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Table of Contents
Year : 2018  |  Volume : 1  |  Issue : 2  |  Page : 58-63

Does cationic incorporation into carbonated hydroxyapatite improve bone repair?

1 Department of Oral Surgery, Dentistry School, Universidade Federal Fluminsnes, Niterói, Rio de Janeiro, Brazil
2 Department of Ungraduated Student, Dentistry School, Universidade Federal Fluminsnes, Niterói, Rio de Janeiro, Brazil
3 Department of Oral Diagnosis, Dentistry School, Universidade Federal Fluminsnes, Niterói, Rio de Janeiro, Brazil
4 Department of Orthodontic, Dentistry School, Universidade Federal Fluminsnes, Niterói, Rio de Janeiro, Brazil

Date of Web Publication3-Aug-2018

Correspondence Address:
Prof. Monica Diuana Calasans-Maia
Universidade Federal Fluminense Faculdade de Odontologia - Rua Mário Santos Braga, n° 28/4° Floor, Centro, CEP 24040-110, Niterói, Rio de Janeiro
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/GFSC.GFSC_18_18

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Background: Calcium phosphate ceramics are a group of materials that have been widely used in bone regeneration, especially hydroxyapatite (HA), because of its biocompatibility and similarity to the main mineral phase component of bone tissue. However, the biological apatite has nanometric dimensions and cationic and anionic substitutions and presents low crystallinity, which differs from stoichiometric HA. Aims: The ionic substitutions in the composition of HA have been done to mimic biological apatite and improve its physicochemical characteristics. Previous studies have demonstrated that strontium-, magnesium-, zinc-, and iron-isolated substitution stimulate osteoblastic activity, as well as reduce osteoclastic activity. Materials and Methods: This study evaluated the osteogenic potential of nanostructured carbonated hydroxyapatite microspheres (cHAMs) containing 5% strontium, 5% zinc, 5% magnesium, 1% iron, and 5% manganese, after implantation in a critical size defect in the rat's calvaria. Two experimental groups were studied: cHA 37°C (nanostructured cHA, control) and cHAM 37°C (metals doped nanostructured cHA). The animals were euthanized after 1, 3, and 6 months, and the samples were histologically processed for histomorphometric analysis regarding the presence of residual biomaterial, neoformed bone, and connective tissue. Statistical Analysis: The averages found were analyzed statistically by the D'Agostino and Pearson analysis and by Kruskal–Wallis test; significant differences were observed for P < 0.05. Results: The test group presented less neoformed bone (P < 0.05) and was less bioabsorbed at 1 and 3 months (P < 0.05). Conclusion: The biomaterials studied were compatible osteoconductors, but the doping with multiple metals did not improve bone repair.

Keywords: Calcium phosphate, carbonated hydroxyapatite, in vivo, metals, rats calvaria

How to cite this article:
Pesce G, Nascimento Barros JR, Resende R, da Silva MT, Uzeda MJ, Novellino Alves AT, Calasans-Maia JA, Calasans-Maia MD. Does cationic incorporation into carbonated hydroxyapatite improve bone repair?. Int J Growth Factors Stem Cells Dent 2018;1:58-63

How to cite this URL:
Pesce G, Nascimento Barros JR, Resende R, da Silva MT, Uzeda MJ, Novellino Alves AT, Calasans-Maia JA, Calasans-Maia MD. Does cationic incorporation into carbonated hydroxyapatite improve bone repair?. Int J Growth Factors Stem Cells Dent [serial online] 2018 [cited 2023 Dec 1];1:58-63. Available from: https://www.cellsindentistry.org/text.asp?2018/1/2/58/238555

  Introduction Top

Bone tissue has excellent remodeling and regenerative capacity.[1],[2],[3] However, in extended bone defects, the regeneration occurred in a limited way and restricted to the bone margins,[4],[5] which compromises the function of individuals affected.[6],[7],[8]

New biomaterials capable of acting as three-dimensional frameworks to enable cell migration, angiogenesis, new extracellular matrix deposition, mineralization, and regeneration of this tissue have been developed.[9],[10] Those composed of calcium phosphate are widely used, mainly hydroxyapatite (HA), due to their similarity to the components of the bone mineral phase.[11]

