Histomorphometric evaluation, SEM, and synchrotron analysis of the biological response of biodegradable and ceramic hydroxyapatite-based grafts: from the synthesis to the bed application

This study aimed to analyze the physicochemical and histological properties of nanostructured hydroxyapatite and alginate composites produced at different temperatures with and without sintering and implanted in rabbit tibiae. Hydroxyapatite-alginate (HA) microspheres (425–600 µm) produced at 90 and 5 °C without (HA90 and HA5) or with sintering at 1000 °C (HA90S and HA5S) were characterized and applied to evaluate the in vitro degradation; also were implanted in bone defects on rabbit’s tibiae (n = 12). The animals were randomly divided into five groups (blood clot, HA90S, HA5S, HA90, and HA5) and euthanized after 7 and 28 d. X-ray diffraction and Fourier-transform infrared analysis of the non-sintered biomaterials showed a lower crystallinity than sintered materials, being more degradable in vitro and in vivo. However, the sinterization of HA5 led to the apatite phase’s decomposition into tricalcium phosphate. Histomorphometric analysis showed the highest (p < 0.01) bone density in the blood clot group, similar bone levels among HA90S, HA90, and HA5, and significantly less bone in the HA5S. HA90 and HA5 groups presented higher degradation and homogeneous distribution of the new bone formation onto the surface of biomaterial fragments, compared to HA90S, presenting bone only around intact microspheres (p < 0.01). The elemental distribution (scanning electron microscope and energy dispersive spectroscopy and μXRF-SR analysis) of Ca, P, and Zn in the newly formed bone is similar to the cortical bone, indicating bone maturity at 28 d. The synthesized biomaterials are biocompatible and osteoconductive. The heat treatment directly influenced the material’s behavior, where non-sintered HA90 and HA5 showed higher degradation, allowing a better distribution of the new bone onto the surface of the biomaterial fragments compared to HA90S presenting the same level of new bone, but only on the surface of the intact microspheres, potentially reducing the bone-biomaterial interface.


Introduction
Autogenous bone is still the 'gold standard' for bone grafts used in reconstructions due to its known properties of osteoinduction, osteogenesis, and osteoconduction [1], which preserve vital cell and growth factors, repairing bone defects.However, drawbacks like an additional surgical site and time, significant postoperative morbidity, complications associated with the harvest, and restricted quantity can limit its use [2,3].
Designing a calcium phosphate-based bone graft for bone tissue bioengineering is complex since its chemical, morphological, and textural properties impact bone repair effectiveness.Tissue engineering requires bioabsorbable materials; hence, nonceramic and nanostructured hydroxyapatite with polymers is becoming more widespread [4], stimulating the development of new synthetic biomaterials to replace autogenous bone in this scenario.Synthetic hydroxyapatite, a calcium phosphate-based material, has been evaluated due to its high biocompatibility and osteoconductive [4] capacity without antigenicity [5][6][7] or genotoxicity [8].Hydroxyapatite shares some characteristics with natural bone as it can change its chemical and physical composition [9,10], affecting its characteristics and crystalline structure.So, hydroxyapatite's physicochemical and biological properties, associated with its ease of manufacture, make it a great candidate for bone substitutes.However, ceramic hydroxyapatite with a calciumphosphate ratio 1.67 tends to present slow degradation under physiological conditions [11].Ideally, synthetic bone substitutes aim to be surrounded by bone tissue, achieving rapid integration and allowing a speeded-up bone ingrowth concomitant for graft degradation [12,13].
In this context, bone grafts produced from the association of polymers and hydroxyapatite synthesized at low temperatures (5 • C-90 • C) have been strong candidates for treating bone repairs due to their in vivo biodegradation.They may be processed as granules, microspheres, nanostructured membranes, and 3D-printed implants [14,15].The polymer gives shape and the mechanics' proprieties to the scaffold, and hydroxyapatite promotes osteoconductivity.Alginate is a naturally occurring anionic polymer with favorable properties, such as good biocompatibility, weak cytotoxicity, broader availability, cost-effectiveness, and ease of gelation in divalent cations [16].Moreover, alginate can provide an inert aqueous environment, which is biodegradable in a physiological environment and has structural similarity to the extracellular matrix [17][18][19][20].Moreover, the biodegradation rate of hydroxyapatite shall be affected by the surface area (e.g.powder > porous solid > thick solid) by decreasing the crystallinity through some ionic substitutions of hydroxyapatite structure (CO 3 , Mg, and Sr) or reducing the grain size, or by the presence of irregular crystallites [21].It is well known that the synthesis's temperature and thermal treatment change the crystallinity, morphology, granule size, and density of the hydroxyapatite powder with a substantial impact on its solubility [22][23][24][25].
Despite the remarkable progress in basic and clinical applications, synthetic hydroxyapatite and related materials are still under investigation to optimize its properties, as the association with metals, anionic groups, and bioactive molecules [26,27].Also, previous studies showed that nanostructured B-type carbonated hydroxyapatite enhances hydroxyapatite biodegradability [28].The absence of thermic treatment during the synthesis process ensures its nanometric characteristics, promoting exciting changes in the material properties, mainly favoring their solubility after implantation, thermal stability, particle size, and morphology in physiology systems [29].However, there are still gaps in knowledge regarding the best combination of synthesis temperature and heat treatment to generate a bioabsorbable, biocompatible, and osteoconductive biomaterial suitable for clinical applications in tissue engineering.
This work produced alginate associated with hydroxyapatite nanoparticles (NPs) microspheres with different crystallinity and dissolution rates.We evaluated the biological response of newly formed bone and the bone-biomaterial interface after implantation in rabbits' tibiae defects.

