Influence of the geometry of nanostructured hydroxyapatite and alginate composites in the initial phase of bone repair 1

Abstract Purpose To analyze, histomorphologically, the influence of the geometry of nanostructured hydroxyapatite and alginate (HAn/Alg) composites in the initial phase of the bone repair. Methods Fifteen rats were distributed to three groups: MiHA - bone defect filled with HAn/Alg microspheres; GrHA - bone defect filled with HAn/Alg granules; and DV - empty bone defect; evaluated after 15 days postoperatively. The experimental surgical model was the critical bone defect, ≅8.5 mm, in rat calvaria. After euthanasia the specimens were embedded in paraffin and stained with hematoxylin and eosin, picrosirius and Masson-Goldner’s trichrome. Results The histomorphologic analysis showed, in the MiHA, deposition of osteoid matrix within some microspheres and circumjacent to the others, near the bone edges. In GrHA, the deposition of this matrix was scarce inside and adjacent to the granules. In these two groups, chronic granulomatous inflammation was noted, more evident in GrHA. In the DV, it was observed bone neoformation restricted to the bone edges and formation of connective tissue with reduced thickness in relation to the bone edges, throughout the defect. Conclusion The geometry of the biomaterials was determinant in the tissue response, since the microspheres showed more favorable to the bone regeneration in relation to the granules.

Influence of the geometry of nanostructured hydroxyapatite and alginate composites in the initial phase of bone repair Santos GG et al. 2 researchers have developed this material on a nanometer scale, considering that the HA nanostructured crystals (HAn) have higher biodegradation due to the smaller size of its particles and larger surface area exposed to the biological environment, which accelerates the speed of formation and growth of the biologically active apatite layer 7,8 . Another way to improve bioceramics is to associate them with natural or synthetic polymers to produce biomaterials of the type composites 2,9 . These materials have, in the same scaffold, physicalchemical properties of the ceramic and polymer, which are improved in relation to the materials when used in an individual way and mimetize the inorganic and organic phases of the natural bone [10][11][12] .
In this perspective, the alginate is a natural polymer widely used, since this material can alter the crystallinity, solubility, network parameters, thermal stability, surface reactivity, bioactivity and adsorption properties of the HA structure 9,13,14 . Therefore, the physicochemical characteristics of the composites vary according to the polymer and its percentage used during the synthesis, as well as the processing of the sample and the final geometry of the biomaterial produced. Thus, these biomaterials, especially in the form of microspheres and granules are configured as a promising alternative to bone substitution, particularly in situations where damage and/ or trauma reach critical dimensions which preclude spontaneous bone regeneration and hinders the functional property or aesthetic of the affected 15,16 .
Given the above, parallel to the need, in worldwide level, to develop new biomaterials, with national technology and affordable cost, most versatile, and promising biological properties for use, especially in cases of extensive bone loss, the present study aims to analyze the influence of the geometry of HAn

■ Introduction
Researchers of the bone tissue bioengineering have sought to develop ideal conditions for the repair and/or replacement of damaged or lost tissue through the use of cellular elements, growth factors, regenerative techniques and biomaterials, in order to provide the scaffold and essential requirements for tissue neoformation [1][2][3] .
The biomaterials can be synthesized from different substrates and processed in different forms of presentation, namely: fiber, membrane, gel, powder, among others; and different geometries such as plates, cylinders, microspheres and granules. Microspheres have attracted great scientific interest due, in particular, to their ability to promote the formation of interstices between them, which allows cell migration, adhesion, proliferation and differentiation, mainly, mesenchymal and osteoprogenitoring, liberation of growth factors, angiogenesis, diffusion of nutrients and new extracellular matrix (ECM) synthesis 4 . By turn the granules, in addition to having the aforementioned properties, are widely used in clinical practice in the filling of defects and tissue lesions with irregular shapes. Both microspheres and granules can be applied through injectable systems in minimally invasive surgical procedures 5 .
Among the substrates most used in the synthesis of biomaterials with the geometry of microspheres and granules, bioceramics based on calcium phosphate (CaP) stand out, mainly, the HA due to its biocompatibility, osteoconduction and bioactivity 6 . However, in spite of these fundamental properties, in the biological interaction with the host, the HA presents a low in vivo degradation rate which, in some applications, may limit its use 7 .
In seeking to improve the physicochemical characteristics of HA, The precipitate resulting was filtered and washed until the pH of the wash water was 7. Soon after, the solid obtained was dried by freeze-drying for 24h and then separated using sieves with a desired mesh aperture. 15g of the obtained solid were weighed into becker and added to a 1.5% w/v solution of sodium alginate, vigorously mixed until obtain a homogeneous paste.
To obtain the microspheres, this paste was extruded with the use of a syringe in 0.15M calcium chloride solution at room temperature. Finally, the microspheres were washed and dried in a glass-house at 50º C and then submitted to the sieve with a granulometric range of 250-425 μm. In order to obtain the granules, the obtained paste was dried in a glass-house and then crushed and sieved in the granulometric range of 250-425 μm. The biomaterial samples were conditioned in eppendorf tubes, properly identified, and sterilized by gamma rays (Fig.  1). Each aliquot was used to fill the bone defect of, approximately, four animals.