However, in general, HAs used clinically are nonbioabsorbable and remain at the site of implantation for extended periods, which limits bone regeneration.[12] It occurs because of the heat treatment at high temperatures, leading to a high crystallinity material losing the nanometric characteristics. The biological apatite has in its composition nanometric crystals as well as different ionic substitutions. The biological apatite has in its composition nanometric crystals as well as different substitutions, such as carbonate (CO3), strontium (Sr 2+), iron (Fe ), magnesium (Mg 2+), and zinc (Zn 2+) among others; further, it has low crystallinity and fast bioabsorption.[13]

Within this context, the objective of improving the physicochemical characteristics of stoichiometric HA (Ca10[PO4]6[OH]2)[14] favored the partial replacement of Ca 2+ by Sr 2+;[1],[15],[16],[17] by Fe ;[18] by Mg 2+;[3],[15] by Zn 2+;[2],[19] by Mn, and by Fe, as well as PO4 by CO3.[20],[21]

In this line of research, the carbonated HA microspheres (cHAMs) mimic the biological apatite of the bone [22],[23],[24],[25],[26] and the objective of this study was to evaluate bone repair of this material comparatively with cHA without metals, specifically to evaluate histologically and histomorphometrically. This study hypothesized that the incorporation of metals in the structure of the HA could improve the biological response after the implantation in rats' calvaria.

  Materials and Methods Top

Synthesis of carbonated hydroxyapatite powders and microspheres

In this study, 425–600 μm microspheres composed of nanostructured cHA containing sodium alginate prepared by a wet precipitation method and containing 6 wt % CO3, with stoichiometry ranging between 1.6< Ca and P < 2.0, were synthesized and characterized in the Biomaterial Laboratory of the Brazilian Center for Physics Research. The cHA was synthesized at 37°C without receiving heat treatment (not sintered), thus maintaining their nanoscale features.

The composite material used in this study is constituted by the association of cHA to the biopolymer sodium alginate in the form of microporous microspheres containing metals. The cHA was synthesized according to good standard manufacturing practices with preparation parameters that of a nanostructured material with a high specific area (>200 m 2/g), low degree of crystallinity, and reduced particle size (d < 20 nm). The syntheses carried out were based on the work of NOUNAH (1993) using calcium nitrate solutions and nitrate of the following metals: strontium, zinc, iron, manganese, and magnesium (with, respectively, 5%, 5%, 1%, 5%, and 5%), dibasic ammonium phosphate and ammonium. The anionic substitution of the (PO4) 3− by (CO3) 2− (Ca10−x [PO4]6−x [CO3]x) (OH)2-x−2y (CO3)y(0 ≤ x ≤ 1.1, 0 ≤ y ≤ 0.2) produces a material with stoichiometry very close to the mineral phase of the biological apatite increasing the biomaterial bioreabsorption in vivo. Inside the reactor the solution containing the anions: (PO4)3- + (CO3)2- were under agitation at 37°C with pH 9. On the outside there was a bottle where it was dissolved all the salts with the metals (cationic solution) pH 5. To mix the two solutions it was used the peristaltic pump with a flow of 10mL/min and mechanical agitation of 240rpm; to maintain the system at pH 9, it was used another peristaltic pump which adds NH4OH; After this addition, it was kept the material under constant agitation and temperature for 4h. Then the solid formed was removed from the reactor, filter and wash until pH7. After dripping the mixture, it remained indigestion for 24 h under the same conditions and then filtered through Buckner's funnel and resuspended in MilliQ water at 37°C until to obtain pH 7.0 in the wash water. The solid obtained was lyophilized for 24 h. The solid was macerated and separated using a sieve with <74 mm mesh opening. After synthesis, the final biomaterial formula that was used in this study was Ca7.84 Sr0.5 Zn0.5 Fe0.1 Mn0.5 Mg0.5(PO4)5.88 (CO3)0.12 (OH)2.