Synthesis of the biomaterials
The calcium phosphate bioceramics synthesis occurred at the Brazilian Center for Research in Physics, Brazil.The hydroxyapatites samples were synthesized through calcium nitrate solutions ([Ca(NO 3 ) 2 .4H 2 O], 0.2 M) by dripping solutions of ammonium phosphate dibasic ([NH 4 ) 2 HPO 4 ], 0.2 M, at 4.5 ml min −1 ), at pH 9.0 with concentrated ammonium hydroxide (NH 4 OH) at 90 • C (HA90) or 5 • C (HA5) under a mechanical flurry of 240 rpm, using a magnetic plate.A portion of the HA5 and HA90 powders underwent thermal treatments at 1000 • C to enhance particle size and crystallinity (table 1).

Sample characterization
The physical and chemical characterization of the biomaterials occurred at the Brazilian Center for Research in Physics, using x-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectroscopy, The FTIR vibrational spectroscopy analysis identified the chemical groups and some substitutions in the chemical composition of hydroxyapatite by the oscillation frequency of vibration in the infrared.The Fourier-Transform Spectrophotometer Shimadzu, IR-Prestige 21 with DTGS KBr detector and KBr beam splitter provided the infrared spectra for powder samples of hydroxyapatites using pellets with the transmittance of 1% KBr in the middle region of the infrared (4000-400 cm −1 ).
SEM micrographs enabled the characterization of the surface topography of the biomaterial microspheres attached on carbon tape and coated with gold, using magnification at 100× for displaying the materials and 1000× for surface evaluation.The images resulted from secondary electrons and observation under low vacuum conditions at the Hertha Meyer Cell Ultrastructure Laboratory of the Federal University of Rio de Janeiro (Brazil), using the SEM-JEOL JSM 5310-the evaluation after the degradation assay was done for the surface topography.
The concentrations of Ca and P in biomaterial microsphere samples were determined using atomic emission spectrometry with inductively coupled plasma on an OPTIMA 3000-Perkin-Elmer instrument (EMBRAPA Soils) in triplicate.Before analysis, 0.05 g of each sample was incubated in 20 ml of buffer (MES or HEPES) using a mechanical continuous gentle flurry on a Kline orbital shaker NT-150 at 37 • C.After 1 and 7 d, 5 ml aliquots of the solutions were collected, centrifuged at 6000 rpm for 10 min using a Centrifuge 6000R-CT-Cientec ® , and the supernatant was pipetted, dissolved in 12.5% nitric acid, and filtered using 0.22 µm membranes (Millipore).

