Biological behavior of magnesium-substituted hydroxyapatite during bone repair

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Introduction
The use of ceramic biomaterials, in order to replace and/or aid in the regeneration of bone tissue, has been documented for more than a century.Although this tissue is able to completely regenerate under physiological conditions, in the presence of extensive tissue losses repair occurs with the formation of fibrous connective tissue (Cardoso et al., 2006;Miguel et al., 2006Miguel et al., , 2013)).This, in most cases, compromises the function, aesthetics, labor and quality of life of the affected individuals.
Among the different types of ceramics used in bone regenerative techniques, calcium phosphates (CaP) are part of the widely used group of materials, especially synthetic HA.This biomaterial is similar to components of the mineral phase of the bone tissue, biocompatible, bioactive, with excellent osteoconductivity and absence of local or systemic toxicity, and with no risk of disease transmission (Vallet-Regí and González-Calbet, 2004;Yuasa et al., 2004).Moreover, it serves as a framework for the cellular events that occurr during tissue regeneration, which make bone neoformation possible in association with its structure.Depending on the method used during the synthesis and processing of the materials, the HA may have a slow or rapid degradation rate, which will determine its clinical applications.
It is known that bone regeneration is a physiological mechanism that depends, among other factors, on the availability of some ions such as calcium (Ca), phosphorus (P), fluorine (F), zinc (Zn) and Mg.Some researchers have performed isomorphic substitutions in the hexagonal structure of synthetic HA through partial exchange of Ca by F, Zn, Sr or Mg (Cazalbou et al., 2005;Terra et al., 2009;Lima et al., 2011;Zilm et al., 2015;Geng et al., 2016;Ribeiro et al., 2016;Tao et al., 2016) in order to improve the physicochemical properties of this ceramic and, consequently, the biological response after implantation in vivo.Among these ions, Mg is of great clinical interest, since it is closely correlated with homeostasis and bone mineralization (Bigi et al., 1992(Bigi et al., , 1993;;Bertoni et al., 1998;Gouveia et al., 2008;Jia et al., 2010;Laurencin et al., 2011;Nabiyouni et al., 2015;Tao et al., 2016).

Synthesis of HA and MgHA powders
The biomaterials evaluated in this study were produced, characterized and supplied by the LABIOMAT researchers of the Brazilian Center for Physical Research (CBPF), Rio de Janeiro, Brazil.The powders were synthesized by wet precipitation method, under a synthesis temperature of 90 °C and Ca/P≥1,67 ratio.For the synthesis, solutions of Ca nitrate, Mg nitrate (molar contents of 1%) and dibasic ammonium phosphate were used.The solution of metals was dripped into the solution of phosphorus ions (P) and the reaction conditions were maintained throughout the process.The material obtained was filtered and washed until pH 7.0, in wash water.The solids were oven dried at 100 °C for 24 h.Finally, the solid was macerated and separated using sieve with an aperture of <74μm mesh.

Processing of HA and MgHA microspheres
The HA powders substituted with 1% by mass of Mg (MgHA) and without substitution (HA), obtained in the previous step (<74μm), were mixed into a solution of 1% wt./v sodium alginate until a ceramic slurry was formed.By extrusion, this slurry was dripped into a solution of Ca chloride (CaCl 2 ) to form the microspheres which, after 24 hours, were washed and oven dried at 70 °C.After this step, the biomaterial was sintered in a heating ramp to a temperature of 1000 °C in a muffle furnace.The obtained microspheres were separated in the granulometric range of 425>ϕ<600µm and aliquoted in Eppendorf tubes for autoclave sterilization.The superficial area of the powders was analyzed by BET method (Micromeritics  -ASAP2010) (Table 1).

Chemical analysis
X-ray fluorescence (XRF) was performed to determine the HA stoichiometry, particularly the Mg/Ca molar ratio and calcium substitutions (Table 2).This analysis was performed through X-ray fluorescence spectrometer (Philips  -PW2400).

X-ray diffraction (X-RD) of HA and MgHA
The X-ray diffraction was performed on the high resolution diffractometer (Zeiss  -HZG4) operating at 30kV and 15mA with CuKα radiation (λ= 1.542Å).The data was collected in the 2θ range of 10-80° with

Biomaterial
Surface area m 2 /g HA 3,445 MgHA 2.283 a step of 0.05° point per second, for 160 seconds.
The spectra obtained was compared with those present in the standard sheet PCPDFWIN 09.0432 of the database (JCPDS -Joint Committee on Powder Diffraction Standards) (Figure 1).

Fourier Transform Infrared Spectroscopy (FTIR) of HA and MgHA
Infrared analysis was performed on the Fourier Transform Infrared Spectrophotometer (Schimadzu  -IR-Prestige 21) with KBr Beam Separator.The analysis was done by transmittance using 1% KBr pellet in the median infrared region (400-4000 cm -1 ) (Figure 2).

