Investigation Of Magnesium And Fluorine Incorporation Biology Essay

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A well-designed three-dimensional scaffold is one of the essential devices to guide tissue formation in vitro and in vivo. Scaffolds for bone tissue engineering purposes should in part mimic the structure and biological function of the extracellular matrix (ECM) [1]. The ideal scaffolds should mechanically support the newly formed tissues as well as deliver inductive molecules or cells to the repair site and provide cues to control the structure and function of the newly formed tissue [2].To fulfill this role, three-dimensional porous architectures have been fabricated using various techniques, such as progen leaching [3], phase separation [4], solvent casting and freeze drying [5, 6, 7].

However, many of these scaffolds do not adequately mimic the structure of the natural ECM in terms of architecture, which may be one of the reasons for suboptimal outcomes in generating functional bone tissues. Biomimetic approaches have been used to fabricate bone scaffolds with the ability to mimic the structure and composition of natural bone. Natural polymers such as collagen, gelatin, and mineral compounds like Hydroxylapatite similar to those in natural bone have been used as biomimetics. The formation and structure of these chemical compounds is of great importance and should come close to bone material while designing bone biomimetics. Most biomimetic synthetic processes occur at physiological conditions (pH value of 7.4 and a temperature of 37 °C) [8-11] that are utmost similar to those of the human body. However, other studies have tried to more closely imitate the body's biological environment. Diffusion-based methods [12-14] and electrophoresis [15] are processes that can be carried out under physiological conditions. Diffusion of calcium and phosphate ions into a gel- like matrix closely mimics the process of bone formation in the body, as the gel simulates the cartilaginous precursor template. In the body, ions move to the bone formation area through the vessels' walls and the extracellular matrix to enter the preliminary cartilage through diffusion. The sedimentation phase, shaped inside the gel on the polymer chains nucleates, grows and has an ideal combination with the matrix. Ergo, composite formed under these conditions are very similar to natural bone. From an engineering point of view, the mineralized phase in this nanocomposite works as a particulate reinforcing phase. In that, the particles show better mechanical properties as they get smaller and form stronger links to the matrix.

Ceramics with the general chemical formula of M10(ZO4)6X2 are called apatite classified within the hexagonal crystal structure (space group P63/m). In this formula, M, Z and X are substituted by certain groups of atoms (Ca, Sr, Ba, Cd, Pb), (P, CO3, V, As, S, Si, Ge) and (OH, CO3, O, F, Cl, Br), respectively. Hydroxyapatite (HA, Ca10(PO4)6(OH)2) and fluorapatite(FA, Ca10(PO4)6F2) are two important members of this family with a crystallographical structure identical to calcified tissues of vertebrates and have been extensively investigated in terms of their physico-chemical properties [16-18].

Fluoride is a vital trace element needed for normal dental and skeletal ontogeny. It has been shown that the presence of fluoride posses beneficial effects on increasing the volume and quality of bone formation in the body [19]. Since the bone mass is increased with F− ion uptake, the fluoride ion has a therapeutic ability for healing osteoporosis [20]. F− ion is known to stimulate osteoblast activity both in vitro and in vivo. In addition, the mineral phase of tooth enamel consists of apatite containing 0.04 wt. % to 0.07 wt. % of fluoride.

F− ions, present in saliva and blood plasma, are necessary for normal dental and skeletal growth. It has been suggested that a fluoride intake of about 1.5-4 mg/day reduces the risk of dental caries dramatically [21]. FA has been extensively studied [20-32] as fluoride is well known for prevention of caries and treatment of osteoporosis.

