Alveolar bone repair with strontium- containing nanostructured carbonated hydroxyapatite

ABSTRACT Objective This study aimed to evaluate bone repair in rat dental sockets after implanting nanostructured carbonated hydroxyapatite/sodium alginate (CHA) and nanostructured carbonated hydroxyapatite/sodium alginate containing 5% strontium microspheres (SrCHA) as bone substitute materials. Methods Twenty male Wistar rats were randomly divided into two experimental groups: CHA and SrCHA (n=5/period/group). After one and 6 weeks of extraction of the right maxillary central incisor and biomaterial implantation, 5 μm bone blocks were obtained for histomorphometric evaluation. The parameters evaluated were remaining biomaterial, loose connective tissue and newly formed bone in a standard area. Statistical analysis was performed by Mann-Withney and and Wilcoxon tests at 95% level of significance. Results The histomorphometric results showed that the microspheres showed similar fragmentation and bio-absorbation (p>0.05). We observed the formation of new bones in both groups during the same experimental periods; however, the new bone formation differed significantly between the weeks 1 and 6 (p=0.0039) in both groups. Conclusion The CHA and SrCHA biomaterials were biocompatible, osteoconductive and bioabsorbable, indicating their great potential for clinical use as bone substitutes.


Introduction
Implant-supported restoration has been increasingly performed by dentists for both aesthetic and functional reasons. However, when infections, pathological processes, extractions, or congenital and traumatic injuries on the maxilla and the mandible lead to bone loss, dental implant installation might not be the best opition 20 . Therefore, to minimize the loss of alveolar bone, or even restore it, different types of alloplastic grafts have been used, and new biomaterials have been the focus of research aiming to develop bone substitutes.
Among these grafts, HA Ca 10 (PO 4 ) 6 (OH) 2 has been widely used as a bone substitute for approximately 80 years 18 . T hi s cerami c i s bi ocompati bl e, osteoconductive 15 , similar to the bone and tooth tissue inorganic portions 1 , bioactive, and allows substitutions in its molecular formula and periodic monitoring via imaging because of its radiopacity. Additionally, it is mechanically tough and bioactive, and it is not antigenic, carcinogenic, or toxic.
However, the clinically used HA is not biodegradable and remains at the implantation site for long periods 23 , which limits bone regeneration. The lack of degradation is probably due to the high temperatures during ceramics production 16 and treatment after synthesis (sintering), which increases the crystallinity and hinders biosorption. With this in mind, nanostructured materials composed of particles smaller than 100 nm with low crystallinity show to be potential alternatives to grafts when produced with non-sintered materials at low temperatures 17 , considering they can imitate biological apatite 12 .
Researchers have chemically modified HA by substituting phosphate groups (PO 4 ) or hydroxyl groups (OH) with carbonate (CO 3 ) to develop a nanostructured carbonated hydroxyapatite at low temperatures 9 . Under these conditions, the produced biomaterial is similar to stoichiometric HA, but with lower crystallinity and higher solubility, which favors rapid bioabsorption and bone regeneration 17 .
Furthermore, the stability and flexibility of the HA structure enables different ionic substitutions 3 .
It is possible to induce the exchange of many cations and anions by modifying the structure of stoichiometric HA to resemble biological apatite 1 .
In these techniques, calcium frequently substitutes strontium (Sr 2+ ) 10 ; despite still being present in lower amounts compared to strontium, calcium alters the crystal structure and some HA properties, including phase stability, solubility, and reactivity 4   The XRD patterns revealed that the microspheres had low crystallinity, as indicated by the broad and poorly defined peaks. However, the XRD pattern of SrCHA ( Figure 1) showed narrower peaks than that of CHA because of the presence of Sr.
The spectra in Figure 2

weeks
In both groups, we observed a discrete chronic inflammatory response, with a few giant cells close to the microspheres, which were evidently fragmented.
The biomaterials were fragmented and bioabsorbed differently. The quantity of remaining material in the SrCHA group was slightly lower than that from the CHA group. Bone formation process replaced the inflammatory cell content in the first experiment at the same time that there was a decrease in mature connective tissue composed by collagen fibers.
The newly formed bone characterized by the thick trabecular bone was similar in both groups and occurred near the remaining bone and in direct contact with microspheres ( Figure 4).
The histological evaluation showed that in the interstitial spaces formed between the microspheres with no bone formation had a highly vascularized loose of connective tissue after one week that showed increased organization after six weeks with newly formed bone. This angiogenesis is essential for bone regeneration because these new blood vessels provide oxygen, nutrients, and cells, all considered essential for bone formation.  However, we observed a higher percentage of newly formed bone in both groups after 6 weeks when compared to1 week (p=0.0039) (Figure 7).