Superficial area
The characterization of the surface area by the BET method (ASAP 2020 -Micromeritics ® ) showed that the HA evaluated in our study has a surface area of 35.9501 m 2 /g, characteristic of manometric biomaterials.

Chemical analysis
The chemical analysis of the X-ray fluorescence (XRF) (PW2400-Philips ® ) demonstrated that HA evaluated in this work presents Ca/P molar ratio of 1.67 (Table 1). Acta Cir Bras. 2019;34(2):e201900203 Influence of the geometry of nanostructured hydroxyapatite and alginate composites in the initial phase of bone repair Santos GG et al. X-ray diffraction (X-RD) analysis The analysis of X-ray diffraction was performed using the high-resolution diffractometer HZG4 (Zeiss ® ) with CuKa radiation (l = 1.5418 Å) and angular scanning of 10-80 o (2ɵ), with step of 0.05/s, time 160 seconds, with reference to the standard PCPDFWIN 09.0432 (International Centre for Diffraction Data -ICDD) 17,18 . The diffractogram evidenced peaks corresponding to the crystalline profile of a standard HA (Fig. 2).

Experimental phase
Fifteen male wistar albino adult rats with body weight between 350 and 400 g were randomly distributed to compose three experimental groups, with 5 animals in each group: MiHA -bone defect filled with HAn microspheres and alginate; GrHA -bone Influence of the geometry of nanostructured hydroxyapatite and alginate composites in the initial phase of bone repair Santos GG et al. defect filled with HAn granules and alginate; DV -bone defect without implantation of biomaterial.

Surgical procedure
The surgical technique used was the same described by Miguel et al. 19 . However, in the present study, the 8.0 mm trephine drill used in the confection of the critical bone defect was with a diameter of ≅8.5 mm and ≅0.8 mm of thickness. After removal of the bone fragment, it was procedered the implantation of the biomaterials, according to each experimental group. In the control group (DV) the bone defect remained empty, without biomaterial. Finally, the tissue flap was repositioned and sutured with simple points (Fig. 4).

Histological processing and histomorphological analysis
After 15 days post surgery, the animals were euthanized and the calvaria removed. The obtained specimens were fixed formaldehyde 4% for 48 hours, included in paraffin and subsequently cut to 5μm of thickness and stained with hematoxylin-eosin (HE), picrosirius-red (PIFG) and Masson-Goldner trichrome (GOLD) and analyzed under an optical microscope (DM1000 -Leica ® ). The images were captured using a digital camera (DFC310FX -Leica ® ). For morphometric analyses, the Leica Application Suite (version 4.12 -Leica ® ) and optical microscope (DM6 B -Leica ® ) were used. To compare the differences between the groups the Kruskal-Wallis test was applied using the software SPSS version 20.0 (IBM SPSS ® ), at a 5% level of significance (p≤0.05).

■ Results
The histomorphologic analysis evidenced neoformation of osteoid matrix restricted to the borders of the bone defect with formation of fibrous connective tissue in the remaining area, in all experimental groups. When compared to the bone edge, the tissue thickness produced in the defect region remained proportional in the groups with implantation of microspheres and granules, and significantly reduced in the group without implantation of biomaterials (Fig. 5). In the MiHA, the biomaterials were arranged in monolayer, with small variation of microspheres size, throughout the extent of the bone defect. The majority remained intact and some presented partial and/or total fragmentation with neoformation of osteoid matrix within the scaffold. In this group, it was observed the presence of mononuclear inflammatory cells and multinucleated giant cells, characteristic of chronic granulomatous inflammatory response, discreetly noticed around the microspheres, especially those located at the periphery of the bone defect.
In GrHA, the granules were distributed in mono and multilayer, throughout the extent of the bone defect. In this group the chronic granulomatous inflammatory reaction observed was more evident than in the MiHA. Most of the granules remained intact, while others presented partial fragmentation, less accentuated in relation to the microspheres, without osteoid neoformation inside of the biomaterials. In both groups with implantation of biomaterials, it was noticed abundant proliferation of blood capillaries around the particles (Fig. 6). The histomorphometric analysis measured the percentage of mineralized linear extension ( Table 2). The Kruskal-Wallis test demonstrated statistically significant difference between the 3 groups (MiHA x GrHA x DV -p=0.007) and between the experimental and control groups -MiHA x DV (p=0.010) and GrHA x DV (p=0.046). In the groups which the biomaterials were implanted the Kruskal-Wallis test did not demonstrate statistically significant difference-MiHA x GrHA (p=1.00).
Influence of the geometry of nanostructured hydroxyapatite and alginate composites in the initial phase of bone repair Santos GG et al.