It is also known that magnesium can influence the bone mineral metabolism, formation and crystallization processes [33, 34]. Consequently, doping calcium- containing biomaterials with magnesium has recently become of great intrest in biomaterial research. It has been reported that Calcium Phosphate (CaP) doped with magnesium can be stabilized, significantly enhancing osteoblast attachment and growth as compared to pure CaP [35, 36]. There has been growing interest on magnesium phosphates bioceramics due to their resorption properties which provide a temporary framework that dissolves as being replaced by the natural host tissue. Magnesium phosphates such as Newberyite (MgHPO4.3H2O) are formed biologically and are known to be degradable and non toxic when implanted in vivo [37]. Also, magnesium apatites have been shown to support osteoblast differentiation and function [38].Additionally, bone cement doped with magnesium showed controllable degradability and great mechanical and biological performance as compared to CaP bone cement (CPC) [39].

Instead of doping calcium -based biomaterials with magnesium, there is a current trend using magnesium-based ones as a potential alternative for orthopedic applications. The deposition of a magnesium coating on metallic implants to induce the formation of a uniform bioactive coating has also been reported [40]. Magnesium phosphate (MgP) that is biocompatible and exhibits a faster biodegradation rate compared to CaP is another area of recent focus [41].

This study aimed to prepare a nanocomposite calcium phosphate/gelatin bone scaffold using a diffusional method. In addition, we intended to evaluate the incorporation of other ions into mineral phase of the scaffold to introduce some kind of enhanced biomimetic gelatin/calcium phosphate nanocomposite scaffold. In order to do that, magnesium and fluorine were added during synthesis process to be incorporated in the structure of calcium phosphates precipitates.

2. Materials and methods

2.1. Scaffold fabrication

Porous GEL/AP nanocomposites were prepared as follows: in the first step, gelatin (Merck, microbiology grade, catalogue number 104070) was added to deionized water, making a 10% (w/v) solution. Then, Hydroxymethylaminomethane (Tris) (Merck, catalogue number 8382) and hydrochloric acid (HCl) (Merck, catalogue number 280211) were added to solution to prepare a buffer gel solution while pH was adjusted to the value of about 7.4. Disodium hydroxide phosphate (1 M) (Merck, catalogue number 106573) and calcium chloride (0.6 M) (Merck, catalogue number 2380) solutions were also prepared and the pH of both solutions were brought to about 7.4 using Tris and HCL. In the next step, the GEL-Calcium containing solution was poured into a cylindrical mold and kept at 4°C for 2 hours until physical gelation occurred. Afterward, the prepared gel was taken out and incubated at 4° for about 48 hours following soaking into the phosphate containing solution. After diffusion of ions into the gel, a white precipitate formed within the gel in a gradient manner from the border to the center of gel.(Fig1a).

Two other series of sample were also prepared to include Mg and F ions within the precipitate. The First one employed Mg along with Ca containing hydrogel and the second one included F along with Phosphorous (PO4) containing solution. Totally, 7 compositions were made as introduced in table1. The resulting nanocomposites were extracted and freeze dried to create a porous structure through solvent sublimation. Scaffolds were incubated in a 1% glutaraldehyde (GA) (Merck, Catalogue number 820603) solution for 24 hours. Samples were then washed with deionized water to remove the remnants of the GA. To achieve a fine powder from fabricated scaffolds for some of the characterization methods, the samples were chopped and milled.

2.2. Porosity measurement

The porosity was measured by liquid substitution method using isopropanol and the following formula:

ε = (V1−V3)/(V2−V1)

Where ε is the porosity of the scaffold; V1 is the volume of isopropanol before submersion of the scaffold; V2 is the volume reading of the liquid after submersion of the scaffold; V3, the volume of isopropanol after the liquid was pressed into the pores of the sample and the sample was taken out of the liquid.

2.3. Characterization

2.3.1. Scanning electron microscopy (SEM)

Investigation of The morphology of the nanocomposites was performed using scanning electron microscopy (SEM; Philips XL30 microscope). The images were obtained by applying an accelerating voltage of 15 kV. All samples were coated with a film of gold by sputtering device (EMITECH K450X, England) before being investigated under the microscope. The obtained images were used to evaluate the pore size and morphology of the nanocomposite scaffolds. Also, higher magnified images were taken to compare morphology of the precipitated minerals within gelatin hydrogel in different conditions.