Experimental groups and surgical procedures
This study agrees with the ethical principles of the National Council for the Control of Animal Experimentation, and it was approved by the Ethics Committee of Fluminense Federal University (Niteroi, RJ, Brazil) (number 003208).This study used 12 New Zealand white rabbits (Oryctolagus cuniculus), adults of both genders, weighing between 2 to 3.5 Kg, maintained in individual cages with galvanized mesh suspended off the floor in the Rabbits Production Laboratory throughout the experiment; water was provided ad libitum through nipple drinkers.The animals were randomly divided into five experimental groups: blood clot (control); HA90S; HA90; HA5S; and HA5.Three surgical bone defects (Ø 2 mm) on the right rabbit's tibia received blood clots, HA90S, and HA90, while two in the left rabbit's tibia received HA5S and HA5.
The animals were deprived of feed six hours before surgery, except water, and weighed on a digital precision scale.They received premedication with ketamine (20 mg kg −1 ) (Clortamina ® , BioChimico) and xylazine (1 mg kg −1 ) (Rompun ® , Bayer).Observing the absence of any reflexes to pain, the animals received 1% isoflurane anesthetic medication by inhalation (Isoran ® , BioChimico), kept until the end of the surgical procedures, and prilocaine 3% with felypressin (2 ml) (Citocaína ® , Cristália Prod.Chemistry.Pharmaceuticals), via intramuscular.The animals were supine with the head hyperextended to install an inhalation mask for maintenance of anesthesia.
A longitudinal incision about 40 mm long was made in the superior medial region of the posterior limb at the proximal tibia using a scalpel cable 3 (Bard-Parker) and blade 15 C (Becton-Dickinson.Ind. Surgical SA).Subsequently, with no compromising muscle tissue, a new incision was made slightly in the periosteum, exposing the bone tissue.Through surgical drills for implantology (Surgical Kit-Compact, Conexão Master) and a specific surgical engine at around 1500 RPM (Driller ® , SIN, Brazil), five bone defects were done for biomaterial implantation.The surgical bone defects were made with a sequence of drills (Ø 2 mm/2-3 mm) with 10 mm of distance between each perforation, under copious external irrigation of sterile saline solution (sodium chloride 0.9%) (Darrow Laboratories SA, supplementary figure 1).
After the grafting procedure of 0.1 g of each biomaterial using the 2/4 Molt Surgical Curette (Fava, Brazil), a single suture directly over the holes with Mononylon 5.0 (ETHICON ® , Johnson & Johnson) relocated the periosteum; the muscle layer and skin were sutured continuously point Mononylon 5.0 (ETHICON ® , Johnson & Johnson).All animals received antibiotics (0.3 mg kg −1 ) (Pentabiotic Vet ® , Fort Dodge) and meloxicam veterinary (0.3 mg kg −1 ) (Maxicam ® , Ouro Fino Bem Estar Animal) in single doses.For elapsed trial periods of 7 and 28 d, the animals were pre-anesthetized as described for surgical procedures and euthanized by anesthetic overdose to remove the samples containing the biomaterials.Euthanasia of the animals occurred at 7 or 28 d after surgery.