The cHA with cationic modifications was prepared with stoichiometry ranging Ca/P = 1.25, containing 3.52 de Zn 2+; 2.46 de Sr 2+; 0.78 de Mg 2+; 2.65 de Mn 2+; and 0.45 de Fe 2+.

cHA was prepared from homogeneous mixtures of the biomaterials with sodium alginate dissolved in aqueous solution. The solution gel electrophoresis of HA/alginate was dripped by extrusion method in chloride solution of calcium for 24 h for the formation of the spherical morphology of the compound and complete ionic Na + by Ca 2+, collected by filtration, and then washed with distilled water. Then, part of the microspheres was heated at 50°C for 24 h for the elimination of water. After all, microspheres with a mean diameter between 400 and 650 μm were obtained.

Before the implantation in the rat's calvaria, the vibrational spectroscopy with Fourier-transformed infrared (Prestige 21, Schimadzu) [Figure 1] was performed to determine the chemical groups present, while the crystalline mineral phases present in the samples and their crystallinity were examined by X-ray diffraction (CuKa Radiation, ZeissHZG4) [Figure 2]. The biomaterials were packaged and sterilized by gamma irradiation (15 kGy/sample – Cobalt 60 irradiator at a dose rate of 19.72 Gy/min for 760 min).
Figure 1: Infrared spectroscopy with Fourier transform identifying the phosphate, carbonate, and water peaks

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Figure 2: X-ray diffraction of carbonated hydroxyapatite containing multiple cationic substitutions

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Animals and protocol design

This project was approved by the Ethics Committee on Animal Use of Fluminense Federal University number 834/16, and the procedures performed followed the CONCEA guidelines of euthanasia practice.

Thirty male Wistar rats of both genders and weighing between 300 and 400 g were distributed into two groups of 15 animals each distributed over experimental periods of 30,90, and 180 days. Each group was distributed into three subgroups of five individuals for each biomaterial and experimental period. The rats were operated under general anesthesia and received as an anesthetic medication a 0.5 ml solution of xylazine 2% (10 mg/kg, Sedazine ®, Fort Dodge, USA), 1.0 ml ketamine 10% (100 mg/kg, Francotar ®, Virbac, São Paulo, Brazil), 0.6 ml 5% midazolam (5 mg/kg, Midazolam ®, Medley, Campinas, São Paulo, Brazil), and 0.2 ml of tramadol 50 mg/ml (2 mg/kg, Tramal ®, Pfizer, Guarulhos, São Paulo, Brazil) diluted in 8.5 ml of sterile 0.9% saline solution (KabiPac ®). Each animal was anesthetized with 1 mL per 100 g of this solution intraperitoneally. After noting the absence of pain reflexes, trichotomy and antisepsis of the calvaria were performed. A semilunar incision was made on the calvaria of each animal. After the incision and subperiosteal detachment, a surgical defect was created using an 8-mm internal diameter trephine drill at intermittent low speed (1200 rpm) coupled to a 16:1 handpiece and microelectric motor with profuse irrigation with 0.9% sodium chloride. The critical size defects created were filled with around 1 g of the biomaterials according to their experimental groups. After filling the defect with cHA or cHAMs, the skin was sutured in a single plane with simple interrupted sutures with mononylon 5.0 (J and J Ethicon ®, São Paulo, Brazil). The wounds were left uncovered, and all animals received anti-inflammatory medication by intramuscular injection of a single dose of Maxicam ® 1 mg/kg (Ourofino Pet, Cravinhos, SP, Brazil). For recovery from anesthesia, the animals were returned to the cages and received food and water ad libitum. After 30, 90, and 180 days and without presenting any inflammatory signs in the operated area, the animals were euthanized with an overdose of general anesthetic (Thiopentax ®, 150 mg/kg; Cristália, São Paulo, Brazil) and the bone blocks were obtained.

Histological preparation

Thirty samples were decalcified and embedded in paraffin, sliced into serial sections of 5 μm thickness transverse to the plane of the implant, and then stained with hematoxylin and eosin. The slides obtained from the decalcified blocks were observed with light microscopy, and images were captured with a digital camera. A descriptive analysis of the tissue response to the biomaterials was performed due to the presence of connective tissue and newly formed bone in the defects beyond the resorption of the biomaterials.