Ethical considerations
The experimental procedures of this study were carried out in the Central Biotério of the State University of Feira de Santana (UEFS) in accordance with the Ethical Norms for Research on Animals (Law No. 11.794 of 2008), after approval by the Ethics Committee on Animal Use (CEUA) of this institution, protocol 004/2011.

Sample
30 adult male Wistar rats, weighing between 350 and 450 g, were randomly assigned to three experimental groups with five animals at each biological point: GI -bone defect filled with HA microspheres; GII -bone defect filled with Mg-substituted HA (MgHA) microspheres; GIII (control) -empty bone defect, evaluated at biological points of 15 and 45 days (Table 3).

Surgical experimental procedures
Prior to the surgical procedure, the animals received analgesia, sedation and anesthesia in a single dose, by intramuscular injection of ketamine hydrochloride, at a ratio of 0.08 mL/100 g body weight and 2% xylazine hydrochloride at a ratio of 0.04 mL/100 g body weight.
The animals were then placed in a prone position, trichotomized in the calvaria and submitted to surgical field antisepsis with chlorhexidine (1%).The surgical technique used in the manufacture of the critical bone defect in rat calvaria was the same as described in Cardoso et al. (2006) and in Miguel et al. (2006).After the creation of the bone defect, the biomaterials were implanted, according to the experimental group.The tissue flap was then repositioned and sutured with interrupted stitches.In the animals of group GIII, the bone defects remained without implantation of microspheres.Throughout the experiment the animals received rat food and water ad libitum, in a fountain appropriate for rats.

Histological processing and histomorphological analysis
After the euthanasia of the animals, at the biological points of 15 and 45 days, the upper portion of the calvaria was removed, reduced in lateral-posterior, posterior and anterior portions, discarding all the surrounding soft tissue.The specimens were then fixed in 4% formaldehyde for 48 hours.After this step, the samples were decalcified in 7% nitric acid, for two hours, under technical supervision, and embedded in paraffin.The serial histological sections of 5μm thickness were stained with hematoxylin-eosin (HE) and examined by light microscopy.

Histological Results
At 15 days, the histomorphological analysis showed discrete chronic inflammation in all three groups.In GI and GII the microspheres remained intact, permeated by cells and surrounded by loose connective tissue.Bone neoformation was noted in the three experimental groups, near the edges of the bone defect.In the GI, some nuclei mineralization was observed adjacent to the microspheres in the region near the center of the defect.In GII, this finding was more evident near the bone edges.In GIII, the remaining area was filled by loose connective tissue (Figures 3 and 4).
At 45 days, discrete chronic inflammation was observed in all three groups.In GI and GII, this inflammation was of the chronic granulomatous type.In the GIII, the scarce presence of mononuclear inflammatory cells was noticed.Most microspheres remained intact, acellular and enveloped by a loose connective tissue denser than the previous period, both in GI and GII.In these two groups, the bone neoformation near the edges was notorious and more evident than in the GIII, in which the mineralization remained similar to that seen at the biological point of 15 days.In the central region of the defect, similarly in GI and GII, a larger amount of newly formed mineralized tissue was observed around the microspheres compared to the previous period (Figures 5 and 6).