Histomorphometric analysis
The bone block sample containing the defects and material grafts was fixed in 70% ethanol before dehydration and then exposed to an ascending series of alcohol fractions (50%-100%) under agitation and vacuum and then impregnated and embedded in methylmethacrylate to prepare undecalcified sections.The embedded blocks were reduced and cut at thicknesses of 300 µm by a device for precision cuts at low speed (Isomet ® , Buehler) to acquire a transverse section of the tibia along the central axis of the bone (Zeiss Axioplan).
An experienced pathologist under light and polarized light microscopy (Zeiss-Axioplan microscope, Germany-Objective 10× of magnification/0.30Acroplan, Neofluar) analyzed the unstained, polished 40 µm sections.A CCD camera captured four non-superposed images near the perforated cortical of each specimen section (Evolution MP Color 5.0, Media Cybernetics, Canada), allowing the histomorphometric analysis with Image-Pro Plus ® (Media Cybernetics, LP, Silver Spring, MD) to compute the volume density of the newly formed bone and to evaluate the connective tissue formation and grafted biomaterials present by measuring the birefringence of bone tissue and collagen fibers in polarized light.Furthermore, the pathologist performed a descriptive histological analysis on the undecalcified sections stained with toluidine blue and acid fuchsin.

Backscattered electron (BSE)-SEM and EDS-SEM microanalysis
Each sample cut into 40 µm thick sections were wrapped in aluminum support (stubs) and attached with a carbon ribbon of high surface area at room temperature of 24.0 • C ± 1.0 • C for analysis at the Department of Microscopy Laboratory from the Brazilian Institute of Metrology in a scanning electron microscope QUANTA 200 (FEI, Inc.) equipped for energy dispersive spectrometry (EDS).The nonmetalized samples were analyzed in low vacuum mode (environmental), performing SEM accompanied by EDS to elemental analyses segmented by area.The microscopy analyses evaluated the bonebiomaterial interface, the implanted spheres' resorption degree, and the environment's observed chemical content.

Synchrotron radiation x-ray microfluorescence (µXRF-SR)
The samples were prepared and cut into 200-300 µm thick, not stained sections.µXRF-SR elemental mapping was carried out at the Brazilian Synchrotron Light Laboratory (LNLS-Campinas, Sao Paulo, Brazil), working at the D09-XRF beamline (Protocols number 8066 and 10108) with a white beam (4 keV min-23 keV max) for sample excitation.Capillary optics allows focusing the x-ray beam, one of the best suits a micrometer XRF instrument, with a conical glass capillary of 20 µm diameter.It used a 45/45-degree geometry, putting the samples at 45 • to the incident beam.A Si (Li) solid-state detector collected the x-ray fluorescent radiation with a resolution of 165 eV at 5.9 keV at this same degree angle.
The measurements were performed in a computed controlled XYZ table, allowing the choice of the region of interest (ROI) at the center of the bone samples.The ROI was chosen based on the interface between bone and implant (control, HA90S, HA90, HA5S, and HA5) allowed by a microscope and a CCD camera.µXRF-SR elemental mapping was performed in several area dimensions according to beam lifetime with an image resolution of 30 µm.For example, the measurement time per point was 10 s, leading to four hours to complete one single scan for an area equal to 1.2 mm 2 .
All the XRF spectra were analyzed using QXAS/AXIL software, which IAEA freely provides.Finally, each element was plotted separately on distribution mapping implemented by the authors with the additional support of the commercial MATLAB ® program (MathWorks, Natick, MA, USA).PyMca (Python multichannel analyzer) fits fluorescence spectra and separates the different elemental contributions [31].This program allowed a batch fitting procedure on each pixel of 2D maps.The µXRF-SR microanalyses assessed the bone-biomaterial interface and the chemical content of the environment at a low detection limit-the high spectral shine results in increased intensity of primary x-rays.

