial stresses" . Silorane has been reported with less polymerisation shrinkage than conventional resin based composites. Asmussen et al carried out a study to investigate the polymerisation contraction of silorane in comparison to conventional methacrylate based composites. They used four methacrylate based resins as control and they concluded that novel silorane based resins has significantly low polymerisation shrinkage than other conventional resin based composites .
Silorane has lower water absorption and decreased water solubility than conventional methacrylate resins leading to higher hydrophobicity .
Ilie et al. compared the mechanical properties of silorane to conventional resins and found them comparable and promising .
Ralf et al. carried out investigation to compare the hydrophobicity, surface roughness and type of matrix of silorane with other conventional resins by adhering them with streptococcal bacteria. They observed low fluorescent intensities for silorane based resins in comparison to other conventional resins. This leads to the conclusion that silorane has high hydrophobicity, lower adhesion potential with bacteria and less surface roughness which may lead to long lasting direct restoration and reduce recurrent caries.
Silorane resins also have better cytotoxicity ratings than methacrylate dental monomers.
Bioactive Minerals - apatites
The most common type of ceramics (biomaterials which are known for their bioactive response in human body environments are apatites .The close resemblance of apatite minerals to dentine and enamel generates excellent osteoconductivity . They have hydroxyapatites and substituted apatites in their composition; which are also part of living tissue. Those ceramics form the desired bonding (apatite layer) between the implant and bone in biological environment; such stimulated body fluid.  Other bioactive ceramics include bioglass, SiC ceramics coated with bioactive glass and A - W ceramics etc. .
Apatites such as hydroxyapatite, fluoroapatite, carbonated apatites are well known for their bioactivity. Phosphocalcic Hydroxyapatite is the main mineral present in the tooth structure ; which is 96 % by weight in enamel and 70 % by weight in the composition of dentine. Hydroxyapatite is a natural mineral form of calcium apatite made up of two crystal units having hexagonal structure . It is well known for its excellent biological behaviour and biocompatibility . It is a bioactive ceramic having very similar composition and crystal structure to the apatite in the human dental structure and skeletal system . They have been proved to be non - toxic and non - inflammatory; showing pyrogenic response or fibrous tissue formation between implants and bone due to their ability to bond directly to the host bone .
Hydroxyapatites were first used in 1981 for periodontal liaison fillings, since then their use has increased in clinically to milestones. They have been used as solid blocks, solid components and films for dental implants afterwards; in the form of porous, dense, granules and coated[39,40]. After root extraction the cavity is left which may lead to bone resorption due to soft tissue deposition. Porous minerals HA and FA can be filled so that bioactive materials regenerate the bone regeneration by osteoblasts by slow mechanism. They provide microporosity for nutrients to pass through. Porosity and chemistry level can be controlled. General mechanical properties of FA include its high stiffness than polymers but less than steel. It is more brittle than metals. HA has been used in bioactive composites, coatings on metal implants and as granular fillings . Apatites are reported to be brittle so they are not very useful for load bearing application such as bone replacement due to their brittleness and lack of strength . On the other hand they can be a good choice to be use with other materials which are good for load bearing applications such as composites. In recent years it has been attempted to incorporate apatite minerals with other dental restoratives and implant materials to tailor make the required characteristics. The microporosity of hydroxyapatite is beneficial for the bone ingrowth after implant.
Fluoroapatite - Ca10 (PO4)6F2
Fluoroapatite Ca10 (PO4)6F2 is another important bioactive ceramic  present as an important constituent of tooth enamel and dentine. It is the natural mineral form of calcium apatite made up of two crystal units having hexagonal structure . It is well known to be combined with hydroxyapatite to be used in biological matrices . FA can be supplied to the tissue via food, toothpaste and supplement water etc. fluoroapatite is much more acid resistant and harder than hydroxyapatite. Hydroxyapatite actively takes part in bone formation by enhancing the physiological processes ; during this process it integrates into bone structure without infringement or dissolving . Fluoroapatite have equivalent bioactivity to hydroxyapatite with more compact lattice structure due to hydroxide ions substituted by fluoride ions containing stronger electrostatic forces hence making more packed structure forming slow dissolution rate and less solubility of fluoride ions [121, 122].
