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ORIGINAL RESEARCH
Year : 2014  |  Volume : 5  |  Issue : 4  |  Page : 155-161

Mechanochemical synthesis evaluation of nanocrystalline bone-derived bioceramic powder using for bone tissue engineering


1 Department of Mechanical Engineering, Khomeinishahr Branch, Islamic Azad University, Khomeinishahr/Isfahan, Iran
2 Department of Materials Engineering, Najafabad Branch, Islamic Azad University, Isfahan, Iran
3 Department of Prosthodontics, Torabinejad Dental Research Center, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Web Publication12-Sep-2014

Correspondence Address:
Amirsalar Khandan
Department of Mechanical Engineering, Khomeinishahr Branch, Islamic Azad University, Khomeinishahr/Isfahan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2155-8213.140606

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  Abstract 

Introduction: Bone tissue engineering proposes a suitable way to regenerate lost bones. Different materials have been considered for use in bone tissue engineering. Hydroxyapatite (HA) is a significant success of bioceramics as a bone tissue repairing biomaterial. Among different bioceramic materials, recent interest has been risen on fluorinated hydroxyapatites, (FHA, Ca 10 (PO 4 ) 6 F x (OH) 2−x ). Fluorine ions can promote apatite formation and improve the stability of HA in the biological environments. Therefore, they have been developed for bone tissue engineering. The aim of this study was to synthesize and characterize the FHA nanopowder via mechanochemical (MC) methods. Materials and Methods: Natural hydroxyapatite (NHA) 95.7 wt.% and calcium fluoride (CaF 2 ) powder 4.3 wt.% were used for synthesis of FHA. MC reaction was performed in the planetary milling balls using a porcelain cup and alumina balls. Ratio of balls to reactant materials was 15:1 at 400 rpm rotation speed. The structures of the powdered particles formed at different milling times were evaluated by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results: Fabrication of FHA from natural sources like bovine bone achieved after 8 h ball milling with pure nanopowder. Conclusion: F ion enhances the crystallization and mechanical properties of HA in formation of bone. The produced FHA was in nano-scale, and its crystal size was about 80-90 nm with sphere distribution in shape and size. FHA powder is a suitable biomaterial for bone tissue engineering.

Keywords: Bovine bone, ball milling, dental implant, fluorhydroxyapatite, mechanochemical, nanoparticle


How to cite this article:
Khandan A, Karamian E, Bonakdarchian M. Mechanochemical synthesis evaluation of nanocrystalline bone-derived bioceramic powder using for bone tissue engineering. Dent Hypotheses 2014;5:155-61

How to cite this URL:
Khandan A, Karamian E, Bonakdarchian M. Mechanochemical synthesis evaluation of nanocrystalline bone-derived bioceramic powder using for bone tissue engineering. Dent Hypotheses [serial online] 2014 [cited 2019 Dec 13];5:155-61. Available from: http://www.dentalhypotheses.com/text.asp?2014/5/4/155/140606