Statistical analysis
The Image-Pro Plus ® allowed the histomorphometric analysis through the segmentation of images to obtain newly formed bone and connective tissue percentages.The programs used were GraphPad InStat v. 3.01 (GraphPad Software, San Diego, California, USA) and GraphPad Prism v.8.0 (GraphPad Software, San Diego, CA, USA) allowed the two-way analysis of means and standard deviations, and Tukey's post-test (p < 0.05).
The non-sintered microspheres are composed of hydroxyapatite NPs associated with alginate acting as an encapsulating element for the hydroxyapatite particles.The high surface roughness of the microspheres is due to the presence of agglomerated NPs of different sizes (figures 1(A)-(D)).Sintering does not significantly modify the surface roughness but eliminates the polymer from the spheres, exposing the hydroxyapatite to the outside environment.The in vitro degradation of sintered and non-sintered microspheres was followed by the release of calcium (Ca) and phosphorus (P) after one and seven days of incubation in HEPES and MES buffers, modifying the surface topography of the HA90, HA5S, and HA5 (figures 1(E) and (F)).Figure 2 shows the measurements obtained for the spheres (425-600 µm).The Ca and P release from microspheres depended on the solution pH, which was lower in the MES buffer, similarizing to the surgical site after the procedure (figure 3).Microspheres containing low crystalline HA5 NPs had higher Ca and P release than those with more crystalline NPs of HA90 or HA90S.The high level of Ca and P release by HA5S microspheres is attributed to the contribution of the greater level of soluble TCP phase present in the HA5S powder.Our results support the ascendent crystallinity level of HA90S < HA90 < HA5S < HA5.

Descriptive histologic analysis
The surgical procedures, conducted under controlled and aseptic conditions, contributed to the stability and health of all animals after grafting throughout the experimental time.Macroscopic analysis of the samples obtained from animals after euthanasia revealed the absence of necrosis or tissue alterations on the control and grafted surgical sites at 7 and 28 d post-surgery.
At a 7 d post-surgery, the polarized lightmicroscopy of the control group evidenced a few trabeculae of newly formed bone growing from the pre-existing bone with the same birefringence as the native bone (figures 4(A) and (B)).At the grafted sites, a loose connective tissue surrounded the HA90S and HA5S microspheres, with initial mineralization between HA90S integral spheres and the fragmented HA5S (figures 4(C), (D) and (G), (H)).HA90 and HA5 spheres fragmented significantly and presented newly formed bone around the fragments with a similar birefringence of the pre-existing bone (figures 4(E), (F) and (I), (J)).
After 28 d, new bone with the same birefringence and density of native bone occluded the defect filled with blood clots (figures 5(A) and (B)).The bone defects filled with biomaterials microspheres presented more organized and mineralized trabeculated tissue than at seven days, confirming the biomaterials' biocompatibility and osteoconductivity.A slight size reduction of HA90S occurred after 28 d, and the spheres were partially covered by connective tissue and new bone, presenting similar birefringence as the native bone (figures 5(C) and (D)).The HA5S spheres also slightly reduced size with new bone and connective tissue onto the material surface highly expanded due to the extensive fragmentation (figures 5(G) and (H)).On the other hand, HA90 and HA5 presented a considerable amount of mineralized tissue permeating and filling the surface cavities of fragmented microspheres.The fragmentation and size reduction of HA90 and HA5 suggest biodegradation since residual HA90 and HA5 are smaller than HA90S and HA5S at the same experimental period (figures 5(E), (F) and (I), (J)).
Histological images of the control group after 7 d (figures 6(A)-(C)) showed small areas of bone formation (trabeculae) at the opening of the bone defect.The morphological analyses evidenced the neutrophilic infiltrate areas granulation tissue reaction around biomaterial into the tibia cortical bone defects seven days after surgery.HA90 (figures 6(G)-(I)) presented fragments of microspheres in opposition to intact HA90S (figures 6(D)-(F)).A similar fact was also observed between HA5 (figures 6(M)-(O)) and HA5S (figures 6(J)-(L)).In addition, centripetally bone formation from the defect's wall and initial osteoid deposition permeating the biomaterials (figures 6(J)-(O)).After 28 d, the control group presented an increased area of new bone from the border to the center of the bone defect (figures 7(A)-(C)).HA90S (figures 7(D)-(F)) and HA90 (figures 7(G)-(I)) presented spaces derived by the fragmentation of the spheres, corroborating the polarized light microscopy images, filled with connective tissue and some areas of the newly formed