Discussion
Calcium ions in biological apatite have been partially substituted with other ions, such as Sr 2+ , Mg 2+, and Zn 2+ . This change affects the crystallinity, solubility, surface energy, and dissolution rate of the material, thus improving its bioactivity. Based on these findings, this study evaluated a promising bone substitute based on a synthesized at low temperature nanostructured carbonated hydroxyapatite containing strontium 10 .
According to a previous study 5 , the sintering of nanostructured hydroxyapatite causes crystal densification and nanostructured features loss, thereby increasing crystallinity and reducing solubility 15 .
Additionally, the sintering process is responsible for the removal of sodium alginate. In this study, we did not sintered the materials, retaining their nanometric characteristics, low crystallinity and sodium alginate content.
The non-strontium-containing hydroxyapatite was used as control group, as we aimed to evaluate the influence of strontium on hydroxyapatite in bone repair. We did not perform the dental socket filling  A previous study observed that at the nanoscale, the biomaterial resembled biological apatite 5 and presented bioabsorption similar to that observed in other studies 15,19 . Such bioabsorption is an essential for a suitable bone substitute because biosorption is important for bone physiology after biomaterial implantation 15 . In areas where the microspheres were bioabsorbed, we observed the formation of mineralized tissue, characterized by more cellular and non-lamellar bone associated with microspheres and at contact with the remaining alveolar bone, delimiting the dental alveolus, which confirms that the nanostructured carbonated HA here evaluated was bioactive, osteoconductive 16,19 and highly crystalline 5,23 .
The use of biomaterial microspheres has been considered preferable because the interstitial space between the implanted spheres provides macropores for tissue invasion and also because microsphere implantation can be performed with minimally invasive surgical techniques. In addition, the spheres do not have surface edges or dimensions that could lead to inflammation. To produce the microspheres, we mixed nanostructured carbonated HA powders with sodium alginate, an inert and biodegradable polymer. The histological results showed that there were interstitial spaces between the microspheres. In areas where there was no new bone formation, we observed a highly vascularized and loose connective tissue after one week that showed increased organization after six weeks. This angiogenesis is essential for bone regeneration because these new blood vessels provide oxygen, nutrients, and cells considered essential for bone formation 24 .
In the histological evaluation, we observed a greater fragmentation of SrCHA compared to the CHA group. We did not sinter the biomaterials used in this study, so they are considered low crystalline materials. However, the incorporation of strontium in the hydroxyapatite by partially replacing it with calcium changed crystallinity, morphology, lattice parameters, crystal size, stability, bioactivity, biocompatibility, and osteoconductivity of CHA. This set of physical and chemical changes alter the fragmentation and bioabsorption of biomaterials 25 . Sr 2+ has been widely used for partial substitutions of HA because of its dual ability to stimulate bone formation and reduce bone resorption. However, in our study, we observed no increase in newly formed mineralized tissue in the SrCHA group, indicating that Sr 2+ did not influence the osteogenic potential of nanostructured carbonated HA, possibly because of the low Sr 2+ content (1.7%) identified by atomic absorption spectrometry after synthesis and prior to the implantation, regardless of an initial theoretical Sr 2+ concentration of 5%. These results are similar to those obtained by other study 8

Conclusion
Our results suggest that both CHA and SrCHA, produced at low temperature and not sintered, were biocompatible, bioactive, ostecondutive osteoconductive, and bioabsorbable, indicating its great potential for clinical use as bone substitutes.
Further studies with a higher content of Sr 2+ associated with nanostructured carbonated hydroxyapatite are necessary to evaluate the effect of Sr 2+ on the biological response.