  Introduction Top


Bioceramics are the most important materials in biomaterials engineering researches. Calcium phosphates (CaP) are the most common family of bioceramics well known for their use in biological and medical applications. [1],[2],[3],[4] It is clear that natural bone (NHA-Bovine Bone) is mainly composed of partially carbonated hydroxyapatite on the nanometer scale and collagen. Furthermore, the nanometer size of bone-like apatite in the inorganic components is considered to play an important role in maintaining the mechanical strength and chemical composition of the bone. [3] Despite these desirable characteristics, synthetic hydroxyapatite (HA) is limited in application due to high in vivo solubility, poor mechanical properties, and poor thermostability. [5],[6],[7] Various amounts of substitutions (i.e. F , Zr 4+ , Br 2+ , Sr 2+ , CO 32−) are present in biological apatites. [8] Among them, F 1− plays leading roles because of its influence on the mechanical and biological properties of HA. [7],[8] In practice, F 1− itself has been widely investigated in dental restoration areas because it prevents dental caries in a bacteria containing, acid environment. Recently, fluorine-doped HA coatings [FHA, Ca 10 (PO 4 ) 6 F x (OH) 2−x ] and fluorapatite (FA, Ca 10 (PO 4 ) 6 F 2 ) on metallic substrates and a bulk have attracted a great deal of attention in areas requiring long-term chemical and mechanical stability. [4],[6],[7],[8] Research shows that FHA has better mechanical properties than calcium phosphate ceramics such as HA. Modern technologies require new biomaterials to match better with natural bone in terms of mechanical properties. There is a high demand for bone regeneration due to various clinical bone diseases such as bone infections, bone tumors and bone loss by trauma. [1],[2] Properties of HA such as bioactivity, biocompatibility, and solubility properties can be tailored over a wide range by modifying the composition via ionic substitutions. [9],[10] In the attempt to find new biomaterials characterized by a greater stability but, at the same time, with good biological performance similar to that of HA, FHA have been proposed. In FHA, the fluorination process has been recognized to be effective in reducing the susceptibility of these materials to degradation in vitro and in vivo. [11],[12] FHA and FA in which the hydroxylic groups (OH 1− ) are substituted by F ions, are considered as alternative biomaterials and have the potential of being used in dental implants [Figure 1]b. [11],[13],[14],[15] Fluorine replacement decreases the solubility, and increases the compression strength. [16] Results have shown that FHA nanoparticles could provide lower dissolution, and comparable or better cell attachment than HA, and significantly improve alkaline phosphatase activity in the SBF solution. [9],[10] F ion improves the crystallization and mineralization of pure HA in formation of bone and teeth. [13] HA coating over metallic implants have been investigated and was found to recuperate the implant's bioactivity and fixation to natural bone (NHA) tissues this in turn increases the implant's functional lifespan. Previous studies on NHA-coated implants (NHA/zircon) have shown good fixation to the host bones. [17] Recently, FHA has been increasingly considered as a clinical restoration biomaterial due to the extensive findings of FHA in bone and teeth and the favorable effect of fluoride ion on bone growth. [11] These mineral particles are combined with collagen into self-assembled complex hierarchical structure in calcified tissue to achieve a remarkable mechanical performance. [18] As a result, many researches have been done to improve calcium phosphate-based, HA, nanostructures such as nanocrystalline HAp and FAp phases, [19],[20] HAp- Al 2 O 3 . [21] Based on the advantages of FHA compared to HA, FHA is a promising and suitable choice as the second phase. The aim of the present survey was to fabricate FHA nanopowder by MC method for dental applications, e.g., coating for metallic implants, restorative composites, and for different clinical applications involving repair of bone defects such as bone augmentation. In the present study, the effects of ball milling time on the fabrication of FHA nanopowders have been investigated. FHA has low solubility, high bioactivity, good biocompatibility, high thermal and chemical stability. Generally, the fabrication methods of calcium phosphate-based materials can be classified into two groups: wet and dry. [10],[22] Several methods have been developed for fabrication of FHA powders and bioceramics, namely, wet precipitation, mechanochemical (MC) method, solid-state reaction, hydrothermal, pH cycling and sol-gel processing. However, so far, most of the studies have noticed on the fabrication of FHA nanopowders. The effects of different milling times on the thermal characteristics of FHA bioceramics have seldom been systematically studied. The performances of nanostructured FHA ceramics in biomaterials applications are strongly dependent on their morphological features. In this study, for a discovering of the effect of MC process on morphological evolution, X-ray diffraction (XRD) and scanning electron microscopy (SEM) analysis are performed on the nanopowders.
Figure 1: (a) FHA microstructure with OH groups and F ions, (b) comparison between HA and FHA microstructures


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  Materials and Methods Top


Bovine bones were boiled with hot water for 12 h to remove flesh and fat. The bones were heated at 110°C for 2 h to remove moisture and impurity. To prevent blackening with soot during heating, the bones were cut into small pieces of about 10-20 mm thick and heated at 600°C named bone ash, for 2 h. The resulting black bone ash was heated for 3 h at 850°C and this synthesis is called thermal decomposition of bone resource to create NHA. The NHA obtained was milled to crash and homogenize in the ball mill for 1 h. The FHA nanopowder were produced by mixing NHA and calcium fluoride (CaF 2 ) by planetary high energy ball milling using porcelain vial and alumina balls with ball/powder ratio of 15:1, rotational speed of 600 rpm, and the time of 2, 4, 6 and 8 h. The relative amounts of NHA and CaF 2 was 1:20. The mass ratio of balls to reactants was 15, whereby the overall ball mass was equal to 160 g. Phase structure analysis was performed by XRD (Philips X'Pert-MPD diffractometer with Cu Kα radiation (λ1 = 0.15418 nm) over the 2θ range of 20-80). The obtained experimental patterns were compared to the standards compiled by the Joint Committee on Powder Diffraction and Standards (JCDPS) which involved card # 09-432 for HA. The crystallite size of prepared powders was determined using XRD patterns. SEM analysis evaluations were performed using a Philips XL30 (Eindhoven, The Netherlands) to investigate the morphology. Transmission electron microscopy (TEM) technique (Philips 100 KV EM208S) was utilized to evaluate the shape and size of prepared FHA. TEM analysis of powders was done after 30 minutes ultrasonic in ethanol to collapse the agglomerated powders. Crystallite size of FHA nanopowders was calculated from X-ray patterns using X'Pert software and modified Scherer equation. The elemental chemical analysis of NHA using X-ray fluorescence (XRF) (Philips PW1606) is shown in [Table 1]. Therefore, we attempt here to consider the morphological features of nanostructured calcium phosphate-based materials.
Table 1: XRF results of hydroxyapatite ball milled for 3 h at 850°C