2.3.2. X-ray diffraction (XRD)

Crystallographic properties of the powder of the scaffolds was analyzed by XRD with a Siemens-Brucker D5000 diffractometer. This instrument uses Cu-Kα radiation (1.540600 Å), with a slit width of 1 mm was set at 40 kV and 40 mA. For qualitative analysis, XRD diagrams were recorded in the interval 20° ≤ 2θ ≤ 70° at a scan speed of 2°/min; step size, 0.02°; and step time, 1 s.

Crystallographic identification of the phases of the synthesized powder was accomplished by comparing experimental XRD patterns to standards compiled by the International Center for Diffraction Data (ICDD). The appropriate cards for HA, dicalcium phosphate dehydrate (brushite, DCPD), fluoroapatite and Magnesium phosphates minerals were used. The average size of individual crystallites was calculated from XRD data using the Scherrer approximation (Eq. 1):


Where t is the crystallite size, λ is the wavelength of Cu-Kα radiation (1.540560 Å), and β1/2 is the full width at half-maximum intensity.

2.3.3. Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy was used to evaluate the formation of calcium and magnesium phosphate within the scaffold using a Bomem MB 100 spectrometer. For IR analysis, 1 mg of powdered sample was carefully mixed with 300 mg of KBr (infrared grade) and pelletized under vacuum. Resulting pellets were analyzed in the range of 500 to 4000 cm-1 at a scan speed of 23 scan/min with 4 cm-1 resolution.

2.3.4. Bulk elemental analysis

Calcium, Magnesium, phosphorous and fluorine contents of the precipitated powders were analyzed using relevant techniques such as titration, atomic absorption and potentiometry. This experiment was carried out to determine level of incorporation of each added element in the final precipitates formed within hydrogel.

Results and discussions

3.1. Porosity measurement

As shown in Fig. 1b, diffusion of Ca+2(or Mg+2) from peripheral solution into the PO43-(or F-) containing hydrogel resulted in formation of white precipitates in a gradient manner. The amount of precipitates formed within the GEL for the F added samples was distinctively more than Mg added samples and the samples with no added F or Mg. Porosity percentage of the synthesized scaffold after being lyophilized was estimated to be about 85%. Results obtained from characterization of this nanocomposite scaffold and precipitates in different conditions are described below.

3.2. Scanning electron microscopy

Typical morphology of synthesized scaffold obtained using SEM was shown in Fig. 2a. This figure illustrates a network of interconnected pores with pore diameters ranging from 200 to 500 µm, which are optimal for bone cell growth. In the micrographs with higher magnification (Fig. 2b, c, d, Fig3 and Fig4), morphology of mineral nanocrystals precipitated within hydrogel is also visible. These minerals have been projected out of the surface of pores wall, after being freeze dried.

In the first sample which contained calcium and phosphate only, budding and crystal growth is observed in the form of rod shaped crystals in closely packed, nearly spherical colonies on the surface. The colonies range from ???? to ???? in size, and rod-shaped crystals have grown with rectangular cross sections about ???? nm in thickness. As Figure 2c depicts, the rod shaped crystals have grown quite separately, and during elongation, steric hindrance has resulted in their tips moving further apart and thus diverging from one another. Consequently, the morphology of each colony is roughly spherical.

Figure 3 illustrates the morphology of minerals formed in Mg containing samples at different Mg/Ca ratios. As the figure depicts, deposits containing magnesium geometrically defined crystals similar to the deposits in the first group. In other words, crystal growth in this group occurs only in the preferred directions, resulting in needle-shaped, planar, and serrated deposits. As shown in Figure 3 (a, b) which pertains to the sample with 25% replacement of calcium with magnesium during synthesis, the shape of the deposits is almost similar to that of the sample lacking magnesium, the only difference being the less regular formation of rod-shaped deposits.