Discussion
Bone regeneration is a mechanism of tissue repair that depends, among other factors, on adequate blood supply, mechanical stability, size of lesion, availability of minerals, vitamins and trace elements, such as F, Zn, Sr and Mg, which are essential for mineralization (Hill and Orth, 1998;Nabiyouni et al., 2015;Geng et al., 2016;Tao et al., 2016).Taking this into consideration, some researchers have mimicked the biological apatite and performed ionic substitutions in the structure of the synthetic HA to alter its physicochemical properties and optimize the biological response, considering that this ceramic presents low rate of biodegradation in vivo (Ribeiro et al., 2016;Geng et al., 2016).Thus, in the present study the biological behavior and the osteogenic potential of Mg-substituted HA microspheres after implantation in a critical bone defect in rat calvaria was analyzed.The creation of critical bone defect results in vascular rupture, leakage of blood cells, clot formation and release of inflammatory cytokines which trigger a chronic inflammatory response (Anderson et al., 2008), as observed in the histological results of the three experimental groups of our study, at 15 days (Cardoso et al., 2006;Miguel et al., 2006;Almeida et al., 2015;Ribeiro et al., 2015).At 45 days, in groups I and II the response was of a discrete granulomatous chronic type, characteristics of the foreign body reaction observed after the implantation of biomaterials (Anderson et al., 2008).These results were also seen by Miguel et al. (2013) and Costa et al. (2016), although these authors have evaluated other types of biomaterials.The absence of rejection by the organism shows that both biomaterials were similarly biocompatible.
In our study, the distribution of the microspheres along the bone defect in both implanted groups, both at 15 and 45 days, provided the three-dimensional (3D) framework essential for cell migration and proliferation, angiogenesis and osteogenesis, fundamental to bone regeneration (Venkatesan et al., 2015).Therefore, the observed tissue formation surrounding and permeating the microspheres was evident and presented thickness proportional to bone edges.On the other hand, the absence of a framework in GIII limited the aforementioned events and resulted in the formation of connective tissue with reduced thickness in relation to the bone edges, mainly in the central region of the defect, at 45 days.Consequently, in this group, the new bone formation was restricted to the borders, throughout the study (Cardoso et al., 2006;Miguel et al., 2006Miguel et al., , 2013;;Tovar et al., 2014;Costa et al., 2016).
Bone neoformation was observed, in a similar way in groups I and II, beyond the borders, in the centripetal direction, in and around the microspheres.This suggests that the incorporation of Mg into the HA structure did not favor a greater osteogenic potential to the biomaterial, corroborating with the findings of Yassuda et al. (2013), which evaluated β-tricalcium phosphate (β-TCP) substituted with Mg in rats, and Sartori et al. ( 2014) who evaluated Mg-substituted HA in sheep, both in critical bone defect.Some studies suggest that Mg inhibits the formation of amorphous CaP within the synthetic HA structure given the introduction of distorted solid structures and the structural lag resulting from inhibition of crystal growth (Bigi et al., 1992(Bigi et al., , 1993;;Bertoni et al., 1998;Laurencin et al., 2011).It is emphasized that the amorphous phase of CaP represents the beginning of the mineralization, which occurs in the gap regions of the collagen fibril and extends parallel to the fibrils, in the inter fiber spaces of the collagen fiber (Chae et al., 2013;Nabiyouni et al., 2015).
The increase in the amount of imperfect crystals and the reduction of crystal size increases the rate of biodegradation of the ceramics (Lu et al., 2002).According to Azevedo et al. (2014) this decrease in crystal size average may be correlated with the decomposition of HA crystals into Tricalcium Phosphate (TCP).
Some studies have shown that Mg can induce the conversion of HA to Mg-containing β-tricalcium phosphate (β-TCMP) during the heat treatment at 1000 °C (Bigi et al., 1992(Bigi et al., , 1993;;Bertoni et al., 1998;Laurencin et al., 2011).However, this depends on the percentage of Mg present in the HA structure, given that at 5% and 10% Mg, this conversion started at 700 °C and at 1% Mg at 900 °C.In the latter case, the percentage of HA and β-TCMP is approximately 90% and 5%, respectively.This occurs similarly with HA without Mg (Bertoni et al., 1998).The MgHA evaluated in our study showed, in the XRF analysis, a concentration of 0.47% Mg, 79% HA phase and 21% β-TCMP, and, nonetheless, the microspheres remained mostly intact at the end of the study, and did not present notorious biodegradation, in consonance with the results found by Yassuda et al. (2013) andSartori et al. (2014), showing that the presence of Mg makes the ceramic biomaterials less bioresorbable, even in reduced concentrations.The microspheres implanted in GI showed a profile of biodegradation similar to GII, even without the presence of the metal.This can be justified by the high crystallinity of these biomaterials (Figure 1).It is worth noting that the materials evaluated in our study (GI and GII) were sintered, a biomaterial processing pathway that favors the fusion of the HA crystals and hinders biodegradation (Bigi et al., 1992(Bigi et al., , 1993;;Bertoni et al., 1998;Laurencin et al., 2011;Xiao et al., 2016).
The results of the present study showed that the percentage of Mg used did not favor a greater bone neoformation in relation to HA without the metal.Therefore, new studies need to be developed in order to evaluate the efficacy of other Mg percentages and their influence on the biodegradation and biological behavior of the new materials.

Conclusion
The biomaterials were biocompatible and osteoconductive, making them promising for use as fillers.Both biomaterials showed similar biodegradation throughout the experiment.The percentage of Mg evaluated did not increase the osteogenic potential of the biomaterials.

Figure 1 .
Figure 1.X-ray diffraction (X-RD) of HA and MgHA.It is noted that the HA has a slightly narrower base of the major peaks in relation to MgHA, evidence for the greater crystallinity of the material.

Figure 2 .
Figure 2. Fourier Transform Infrared Spectroscopy (FTIR) of HA and MgHA.It is observed that the spectra revealed characteristic bands of HA for phosphate (PO 4 3-), water (H 2 O) and hydroxyl (OH 1-), even for the material substituted with Mg.

Table 1 .
Surface area of HA and MgHA powders, BET method.

Table 2 .
Chemical analysis of HA and MgHA powders, values obtained by the average of the three samples.