Liu et al. carried out a study in 2010 to investigate the effect of fluoroapatite surfaces on osteoblast-like cell adhesion, growth, and mineralization. They grew MG-63 cells on metals and coated with fluoroapatite. They observed the growth of the cells with and without OI supplement. They concluded that "MG-63 cells grown on FA crystal surfaces start to differentiate and mineralize, suggesting that the FA crystal could be a simple and bioactive implant coating material" .
Incorporation of Apatites in Dental Restoratives and implant materials
In natural composites such as dentin minerals are the main constituent of the composition. The ability of hydroxyapatite to assimilate with the bone helps for the bonding between bone and implants. It has been attempted to try and incorporate hydroxyapatite in dental restoratives to enhance bioactivity [2, 9].Attempts have been made to incorporate HA in glass ionomer cement and other materials. With GIC it has been found to interact chemically with polyacid (carboxylic acid functional groups); which may alter its properties and it does not improve the biocompatibility. On the other hand it improves the mechanical properties and bond strength. Hydroxyapatite have been reported to be attempted to incorporate with other load bearing implants such as zinc, titanium, strontium, zinc, ,manganese etc. to enhance its mechanical strength . Lucas et al. investigated fracture toughness of GIC with incorporation with hydroxyapatite and they found out that glass ionomer cements incorporated with hydroxyapatite increases the fracture toughness of the cements while maintaining long-term bonding to dentin . Gu et al. also that reported that glass-ionomer cements containing 4%by weight hydroxyapatite enhance mechanical properties in comparison to commercial glass - ionomer cements . Rehman et al. carried out test for the incorporation of hydroxyapatite and fluoride substituted apatite with GIC cements by acid base reaction and they concluded that it improves the crystallinity and hence chemical stability of the final structure; giving better results for FA than HA . The mechanical properties (tensile strength and biaxial flexural strength) also improved. Nano - FA showed lower dissolution rate than that of nano - HA in distilled water. FA provides stiffness and brittleness to tooth structure and also provides acid stability to it to prevent dissolution of enamel and dentine. It is reported that it improves mechanical properties and bond strength of the dental materials . Apatites provide greater possibility for additional bonding such as hydrogen bonding due to the presence of hydroxyl, fluoride and phosphate ions in the matrix. It is certain that the stronger the bonding between organic matrix of the composite and mineral is, the stronger the matrix will be, and hence there will be better the mechanical properties .
HA has also been investigated with used with incorporation with polymers such as polyethylene (HDPE) to make commercially well-known composite HAPEX and with other polymers including polyacrylates, polyethylene and PLA to make to enhance the mechanical properties and bioactivity of the composites . The main problem faced in such incorporations has been the week mechanical bonding formed at matrix and filler (HA) particles junction. Saba et al. attempted to improve the bonding between two phases by chemical means by adding bonding agents or surface grafting .
Polyhydroxyalkanoates (PHA) such as Polyhydroxybutyrate (PHB) is thermoplastic polyester that uses a renewable feedstock such as glucose . These biodegradable polymers have been used as grafts due to their biocompatibility, ease control of degradation, non-toxic degradation and its degradation product (hydroxybutyric acid) is a common metabolite in human body . Reinforcement of hydroxyapatiteÂ and of tricalcium phosphate into this polymer has been used to increase Young Modulus and micro-hardness and bioactivity .
Two main reasons for the failure of the dental restoratives are secondary caries and restorative . Fluoride ion containing dental materials show more resistance to caries. Such as GIC releaseÂ fluoride which can be "recharged" by the use of fluoride-containing toothpaste, which is used to prevent carious lesions . Fluoride-releasing materials act as aÂ fluorideÂ reservoir to increase theÂ fluorideÂ level inÂ natural tissue and saliva. Annette at el. reviewed theÂ fluorideÂ ion release, recharge capabilities, and antibacterial properties ofÂ fluoride-releasing dental restoratives. They found the dependence of fluorideÂ release on the matrices, fillers andÂ fluorideÂ content, setting reactions and environment of restoratives .Â In very recent studies by Jennifer et al. used Â three resin-modified glass ionomers (Vitremer, Fuji II LC, and Ketac Nano), one flowable compomer (Dyract Flow), and one composite (Heliomolar).Â They reported that the fluoride ion release increases at acidic, cariogenic pH. They also reported that the Fluoride releasing restoratives degraded significantly in immersion . Ming et al. developed a continuous flow system for assessing fluoride release/uptake of fluoride-containing restorative materials to mimic the kinetics of salivary flow. They observed the immediate decrease in rate of fluoride release after a brief initial burst of Fluoride release. They recommended the continuous flow method to be used to determine the rate of release and uptake by the material.