Bone-biomaterial interface microanalyses
The analyses of SEM micrographs with BSEs at seven days confirmed the histological findings: the newly formed bone tissue from the border of the cortical bone towards the spheres surrounding them (figure 8), especially in non-sintered materials.At 28 d, the mineralized tissue surrounding the HA90, HA90S, and HA5 spheres increased significantly compared to seven days; HA90S and HA5S showed slight size reduction, but a discrete new bone formation occurred onto HA5S spheres (figure 9).The fragmentation of HA90 and HA5 benefited the osteoconduction e allowed the newly formed bone deposition around the biomaterials fragments.(figures 8(E) and (M)).
As expected, the scanning electron microscope and energy dispersive spectroscopy (EDS-SEM) microanalyses found similar densities of calcium and phosphorus in pre-existing bone, new bone, and biomaterials of all samples at both experimental periods (figures 8 and 9).Carbon predominated in all areas with connective tissue (figures 8 and 9).
Elemental analysis of the bone-biomaterialconnective tissue interface conducted by µXRF-SR detected Ca and P as the main elements besides low Zn, Fe, and Sr ions concentrations (figures 10 In vitro degradation assay of HA microspheres.HEPES Buffer pH 7.4.Calcium release was higher than P in all groups, and HA5 and HA5S presented higher solubility than HA90 and HA90S.MES Buffer pH 5.9.The calcium and phosphate release followed the same pattern as in the HEPES buffer, with Ca being more released than P, mainly for HA5 and HA5S.HA90S presented the lowest release of calcium and phosphate at pH 5.9 ( * , p-value with significant level).area corresponding to the pre-existing bone, and the intensities of Ca and Zn in the region between the spheres and the native bone are minimal, characterizing that there is no or low presence of mineralized tissue during this period.At 28 d, a high intensity of calcium and zinc was observed among the particles HA90S, HA90, and HA5, compatible with the mineralization process.Note the significant fragmentation of HA90 and HA5 in the calcium intensity image; however, the presence of calcium around HA5S is very discreet, indicating low bone formation.On the other hand, H5 at 28 d shows intense microsphere fragmentation and high calcium intensity between the particles.

Histomorphometric analysis
Figure 13 summarizes the results of the histomorphometric analysis of new bone formation (figure 13(A)) and connective tissue (figure 13(B)).From seven to 28 d, the density of newly formed bone increased significantly for all experimental groups (two-way ANOVA and Tukey post-test, F = 111.75.DFn = 1, DFd = 40, p < 0.0001).As expected, at each experimental period, the density of new bone was higher in blood clots compared to all biomaterials.As observed in previous studies [32,33], the presence of the biomaterial in the bone defect reduces the space available for the new bone, in opposition to the group filled with the blood clot, which is completely removed during bone repair.In defects filled with non-resorbable or partially absorbable biomaterials, there existed less space to be filled by the new bone.The density of new bone for blood clot, HA90S, HA90, HA5S, and HA5 increased overtime, respectively, 1.7×; 2.1×; 3.2×; 2.4×; 2.0×, confirming the biocompatibility of the biomaterials evaluated.In opposition to the sevenday period, where the bone density is similar among the grafted groups, at 28 d, the density of the bone in the HA90S, HA90, and HA5 groups is quite similar but significantly higher than HA5S.The connective tissue occupied a similar area at seven and 28 d, roughly 20 for HA90S and HA5S, but 40%-50% for HA90 and HA5 at seven days.After 28 d, there was a slight reduction in connective tissue density, around 17%-37%, considering all experimental groups.