In the sample with 50% replacement of calcium with magnesium during synthesis, the deposits tend to lost their square-section rod shape and grow into an asymmetrical, serrated plane. Here, crystal layers are less regular or symmetrical compared to previous samples.

In the sample with 75% replacement of calcium with magnesium during synthesis, deposits with geometrically defined shaped are found in only a few locations, and the majority of deposits have grown in a completely irregular fashion without any geometric shapes.

Samples where fluorine ion was added along with phosphate yielded deposits with morphologies completely different from previous samples. In all samples where fluorine ion replaced phosphate at 25%, 50% and 75% ratios during synthesis process, the deposits formed nearly spherical particles consisting of smaller crystals with uneven (rough) surface scattered over the gelatinous bulk material. Therefore, this is an indication that adding fluorine influence crystal growth profoundly and prevents the formation of crystals with well-defined directions or geometry.

3.3. X-ray diffraction analysis

Crystallographic properties of precipitated phase within the GEL matrix were analyzed using XRD. Figures 4 and 5 illustrate the diffractogram from deposit samples with different states of fluorine and magnesium replacement alongside a sample lacking any fluorine or magnesium, for comparison.

As evidenced in Figure 4a or 5a, the diffractogram pertaining to the sample without fluorine or magnesium has clearly defined peaks, indicating its relatively crystalline nature. As marked in Figure 4, the peaks obtained from this sample mostly pertain to hydroxyapatite and octacalcium phosphate. Diffractograms b, c, and d, pertaining to samples with 25%, 50% and 75% magnesium additional ratio, respectively, depict peaks of a completely different nature; here, in addition to the few peaks related to OCP, new peaks pertaining mostly to compounds of magnesium phosphate have appeared.

Figure 5 presents the diffractogram of samples containing fluorine compared to the sample without magnesium or fluorine. Graphs b, c, and d pertain to samples with 25%, 50% and 75% phosphate additional ratio with fluorine, respectively. As the Figure shows, addition of fluorine to during synthesis process has caused the relatively crystalline structure of the initial sample to shift towards an amorphous compound with increasing ratios of fluorine. The peaks appearing in the diffractogram all pertain to hydroxyapatite and fluorine apatite. In other words, addition of fluorine to the sample lacking magnesium and fluorine changes its OCP and HAp to HAp and FAp.

These peaks can be ascribed to both HAp and OCP (marked in this figure), according to the ICCD database.

These results indicate that there is a clear transition from well-defined crystalline compound with sharp diffraction peaks to an amorphous or nanocrystalline compounds with broad diffraction peaks.

The average size of individual crystallites was estimated to be between 40-80 nm using the Scherrer approximation.

3.4. FTIR analysis

Chemical composition of the prepared nanocomposite scaffolds was analyzed using FTIR analyses. Fig. 7 shows the FTIR spectra of the scaffolds with different precipitates including those without any added F and Mg, with 50% Mg additional ratio and with 50% F additional ratio in synthesis procedure. Table 2 shows detected chemical bonds with related wave numbers. For all of the samples, most of the peaks appearing in these spectra were almost identical. A category of peaks are related to GEL matrices. Second category ascribes to the minerals formed within GEL. Peaks at about 1240, 1456, 1540, 1652, 2930 and 3070 cm-1 are known as typical peaks for GEL and peaks at about 567, 605, 630, 870, 1040 and 1400 cm-1 are due to mineral phase formed within GEL including HA, DCPD, OCP, MgP and FA. In addition, a peak appeared at approximately 1340 cm-1 corresponds to a chemical bond formed between the GEL matrix and the precipitated calcium containing minerals. This peak indicates formation of a linkage between a carboxyl group from GEL and Ca+2 ions from precipitate which is also consistent with our previous studies [5, 6, 42].