Histomorphometric evaluation and statistical analysis

The histomorphometric evaluation was performed using Image-Pro Plus ® 6.0 (Media Cybernetics, Silver Spring, MD, USA). Quantitative records of this information were stored in a database developed in Microsoft Excel ® spreadsheet software and transferred to Prism ® 6.0 software (GraphPad Software, Inc., CA, USA) for statistical analysis. The averages found were analyzed statistically by the D'Agostino and Pearson analysis. Outliers were removed using the Rout equation with a value of Q = 1%. After the Kruskal–Wallis test and posttest of multiple Dunn's comparisons, significant differences were observed for P < 0.05. Statistical analysis of experimental and control groups was performed by comparing the amount of area of newly formed bone and connective tissue in the critical defect and the presence of the remaining biomaterial for each experimental period.

  Results Top

Descriptive histological analysis of calcium hydroxyapatite microspheres and carbonated hydroxyapatite

Calcium hydroxyapatite microspheres

At 1 month, small particles of biomaterial were observed, sometimes enclosed by multinucleated giant cells, presence of fibrocellular connective tissue, moderate mononuclear infiltrate, and presence of congestive vessels. On the margins of the defect, the presence of neoformed bone tissue toward the center of the defect [Figure 3].
Figure 3: Photomicrographs of calcium hydroxyapatite microspheres(a, c, and e) and carbonated hydroxyapatite Group (b, d, and f) after calvaria implantation. (a) Calcium hydroxyapatite microspheres after 1 month; (b) carbonated hydroxyapatite after 1 month; (c) calcium hydroxyapatite microspheres after 3 months; (d) carbonated hydroxyapatite after 3 months; (e) calcium hydroxyapatite microspheres after 6 months; (f) carbonated hydroxyapatite after 6 months. Observe the presence of minimal biomaterials fragments at the carbonated hydroxyapatite group after 1-month experimental period and a large amount of NFB. Observe the presence of biomaterials at the calcium hydroxyapatite microspheres group after all the experimental periods and a large amount of connective tissue. Stain: H and E; Scale: 50 μm. B: Biomaterial, NFB: Newly formed bone and CT: Connective tissue

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At 3 months, small particles of the biomaterial were present, surrounded by multinucleated giant cells, areas of neoformed bone involving biomaterial, interspersed with connective tissue showing areas of moderate inflammatory infiltrate with a predominance of macrophages in their totalized foam. A greater amount of neoformed bone was observed on the margin of the defect.

At 6 months, the area of the defect was filled with connective tissue showing foci of moderate inflammatory infiltrate with a moderate amount of foamy macrophages, absence of giant cells, and minimal presence of neoformed bone. The presence of neoformed bone is observed in the distal extremities.

Carbonated hydroxyapatite

After 1 month of implantation, the area of the defect is filled with connective tissue showing fragmented biomaterial particles and a large amount of macrophagic infiltrate (foamy macrophages). Little neoformed bone is seen at the center of the defect and the extremities. The presence of multinucleated giant cells was observed.

After 3 months of implantation, the defect is filled with connective tissue showing a moderate amount of neoformed bone from the periphery to the scarcely inflammatory infiltrate center.

After 6 months of implantation, a defect filled by a delicate band of connective tissue, scarce inflammatory infiltrate, and presence of neoformed bone in the periphery of the defect toward the center and islets of neoformed bone close to the periphery of the defect is observed.

Histomorphometric analysis of calcium hydroxyapatite microspheres

Newly formed bone

After histomorphometric evaluation, we observed a statistically significant difference between the cHA and cHAM groups after 1 month of implantation (P < 0.05); however, when compared between 1 and 3 months and between 1 and 6 months for the cHAM group, there was a statistically significant difference (P > 0.05). In cHA group between the periods of 1 and 3 months and between the periods of 1 and 6 months, there was no statistical difference (P > 0.05). After 3 months of implantation in the cHAM group, we observed that when compared to the cHA group, there was a statistically significant difference (P < 0.01), but when the two groups were compared after 6 months of implantation, there was no significant difference. When comparing the periods between 3 and 6 months for both groups, there was no statistical difference (P > 0.05) [Figure 4].
Figure 4: Histomorphometric evaluation of the bone repair in the rats' calvaria considering the volume density of (a) newly formed bone; (b) connective tissue; and (c) residual biomaterial particles