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  Results Top


Characterization procedure

The crystalline size of the prepared powders were determined using XRD patterns, and the modified Scherrer equation (Eq. 1). [23] In the current study, crystal size was measured according to the procedure described by Karamian et al. [24] This is equivalent to lnβ = ln(1/cosθ) + ln(kλ/L) (Eq.1). [25]



Chemical analysis (XRF Results)

[Table 1] shows the composition of NHA which is achieved from bovine. XRD pattern demonstrates that F ions have had diffused into NHA microstructure and doped and influence the HA particles.

SEM analysis

[Figure 2] illustrates the graphs of FHA powders with different ball milling time. In all the samples we found nanoparticles HA crystals with different dimensions which agglomerated. Morphology of particles is sphere and semi sphere. [Figure 2] (a and b) reveals that the microstructure and morphology of FHA nanopowder samples for different time ball mill duration. The morphology of the nanopowders indicates that it is composed of agglomerates with wide particle size distributions. As can be seen, these samples are formed by particles of very irregular shape and dimensions. The SEM photomicrograph of FHA powder in lower magnification is shown in [Figure 2]a.
Figure 2: SEM observation of FHA nanopowder after (a) 2 h, (b) 8 h MC method

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F substitution

As shown in [Figure 1], The hydrogen (H + ) ions (the smallest spheres in the figure) HA were arranged in the atomic interstices neighbouring to oxygen ions (O 2− ) ],[ forming OH groups and were oriented randomly, which conferred a certain degree of disorder to the crystal microstructure of HA [Figure 1]a. Once the OH groups were partially substituted by the F ions, the existing hydrogen ions of the OH groups were affinity of the F ions with respect to oxygen ions, producing a quite well-ordered apatite microstructure, which caused the increase of thermal and chemical stability of HA matrix.

TEM analysis

The morphological shape and size of FHA nanopowder obtained from TEM are shown in [Figure 3]. The TEM image reveals that nanopowders are formed by particles ranging from 70-100 nm. This is in agreement with the particle size of 80-90 nm powders calculated using XRD data. This micrograph confirms that nano-sized FHA particles were fabricated by MC method. The produced FHA nanoparticles have both spherical and rod-like morphology. Bioceramics with a crystal size lower than 100 nm have superior mechanical, chemical and biological properties. [24] Also, It has been indicated that the dissolution rate of the bioceramic nanopowder (FHA) is higher than that of conventional powder (NHA), and thus, apatite is formed more easily. [19],[20],[24] Nanometer size characteristic for obtained powders renders them similar to natural bone (biological bone). In addition, the FHA materials are expected to have better bioactivity and biological properties than pure HA by reviewing several other studies. [26] The morphological shape and size of FHA powder obtained from TEM micrograph.
Figure 3: TEM graph (100 nm) of the prepared FHA powder obtained after 8 h of ball milling

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Crystal size and lattice parameter calculation

In addition, crystallinity (X c ) of FHA is calculated using XRD data and equation 2 as follows:



Where I 300 is the intensity of the (300) diffraction peak, and V 112/300 is the intensity of the hollow between the (112) and the (300) diffraction peaks of the produced powder. [24] Xc is equal to 38%.


  Discussion Top


Despite these desirable characteristics, synthetic HA are limited in application due to poor mechanical. The aim of this study was to prepare Nano FHA by MC reaction to use as bone tissue engineering application to increase HA mechanical properties. Therefore, various amounts of substitutions F enhances the crystallization of HA in formation of bone. The incorporation of F ions into the HA lattice caused the decrease in the lattice parameters. Nanostructured bioceramic materials have been synthesized by various (Mechanochemical) MC reactions. Compared to various synthesis processes, MC synthesis method presents a number of advantages such as high efficiency, enabling to synthesis a wide range of novel advanced and economic biomaterials. Many attempts have been made to enhance the mechanical and biological properties as well as structural features of bioceramics through the incorporation of ceramic second phases. As a result, many researches have been done to improve calcium phosphate-based, hydroxyapatite (HA), structures such as nanocrystalline HAp and FAp phases, [18],[21],[27] HAp-Al 2 O 3 , [28] HAp-ZrO2, [29] HAp-TiO 2 , HAp-Ti, FAp-TiO2. [12]