Techniques for preparation of apatites - Sol - gel technique
Many methods have been used in the past and in recent years for preparation of apatites. Sanosh et al, 2009 reported the use of waste biomass, egg shells for the synthesis of nano crystalline Hydroxyapatite. . Apatites formed by this technique were economically cheap. They were characterised by X-ray diffraction, Transmission electron microscope, particle size distribution analyser and Fourier transform infra-red spectroscopy techniques. The results showed the formation of polycrystalline HA with the average particle size of ~35 nm. In history several methods have been used for the synthesis of apatites range from 5 to 90 nm.
Those techniques include;
Wet chemical method:
Including Precipitation method , Hydrothermal technique , Hydrolysis  and Sol - gel method [15, 1, 2, 45]
Dry solid state reactions .
Mechano - chemical method [46, 47].
Spray Pyrolysis .
Following different preparation route leads to different morphology, stoichiometry level and level of crystallinity of the ceramic apatites .
Sol gel Method is wet chemical technique used for material's fabrication and deposition in which 2 mono - phasic solutions are used as precursors to produce and integrated biphasic network (gel). It was first employed by Sakka et al. in 1990 for the preparation of hydroxyapatite; by using hydrolysis and polycondensation of calcium diethoxide and phosphorus triethoxide monomers in neutral and acidic solutions .In the process, change in morphology of substances takes place such as change from discrete particles to continuous polymer network. Sol gel technique is selective for in this research project to prepare fine grains of nano - fluoroapatite. Ethanol is selected to be used as solvent for the precursor materials of the nanobioceramics. EDTA can be used as chelating agent to prevent the immediate precipitation of calcium ions during the course of gel formation .
The general mechanism of apatite preparation via sol - gel method is given below;
Mixing - Solution
Solution of Calcium precursor in ethanol
Solution of Ca (NO3) . 4H2O in ethanol
Excess Solvent is removed.
Aging: Cross-linked structure of molecules increases during aging. As aging time increases, cross-linking also increases.
Gelation: Polymerization occurs at this step to form cross-linking of the molecules making up the skeletal structure.
Apatite mineral (Nano sized)
Sintering: Porous structure is eliminated, the residual organisms are removed, and the minerals are crystallized.
Fig 9: Schematic diagram for the main steps in sol - gel Technique 
One of the advantage reported for the sol gel technique is high cost of the raw materials but the precursor materials used in this project are low cost expected to yield extraordinary fine grains of nano - fluoroapatite. Sol - gel is a very well-known technique to process ceramics giving products with following advantages.
High purity of the nano-sized highly crystalline product [9, 49].
High reactivity of sol gel product allows a reduction of processing temperature and any degradation phenomena occurring during sintering. 
Gel formation without use of catalyst.
Simplicity of the experiments .
Better homogeneity of the final product .
Limitations of sol gel method and their possible solutions are as follows;
Possible hydrolysis of phosphates .
Strict pH control.
Long time may be required for hydrolysis - Involves polycondensation reaction (hydrolysis).
For the preparation of apatites calcium tetrahydrate and diammmonium hydrogen phosphate are used as precursors for calcium and phosphorus .