Discussion
In bone tissue bioengineering, the design of a calcium phosphate-based bone graft remains tricky.A graft's chemical, morphological, and textural qualities determine how effective it will be in the bone repair process.Each clinical application requires a unique setup of the biomaterial's composition, crystallinity, surface topography, micro-and nano-porosity, and bio-disaggregation [34].With advances in bioengineering, the development of devices and new biomaterials has improved and favored the treatment of bone defects.Among the ceramic biomaterials, hydroxyapatite is probably the most evaluated material for in vitro and in vivo research and the most indicated as a bone substitute in clinical situations   [35].The growing development of tissue engineering depends on bioabsorbable materials, increasing the interest in non-ceramic and nanostructured hydroxyapatite associated with polymers [4].
In this study, we compared the efficacy of alginate microspheres associated with hydroxyapatite NPs synthesized at 5 • C and 90 • C, sintered at 1000 • C, or not, on the bone repair.The four samples chosen for the work had different structural characteristics and in vitro and in vivo Ca and P degradation rates: (i) alginate microspheres with crystalline NPs (HA90) and moderate dissolution rates, (ii) microspheres with high crystalline NPs (HA90S) and low dissolution rates, (iii) microspheres with low crystalline NPs (HA5) and high dissolution rates and (iv) microspheres containing high crystalline hydroxyapatite and TCP NPs with high dissolution rates.
Previous studies published by our group using biomaterials with similar characteristics demonstrated favorable cell viability and biocompatibility [30,36,37].Therefore, to evaluate the biological effect of samples on bone repair, we used tibial defects in rabbits considering the animal size, low cost, and easy management, with a considerable scientific background supporting their use to study biomaterials and the similarity with the mineral bone density from humans [38].The non-critical bone defect of 2 mm diameter in tibiae followed the ISO 10993-6/2016 since a previous study showed similar repair of a tibial defect of 1.0, 2.0, and 3.0 mm after 45 d [39], supporting the use of 2.0 mm diameter defect.The micrometric spheres (425-600 µm) dispersed quickly in the surgical site, mainly the HA90 and HA5, quickly fragmented by the alginate dissolution, the strong interaction of particles with interstitial fluid and local cells with clastic activities present at the site of perforations.The microsphere fragmentation increases the surface area for osteoblasts' attachment to the biomaterial, improving the bone repair process and osteoconduction [34].
The present characterization of HA90S, HA90, HA5S, and HA5 confirmed the effect of hydroxyapatite's structural and chemical properties on the biological response after implantation in the noncritical bone defect.Both sintered and non-sintered hydroxyapatite remain biocompatible [8,[40][41][42], and nanostructured hydroxyapatite has been evaluated in vivo in association with bioactive molecules [43,44] and clinically with a growth factor [26].In addition to being biocompatible, hydroxyapatite should be biodegradable to increase the bone and dental implant interface in oral rehabilitation procedures [45].
The degradability of HA90 and HA5 occurred in vitro (acidic pH) with a significant release of calcium and phosphate as expected [46] and in vivo by a significant fragmentation of the microspheres, increasing the area of contact with the connective tissue and conducting the newly formed bone from the native bone through the spheres and just not around them as observed here for HA90S and HA90 and in previous studies [47,48].Moreover, µXRF-SR showed no presence of calcium in the connective tissue in opposition to higher concentration in bone and the grafts, suggesting that calcium release in vivo was not significant and those morphological and chemical properties of the spheres of HA90S, HA90, and HA5 were more relevant to bone osteoconductivity.
Recently, Martinez-Zelaya et al [34] found that the disaggregation of nanostructured cHA/alginate microspheres had no impact on the growth of inherent topological motifs of trabecular-type microarchitecture during the healing process of bones.In the present study, we confirmed Martinez-Zelaya's observations.From a clinical point of view, we can infer that although HA90S and HA90 present levels of new bone similar to HA5, the fact that the bone is permeating the fragmented microspheres instead of adjacent to the intact spheres can provide a greater bonedental implant interface when installing implants in bone grafted with HA5.
An intriguing result refers to the low density of new bone for HA5S, and further studies shall be conducted to address this question better.We hypothesize that TCP in the microspheres significantly increases the Ca and P release in the implanted defect, inducing an intense inflammatory response and delaying the new bone growth.Our histologic results reinforce this hypothesis since we observed a discrete but persistent inflammatory infiltrate after 28 d, possibly slowing bone healing.Synthetic b-TCP ceramics are osteoconductive biomaterials with a high reabsorption capacity and good clinical and histological results in animals and humans [49].However, fast TCP dissolution increasing Ca concentration in the tissue contributes to the delay of bone repair in vivo [50] and clinically [51].
A limitation of the present study was the lack of a histomorphometric analysis of the biomaterials tested.On the other hand, the analysis of the bone-biomaterials interface allowed a better understanding of the dynamics of microsphere degradation and bone apposition directly onto the surface of the hydroxyapatite sintered or not.Moreover, there was no micro-CT analysis comparing the groups.We suggest this analysis for future studies.
Altogether, the results provide direct evidence that fragmentation of the HA5 and, partially, of the HA90 promoted a better-distributed newly formed bone tissue onto the surface of biomaterials compared to bone present just around the intact HA90S microspheres.The clinical relevance of HA90 and HA5 for implantology is the potential increase in bone-dental implant contact during osteointegration of dental implants installed in bone defects filled with these biomaterials, promoting better dental implant stability.Further clinical studies are required to test this hypothesis.The rationale involved in this study was that lower temperature would quicker degrade and might improve the new bone formation.The degradation had positive results according to the temperature; however, our biological results (histomorphometric analysis) demonstrated that both temperatures (5 • and 90 • ) had similar and statistically nonsignificant results for bone formation.Therefore, sintered biomaterial had a lower bone formation than non-sintered.