■ Discussion
The bone repair mechanism is a dynamic, temporal and complex phenomenon that depends on the presence of a threedimensional scaffold and on the manner in which the bone, undifferentiated mesenchymal and endothelial cells interact with each other and with the microenvironment around them. Under the physiological conditions, this event is consolidated by regeneration. On the other hand, when the tissue loss presents critical dimensions, extension and morphology, there is impairment of this mechanism and the tissue repair occurs by fibrosis 19,20 . Under these conditions, bone regeneration becomes limited, as observed in the control group (DV) of our study and in other studies [19][20][21][22] . These results validate the simulation of extensive bone loss as it occurs in cases of congenital pathologies, extensive surgical resections, trauma and severe inflammatory diseases. In view of these inhospitable conditions, it is evidenced the need for the use of bone substitutes that make feasible the complete tissue regeneration.
The spatial arrangement of the biomaterials and the neoformed tissues between and around the particles, in the two implanted groups showed that the microspheres and granules acted as scaffolds suitable to applications as fillers materials. However, in view of the almost complete reduction of the neoformed interstitium between the granules, it is noted that the spatial arrangement of these biomaterials, similar to the mosaic, interfered in the migration of the cells between the particles during bone repair. These findings were consonant those were evidenced by Ribeiro et al. 22 , who evaluated granules of HA and alginate in the same biological point.
The evident presence of neoformed blood vessels surrounding the biomaterials demonstrates that the materials evaluated were biocompatible and osteoconductive, independent of the geometric shape, and provided a structure favorable to migration, adhesion and proliferation cellular, the release of growth factors, angiogenesis and tissue neoformation 4,12,23 . Thus, the neoformed osteoid matrix was similarly consolidated in both groups with implantation of biomaterials (p>0.05).
The partial biodegradation of the biomaterials evaluated in this study, most noticeable in the MiHA, demonstrated the influence of alginate on the materials that, when coming into contact with fluids and living tissues, was dissolved by local enzymes and biorreabsorbed. In this way, it induced the gradual release of the inorganic components of the composite, mainly ions of Ca and PO 4 , contained in the crystals of HA. These results contradict those observed by Barreto 24 , in which the microspheres of HA and alginate did not undergo evident biodegradation at the same biological point. This occurred due to the removal of the polymer from the structure of the microspheres during the calcination process. This finding can be attributed, in sum, to the size of the microspheres used by Barreto 24 in relation to those used in our experiment, 400-600 μm and 250-425 μm, respectively, because the smaller the particle of the biomaterial, the larger the superficial area of the material exposed to the biological environment. In view of these findings, it is noted that the calcination and sintering processes increase crystallinity, since it favors crystal fusion, aggregation and growth particle, making them resistant to biodegradation 24 .
Corroborating with this analysis, in the study by Paula et al. 25 , non calcined microspheres with 400μm showed partial biodegradation with deposition of collagen fibers inside the particles. Conversely, in the study realized by Rossi et al. 26 . the sintered HA microspheres, with the same size as those used in our study, did not present expressive biodegradation, at the same biological point.
One of the technological innovations presented by the biomaterials evaluated in this study, in both geometric shape, was the conception of the HA at nanometric scale. As observed in the MiHA, according to Valenzuela et al. 8 , the nanostructured HA crystals tend to dissolve (biodegrade) faster because of the smaller particle size and the larger surface area exposed to the biological environment.
In our study, the chronic granulomatous inflammation, noted in both groups in which biomaterials were implanted, was compatible with that expected every time a biomaterial is implanted in the living organism 27,28 . This finding proves the biocompatibility of the biomaterials, since there was no rejection by the organism, characterized by acute exacerbated inflammation 27,28 . This potentiality can be attributed, mainly, to the physical-chemical composition of the materials that mimic the inorganic and organic phases of the natural bone tissue, by HAn and alginate, respectively. On the other hand, the more evident chronic granulomatous inflammation in the GrHA, in comparison to the MiHA, reveals that the irregular surface of the granules modulated the cellular response in the interstice between the particles of the biomaterial 27,28 . These findings reveal that the geometry of the biomaterials interferes directly in the tissue response to the presence of the particles 5,29 .
In the study by Ribeiro et al. 22 the microspheres acted better as filling scaffold and the granules presented superior osteoconductive potential, contrasting our results. It should be emphasized which, in that study, the biomaterials had a diameter of 425-600 μm; the granules contained 1% of alginate; and the biomaterials were synthesized by another way. In our experiment, the percentage of alginate used was 1.5% and diameter of 250-425 μm, which may have influenced the tissue response to granules 22 .
Considering that the results of this study are related to the initial phase of the bone repair mechanism (15 days) and can influence, significantly, subsequent cellular events, it becomes pressing the need to develop new studies to observe this response in the long term.

■ Conclusions
In the initial phase of bone repair, the geometry of the biomaterials influenced the tissue response to the implantation of HAn and alginate composites. Both biomaterials exhibited neoformation of osteoid matrix, although the microspheres exhibited histological characteristics more favorable to bone regeneration than granules.