10Ca (NO3)Â 2.4H2O(s)Â + 6 (NH4)2HPO4(s)Â + 8 NH3 (aq)Â + â†’ Ca10 (PO4)6(OH) 2(s)Â + 20 NH4NO3 (aq) 2 H2O (l)
Wang et al. used sol gel technique for the preparation of hydroxyapatites and they investigated effect of sintering temperatures on degree of crystallinity and hydroxyapatite phase composition . They also varied the aging times and observed the effect of it on crystals size of the nano - hydroxyapatite. They examined the variation by X - Ray Diffraction and T - Electron Microscopy .Â Â They found out that varying the sintering temperature affects the crystallinity of hydroxyapatite. Increasing the sintering temperature from 600 to 900 Â°C increases the degree of crystallinity for hydroxyapatite powder. But they also observed the appearance of additional crystalline phases of Î²-TCP and calcium oxide at 800 and 900 Â°C, and no presence of crystalline phases for hydroxyapatite at 600 and 700 Â°C. So HA may decompose into Î²-TCP and calcium oxide if the sintering temperature is kept at 800 Â°C or increased above 800 Â°C following this decomposition reaction route; 
Ca10 (PO4)6(OH) 2 3 Ca3 (PO4)2+CaO+H2O
So to obtain the pure HA, the sintering temperature should be below 800 Â°C . To investigate the effect of aging time on the formation of pure hydroxyapatite, Wang et al. aged the samples for 4, 48, and 72 h and sintered them at 700 and 800 Â°C, respectively. They investigated the results by XRD and found that there is no difference of purity for the powders which were sintered at 700 Â°C; and those powders which were sintered at 800 Â° were affected due to additional crystalline phases. So they concluded that the aging time has less effect on formation of pure hydroxyapatite than the effect of sintering temperature .
Hydroxyapatite powder prepared by Wand et al. at 700 Â°C (sintering temperature) was then examined by TEM for their particles size and morphology. They used samples aged at different aging times and they concluded that they all show nano -sized ellipse-like morphology . The samples aged for 4 h were only 10-15 nm size, samples aged 48 h were 15-25 nm in size, while the samples aged 72 h were 50-80 nm and they were not well dispersed. Hence they concluded that the hydroxyapatite powders aged for long hours were larger in particle size than those aged for short time concluding that the aging could contribute to powder growth and agglomerate. Hence the best conditions they suggested for sol gel technique for hydroxyapatite were sintering at 700 Â°C followed by aging at 4 hours .
In substituted apatites precursors for the substituted ion source is also added. For example for fluoroapatite, ammonium fluoride is added as fluoride ion source . Low temperature fusion and formation of apatite crystals have been the main advantages of the sol gel method in comparison with other conventional methods for nano-ceramics synthesis .
Stimulated Body Fluid
Human body environment is diverse and violent. Any biomaterial that is implanted in the body faces this impact and has to withstand these changes. Physical and chemical environment of the human body may lead the biomaterial for loss of its stability by different ways such as corrosion and degradation (which lead to loss of mechanical efficiency) . A biomaterial is only suitable for implantation if is able to maintain its properties in adverse biological and chemical changes. Stimulated body fluid (SBF) is a mixture of salts containing ion concentration and pH similar to that of human blood plasma .
Simulate Body Fluid
Table 2 Ion concentrations of SBF and blood plasma 
An example of human body environment can be human body fluid which generally comes in contact with the implant and is known to be effective on biomaterials for their surface modification . In vitro stimulated body fluid can be used to observe the response of the implants in biological conditions . The first study done on effectiveness of SBF on implants was carried out by Aza et al.; they had to encapsulate the ceramics in fibrous tissue to form bond with the defective tissue .
SBF is prepared by dissolving following salts in distilled water; Sodium chloride (NaCl), Sodium bicarbonate (NaHCO3), Hydrated magnesium chloride (MgCl2.6H2O), Calcium chloride (CaCl2), Sodium sulphate (Na2SO4), Potassium chloride (KCl) and precursors for Phosphate ions. The pH of the solution is controlled by tris - hydroxylmethylaminomethane buffer and hydrochloric acid at 7.4[4, 18, and 19].
Analysis of Bioactive Behaviour of Restoratives and Implants in Stimulated Body Fluid
Some biomaterials especially some ceramics do give bioactive response in biological environment; such behaviour of those materials is known as Bioactivity. Upon implantation in vivo or when incubated in vitro in simulated body fluid (SBF), apatite layer forms on the surface of the implant that is considered to be needed for the nucleation of biological apatite .The biological apatite layer is thought to be essential for protein adsorption and cell adhesion. This helps the implant to bond with the surrounding tissue . The bonding ability of bioactive material to bone is evaluated by ability of apatite to form on material surface in a simulated body fluid (SBF) at biological pH . Kokubo et al. carried out experiments for the formation of apatite layer on organic polymers and they reported the positive results. They have reported the thickness of apatite layer to be dependent on time of immersion. The rate of growth of apatite layer is said to be effected by temperature and saturation of the solution. In the experiment Kokubo changed the type of polymer matrix and concluded that the adhesive strength of apatite layer is different for different polymers .