Table1- 1



Applied in synthesis

Precipitated mineral

Mg Substitution%




Mol ratio


Mol ratio


Mol ratio


Mol ratio


Mol ratio


Mol ratio

Without Mg or F










Mg substitution




























F substitution




























Note: Molar Ratio (Ca+Mg)/(PO4+F) is 1.67 for all samples

Substitution% for Mg is calculated base on the ratio of applied Mg/Ca in synthesis over the same ratio for precipitate, So is the for substitution% of F.

F/Ca ratio was used for evaluation of OH substitution with F because only 2 kind of mineral were seen including HA and FA.


Infrared frequency (cm-1)



567, 605

PO4-3 bend ν4









PO4-3 bend ν3




GEL (Amide III)






Precipitate(MgP/ DCPD)





N-H bend

GEL (Amide II)


C=O stretch

GEL (Amide I)



GEL (Amide B)


C-H stretch

GEL (Amide B)




Bulk elemental analysis

To determine molar percentage of calcium, magnesium, phosphorous and fluorine in the minerals precipitated within the GEL, elemental analysis was done. This analysis was done to get more information about the precipitated phase and also to investigate the amount of Ca and OH substitution by Mg and F in the structure of HA. Before the analysis, precipitates were washed to remove remained unreacted ions trapped within the GEL. Obtained results can be seen in table 1. In order to have a better comparison, atomic molar ratios in two situations including, before precipitation (applied in synthesis) and after precipitation were used. This especially helped us to determine the exact substitution that has been occurred.

As table 1 shows, three Mg/Ca ratios including 0.33, 1 and 3 were applied during synthesis process. The corresponding ratios were 0.07, 0.16 and 0.61 for precipitated minerals which indicates that with increasing primary Mg/Ca ratio, the same ratios increased for the precipitated minerals.

Dividing the ratio after sedimentation by the ratio before sedimentation and multiplying the product by 100 yields the number presented in table 1 as substitution percent.

Substitution percent for Mg for all of the applied ratios were about 20%. It means that there is no more than 20% of calcium atoms can be substituted by Mg atoms in the structure of HA or DCPD.

In order to evaluate the rate of F atom entering the sediment's structure, we used the F/Ca ratio before and after sedimentation. As the results of XRD analysis indicate the presence of FA and HA in the deposit phase, this ratio can be used for this purpose. The F/Ca ratios for the three designed samples of OH replacement with F atom in HA structure were 0.25, 0.50, and 0.75. Analysis of sediment reveals that the relevant ratios in the final deposit were 0.16, 0.25 and 0.36. This finding indicates that in lower ratios of F addition, all the F atoms enter the deposit structure. However, as the ratio rises, smaller amount penetrate the final sediment. In addition, as the F/Ca ratio in the FA compound equals 0.2, it may be concluded that some other compound other than FA with higher F/Ca ratio must be present in the final sediment so that the final ratios make sense. Considering the type of reactants, the only other compound presumable is CaF2; this is consistent with the results of XRD analysis. In other words, with increasing amounts of F added, more CaF2 sediment is produced alongside HA and FA.

4. Discussion

In recent years, to mimic bone structure better, diffusional methods have been used for synthesis of polymer/ HA(CP) composite scaffold[12-14, 18-20, 43]. In addition, formation of amorphous CP phase within the mentioned composite scaffold was aimed in some researches [44-48] to produce a mineral phase similar to the natural bone.

In this study, addition of Mg and F atoms inside the CP ceramic phase was applied using the diffusional method.

To obtain an amorphous CP within GEL, the diffusion experiment was done at 4°C.

For the sample without any Mg or F atoms, data obtained from various analyses revealed that the mineral formed within GEL has a low crystalline nature, consisting of HA and OCP.

Result obtained from XRD showed distinct change of precipitated phases after addition of Mg and F ions to the synthesis process. While addition of new atoms, competitive reaction between atoms will determine types of mineral to be precipitated.