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After 1 and 3 months between the two groups, cHA and cHAM, there was a statistical difference (P < 0.01); however, after 6 months of implantation, there was no statistical difference (P > 0.05). When comparing within month group (cHA) between the periods of 1 and 3 months and between the periods of 1 and 6 months, no statistically significant difference was observed (P > 0.05). In the cHAM group between the periods of 1 and 3 months, there was no statistically significant difference (P > 0.05), but between the periods of 1 and 6 months, there was a difference (P < 0.01).

Connective tissue

Between the two groups after 1 month of implantation, a statistically significant difference was observed (P < 0.001). After 3 and 6 months, the statistically significant difference between the groups was also observed (P < 0.0001). Within the cHA group between the periods of 1 and 3 months and between the periods of 1 and 6 months, there was no statistical difference (P > 0.05). In the cHAM group between the periods of 1 and 3 months and between 1 and 6 months, there was no statistical difference (P > 0.05).

  Discussion Top

To develop the in vivo experiment as efficient and ethical as possible, the test material was previously analyzed in in vitro studies to confirm its cytocompatibility and to avoid adverse responses in the animals. It has been demonstrated, therefore, that the biomaterial is as cytocompatible as the control material being feasible for its animal experimentation.

Biomaterials are the most studied option for bone repair as they are seen as a viable possibility for the replacement of autogenous grafts. Among the various biomaterials, calcium phosphates are distinguished by properties such as biocompatibility, osteoconduction, and bone neoformation. Cationic and anionic substitutions in calcium phosphates have been reported in the literature as an alternative to mimicking the biological apatite of the bone tissue that contains trace elements. The present study incorporated cationic substitutions to the cHA (zinc, strontium, manganese, magnesium, and iron) to obtain improvements in its properties since it aims to obtain similarities ever closer with the natural HA present in the inorganic part of the human bone. Previous studies incorporating zinc exclusively into calcium phosphates found that the presence of zinc improved bone repair [27],[28] while other studies have shown that there was no difference when compared to nonzinc materials.[2] However, in a systematic review, Cruz et al. evaluated that the zinc concentration may influence this repair of 7%–8% more.

The incorporation of strontium (Sr 2+) to the calcium phosphates reported in previous studies showed that when combined with CHA, it was favorable to the osseous repair because of its good properties such as inducing bone formation.[1],[17],[29]

Previous studies using magnesium reported that once incorporated into the biomaterial, it provided an improvement of 4–5 times regarding bone repair when compared to the clot control group.[3] Manganese and iron were incorporated into the test material to simulate the biological apatite, but no previous studies have used these metals incorporated into calcium phosphate.

In this study, we did not observe improvement in the bone repair with metals containing cHA. The association of each ion to biomaterials showed to help in bone neoformation as shown in some studies; however, when adding all ions together, these results become negative as shown in the present study.

From the results obtained, it is understood that HA carbonated at 37°C with ionic substitutions showed a greater amount of connective tissue and consequently less neoformed bone when compared to the control group. There was also a low biomaterial presence in the two test and control groups during the three experimental periods, which may demonstrate that its reabsorption occurred rapidly. In the control group, this was replaced by bone tissue, whereas in the test group, this substitution did not characterize bone neoformation, but rather the formation of more connective tissue.

A possible explanation for the different results is the fact that the test material is unstable in organic place and may have been reabsorbed before the time to perform its function in the tissue place.

  Conclusion Top

It was concluded that the nanostructured cHAs with cationic substitutions are biocompatible, but very reabsorbable, and therefore, they have low osteoconductive potential while nanostructured cHAs synthesized at 37°C without cationic substitutions are bioactive biomaterials that have optimal activity and biocompatibility and can be used as a bone substitute.

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Conflicts of interest

There are no conflicts of interest.

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