Kazuhiko et al, reported hydroxyapatite to have extraordinary affinity for proteins and considered it as most stable calcium phosphate under physiological conditions. They synthesised positively charged nano hydroxyapatite and analysed the behaviour of their adsorption with proteins. Positively charged nano HA was adsorbed to Î²-alanine. FTIR and TG-DTA reported that Î’-alanine protein was absorbed on the surface of hydroxyapatite forming a bioactive layer .
The apatite layer on ceramics surfaces is formed by Si - OH group's nucleation and is tempted to grow by consumption of the calcium and phosphate ions from the body fluid as those ions are also part of apatites and natural tissue composition [19, 22]. Kontanasaki et al. reported that the apatite layer is formed on the ceramics surface in presence of SBF in 18 hours and then it converts to inorganic crystalline layer of hydroxyapatite in later 24 hours. It is important to note that hydroxyapatite is main inorganic component of the hard tissue composition such as dentine .
In the study carried out by Magdalena et al. they treated a set of polymers with stimulate body fluid for 52 days and they observed the formation of apatite layer forming on their surface and conversion of rubber like materials into semi rigid materials due to inorganic layer formation. This leads to enhancement in mechanical properties and lower creep for polymers treated with SBF .
Jacqueline et al. used microporous hydroxyapatite to analyse their influence on the mechanical properties scaffolds. Microporous HA samples were tested for bend, compression and ultrasound tests. They observed the increase in strength with decrease in micropore size, in bending and compression tests. Pore size did not show effect on elastic properties of HA. They prepared hydroxyapatite scaffolds with multi porosities to get strength closer to trabecular bone. They suggested that the elastic properties of scaffold can be tailored using the above method125].
Surface Characterisation techniques
2.14.1. FTIR Spectroscopy
FTIR spectroscopy is a vibrational spectroscopy technique which is sensitive to absorption of radiations by dipole vibrations of polar bonds such as O - H, N - H, C = O etc. It is reproducible non - destructive analysis; used for advanced structural characterisation and surface analysis for natural and synthetic materials (including polymers, ceramics and composites etc.). .
GAS INLET / OUT LET
TO SIGNAL PROCESSOR / CPU
Modulated IR radiations from interferometer
Fig 10: Diagrammatic representation of infra - red photoaquastic cell 
2.14.2. Raman Spectroscopy
Raman spectroscopy is fast vibrational spectroscopy technique which is sensitive to emission of scattered laser light by dipole vibrations of polarizable bonds such as aromatic compounds, C = C etc. It is reproducible non - destructive analysis; used for advanced structural characterisation and surface analysis for natural and synthetic materials (including polymers, ceramics and composites etc.). No sample preparation is required for this technique and it characterises glass, plastic or aqueous samples also . Nicolet Almega XR Dispersive Raman utilises visible laser sources with high sensitivity .
2.14.3. X - RAY Diffraction (XRD)
XRDA9154 x - ray diffractometer was used. . XRDA9155 is the plasticine used to hold all the samples. So there is no contribution to either of the samples from the plasticine. Structural data files from the ICDD database that is commonly used to identify the phases present are attached in the appendices. Dimensions of crystal lattice and elemental composition of the geometry of the crystal material is done by bombardment of X rays of the sample surface. (Information obtained by x ray specialist Rolley Wilson in XRD lab (QMUL) at the time of use of the machine).
2.14.4. Scanning Electron Microscopy
SEM (Scanning Electron Microscope) - oxford instrument; SEI inspect F; With Energy dispersive X - ray analysis (EDS) detector - Oxford instrument; Inca X - act. will be used for taking high resolution morphological images and under high vacuum and pressure. EDS detector will be used for elemental analysis of the surface to find out the presence of apatite layer. The general working scheme for the microscope is given as follows;