In the study of Fathi et al., [26] they have prepared FHA by mechanical alloying (MA), as a solid-state simple method for powder to enhance kinetic reactions between solids. The mechanical and biological properties of FHA with different composition have been studied by several researchers. [26],[30] The MC processes reported by Fathi et al. [26],[30] have evaluated effect of ball milling parameters on the synthesis of FHAp powder. In addition, the effect of fluoridation on bioactivity of bone-like apatite was studied. The results showed that the size and number of balls had no significant effect on the synthesizing time and grain size of FHAp. [26] Many data found in the literature suggest that bulk bioceramic greatly and positively affect bone growth and attachment to an implant. [29] Regarding bulk bioceramic HA is by far the strategy most frequently adopted to enhance osseointegration and has been widely used in clinical care of osteoporotic patients. [22],[31],[32] However, several important questions still remain unresolved regarding the HA-metal substrate adhesion and HA stability and dissolution. [31],[33]

In addition, FHA has also been investigated as an alternative bioceramic coating because its osseointegration properties are similar to those of HA and its biostability appears to be enhanced as compared to the other ceramic coatings [31],[34] as can be seen in the [Figure 4]. It is found that the calcified tissues, such as bone or dentin contain ~70 wt. % apatite and ~20 wt. % collagen. These mineral powders are combined with collagen into self-assembled complex hierarchical microstructure in calcified tissue to achieve impressive mechanical performance. [18]
Figure 4: FHA using for bone tissue engineering and scaffold

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By investigating other research results the authors come into conclusion to consider FHA differently:

This study was focused on the characterization of FHA by XRD, SEM, and TEM. Fluoride ions are incorporated into bone (of the developing infant) substituting hydroxyl groups in the carbonate-apatite structure to produce FHA, thus altering the mineral microstructure of the bone and teeth. Fluoride is a chemically active ionized element, it may affect oxygen metabolism and induce oxygen free radicals which appear to play a role in diminishing cognitive ability processes such as learning and memory. [21] SEM and XRD analysis can be tailored over a wide range by modifying the composition and properties of prepared biomaterials.

The SEM graph of prepared FHA 8 h shows a relatively uniform distribution of the FHA nanopowder observations [Figure 2]. Some FHA agglomerates can be seen in the observation from the TEM photomicrograph [Figure 3]. The solubility of FHA can be improved with making its crystallinity degree (38% calculated by Eq. 2) similar to biological HA (70-80%) and decreasing its crystal size to nano-level. [26] [Figure 2]b shows that the ball mill time plays an important role on the formation of FHA. XRD patterns of FHA 2 h and FHA 8 h powders subjected to MC are shown in [Figure 5] (a and b). Consideration of FHA by XRD patterns reveals that additional crystalline phases appear at 6 h, and no other crystalline phase presents besides FHA.

No other peaks except for characteristic peaks of FHA in [Figure 5]b was observed. By comparing the XRD pattern of FHA 2 h with FHA 8 h sample, it is clear that some set of FHA characteristic peaks (reflections) gradually shifted to the right hand side. The slight shift of characteristic peaks to the right side of the 2θ axis, which also was observed in other researches, [35] was related to the reduce of the a-axis length of the hexagonal HA crystals lattice due to lower ionic radii of F 1− than OH 1− which caused introducing distortion in the lattice with incorporation of fluorine ions instead of hydroxyl groups in the apatite structure. It was noticed that the relationship between increase of the crystallinity (Xc ) and time was linear. It has found that the position of FHA peaks shifted slightly to higher angles with increasing the ball milling time. It is clear that the lattice parameters of NHA reduce as a result of the substitution of Ca 2+ with F ions in the HA lattice. [30] On the other hand, since F ionic radius (0.065 nm) is smaller than that of OH ions (1.32 nm), the substitution of OH 1− by smaller F ions resulted in a contraction of the cell parameters of HA [Figure 1]. In this study XRD patterns revealed that after 2 and 4 h ball milling times, FHA has not created, though after 6 h ball milling the peaks shifted to the left side and finally after 8 h FHA patterns was seen in the XRD patterns.
Figure 5: (a) XRD pattern of NHA nanopowder, (b) NHA and CaF2 ball milled for 2 h, NHA and CaF2 ball milled for 4 and 6 h, mixture of NHA and CaF2 ball milled for 8 h

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Properties of HA or FHA, such as bioactivity, solubility properties can be tailored over a wide range by modifying the composition via ionic substitutions. [19],[23] The FHA was produced in nano-scale and the particle size of the nanopowders was about 80-90 nm with sphere distribution in shape and size. FHA nanopowder seems to be a good candidate for bone tissue engineering dental applications. However, further in vitro and in vivo studies need to be conducted to explore the applicability of these bioceramics as a bone tissue material.


  Acknowledgment Top


The authors would like to extend their gratitude for the supporting provided by Khomeinishahr Branch, Islamic Azad University, and Isfahan, Iran.

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1]


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