The Addition of Mg, cause formation of MgPO4 along with HA. It is clearly shown that, Mg was substituted with Ca in the structure of precipitated mineral but the level of substitution was not more than 20%. It seems that the tendency of the system render substitution more than this value

Addition of F to the synthesis process, causes formation of FA and CaF2 along with HA, while there was no OCP or DCPD. Since, addition of F to the synthesis was done with a ratios of F/Ca which were more than stoichiometric ratio in the FA compound (equal to 0.2), it is expectable that some other compounds with the F/Ca ratio more than 0.2 should be precipitated such as CaF2. Ratios F/Ca obtained from elemental analysis for precipitated minerals are also in agreement with this fact.

Effect of Mg and F atoms substitution within precipitated minerals on the morphology of minerals was also very intense and interesting. Morphology of minerals for the sample with no Mg of F was in the form of oriented nucleation and growth which resulted in minerals with rod-like structure with rectangular cross section. From the morphological point of view, Major difference between these observation with the reports[11, 12, 49-54] published earlier was in the form of rod-like or needle-like minerals of CP or HA and also size of mineral clusters, as here formation of bigger clusters was seen.

Morphology of precipitated mineral will be influenced by various parameters. In several studies related to precipitation of a CP compound, different morphologies have been observed [49, 50, 51]. Morphology may be affected by chemical functional groups of the substrate used for mineral deposition or even with the place of precipitation, inside or on the surface of a gel (or hydrogel).

((Substrate …….))

Liu and colleagues investigated the growth of hydroxyapatite (HAp) crystal in the presence of hexadecylamine via soaking in a supersaturated calcified solution. They observed large amount of plate-like crystals, with a width of about 2-5 micron and a thickness about 50 nm. The platelet grows to be strip-like, that is, the growth rate along the longitudinal direction is much faster than those along the other two directions. So it is reasonable to conclude that the HAp crystals grow with the (0001) plane parallel to the surface of the organic layer[50].

Tarasevich and colleagues investigated nucleation and growth of calcium phosphate in the presence and absence of amelogenin. It was concluded that nucleation was promoted in the presence of protein compared to solutions without protein. For the amelogenin containing they observed that mineral particles are spherical and contain plate-like substructures after 24 h, while there were very few visible crystals formed in the non containing protein solution. The average particle size is about 47 µm. In general, the particle sizes varied directly with supersaturations level of solutions [51].

Eiden-Aßmann and colleagues studied the effects of amino acids, such as aspartic acid, glutamic acid, and serine on HA morphology in a double diffusion experiments. They concluded that, the presence of the amino acids results in spheres with 2micron in diameter consisting of randomly distributed needles, blades or plates depending on the reaction system. It was shown also that, HA was only formed when the pH was around 7.4 or higher during reaction. A decrease in pH resulted in the transformation of hydroxyapatite to octacalcium phosphate. [49].

Method of mineral formation or the procedure that ions react with each other is another important parameter. Two kinds of procedures including diffusion controlled and non-diffusion controlled may be considered.

One of the simplest methods for preparation of a biomineralization is in situ synthesis of HA via addition of reactant in the presence of a polymeric phase like collagen.

Li et al. [43] aimed to synthesize ACP through drop-wise addition of a phosphate solution into a polyethylene glycol (PEG, a stabilizer) containing calcium solution at 5°C and investigated the effect of ageing, pH, and Ca/P atomic ratios on the final product. Although they obtained ACP under different conditions, they observed some crystalline peaks at 2Ó© = 11.6, 20.9, 29.2, 30.4, and 34.0 in XRD diffractograms. The authors ascribed these peaks to DCPD that appeared in the sample, which was synthesized at an initial pH of 6.

Soaking when a polymeric film or a hydrogel is exposed to a supersaturated solution including Ca and PO4 ions is also another method of biomineralisation.