Conclusions
Within the limitation of this study, the synthesized biomaterials are biocompatible and osteoconductive.The heat treatment directly influenced the material behaviors, where HA90 and HA5 (non-sintered) showed higher degradation, allowing a better distribution of the new bone onto the surface of the biomaterial fragments compared to the HA90S presenting the same level of new bone, but only on the surface of the intact microspheres, potentially reducing the bone-biomaterial interface.Also, EDS-SEM and µXRF-SR mapping techniques showed the elemental distribution of Ca, P, and Zn in the newly formed bone similar to the cortical bone, indicating bone maturity at 28 d.This study opens a range of possibilities for developing new biomaterials using synthetic bioceramics based on hydroxyapatite as bone substitutes, such as diversifying a fragmentation ratio and bioabsorption of nanostructured grafts.Further clinical studies are required to test this hypothesis.

Figure 1 .
Figure 1.The scanning electron microscopy (SEM) analysis of the microspheres shows the irregular surface of biomaterials before (A)-(D) and after in vitro degradation assay (E)-(H) with 1000× magnification.Note the differences in the surface topography and rugosity among samples being more evident in the non-sintered materials (B) and (D).Degradation assay in MES buffer modified the surface topography of HA, except for HA90S.

Figure 2 .
Figure 2. The scanning electron microscopy (SEM) shows the microspheres' measurements for each group.

Figure 3 .
Figure 3.In vitro degradation assay of HA microspheres.HEPES Buffer pH 7.4.Calcium release was higher than P in all groups, and HA5 and HA5S presented higher solubility than HA90 and HA90S.MES Buffer pH 5.9.The calcium and phosphate release followed the same pattern as in the HEPES buffer, with Ca being more released than P, mainly for HA5 and HA5S.HA90S presented the lowest release of calcium and phosphate at pH 5.9 ( * , p-value with significant level).
Figure 12  presents the µXRF-SR 2D elemental map of the evaluated samples.In all periods, calcium intensities were detected in all groups, with

Figure 12 .
Figure 12.Elemental analysis of the bone-biomaterial-connective tissue interface by µXRF-SR.The region of interest (red rectangle) of undecalcified samples of HA90S, HA90, HA5S, and HA5 in the tibial bone defect after seven and 28 d post surgery were submitted to µXRF-SR for detection of calcium and zinc; each elemental map posses its concentration gradient.(From blue [the lowest concentration] to red [the greatest concentration], it shows the value for the element analyzed).

Table 1 .
Synthesis and sintering temperature of hydroxyapatite powder.