Zhai and colleagues investigated formation of nano-hydroxyapatite on recombinant human-like collagen fibrils through in situ synthesis of HA. For this aim they added NaH2PO4 solution slowly to a CaCl2 containing collagen solution while the pH was adjusted to 7.0. Results demonstrated the flower-like morphology for the mineral formed on collagen fibrils and the leaves of the flower were entangled and perpendicular to the surface. [52].


Hutchens and colleagues fabricated a composite material consisting of calcium-deficient HA deposited in a bacterial cellulose via alternating incubation in Ca and PO4 solution sources. SEM images confirmed that uniform solid spherical HAp particles with a size of about 1 µm like a network of nanosized lamellar crystallites. They reported that orderly clusters become rough and irregular in shape as more HA was formed in the matrix.[53].

Diffusion based methods are also used for mineralization of HA within a matrix. Furuichi and his colleagues prepared a CP/polymer composite by calcification of hydrogel via diffusion of Ca ions into the PO4 containing poly(acrylic acid) and GEL hydrogel. By using TEM they observed formation of semi spherical crystal clusters with about 1µm in diameter consisting as if needle like minerals projecting out from the center of each sphere. [11].

In a study by Manjubala and colleagues mineralisation of chitosan scaffolds with nano-apatite formation by double diffusion technique was investigated. Results showed clusters of apatite with a size ranging 1-2 µm and the needle-shaped apatite distributed homogeneously over the surface layer [12].

Karsten Schwarz and Matthias Epple investigated biomimetic crystallization of HAp and FAp crystallization by a double diffusion system in a porous poly(hydroxyacetic acid) (polyglycolide) matrix. They suggested that specific interactions between the growing aggregates and the polymeric matrix (nucleation and growth of crystals) occurred during their experiment.

Fluoroapatite gave elongated hexagonal prisms developing multiply branched edges that finally closed into spheres. The results showed that calcium phosphate phases are formed in specific biomimetic morphology by diffusion of two solutions into a porous polyglycolide matrix.[54].

Teng and colleagues used gelatin as a precursor for formation of calcium phosphates with a diffusion system. They concluded that by varying pH values and concentrations of calcium and phosphate solutions, plate-like OCP crystals (at lower pH), spherical ACP particles and flower-like HAp aggregates (at higher pH and concentration) can be successfully synthesized[45].

Considering results obtained from this study and previous reports it can be concluded that the natural tendency of CP minerals especially in HA and FA is towards formation of minerals in an ordered orientations and shapes. But the condition of environment usually does not allow to the crystals to be grown orderly. For example, in a simple precipitation method, high rate of exposure of reactant in the medium inhibits formation of oriented crystals. While application of diffusion method for precipitation within hydrogel provides a condition in which ions react with each other slowly. Therefore atoms have more chance to be allocated in the particular crystallographic positions and thus form oriented crystals.

The reason of obtained result in this study and similar studies could be due to the growth of the precipitate within the hydrogel caused by diffusion process.

It should be noted that the addition of F in to the structure causes destruction of this growth and affects the geometry of crystals .This added ion has also a noticeable impact on the formation of more amorphous phase compared with the crystalline phase.

While for the Mg containing samples, it was shown that the precipitates tend to be more crystalline rather than the sample with no F and Mg.

Precipitation of calcium phosphate mineral via the methods described can give a gradient composite structure, higher density from border to the center, and better adhesion of ceramic particles with GEL matrix compared with conventional mixing of previously prepared ceramic particles with GEL [8-13].

In our previous work, we showed that if the GEL/ACP scaffold is implanted in the body, it will ultimately be converted to HA crystals through a process similar to the natural bone formation in the body. However, one can incubate the GEL/ACP scaffold in the SBF solution prior to implantation into the body, but after conversion to the GEL/HA, as discussed earlier.(no meaning??)

Thermodynamically, all of the calcium phosphates should finally be converted to HA in the body environment [51].