Physicochemical Characterization and Effects of Monetite Obtained from Titania-Reinforced Eggshell on Bone Repair: A New Possibility for Tissue Bioengineering?

Physicochemical Characterization and Effects of Monetite Obtained from Titania-Reinforced Eggshell on Bone Repair: A New Possibility for Tissue Bioengineering? | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Physicochemical Characterization and Effects of Monetite Obtained from Titania-Reinforced Eggshell on Bone Repair: A New Possibility for Tissue Bioengineering? Laisa Kindely Ramos Oliveira, Conrado Dias do Nascimento Neto, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4807871/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Clinical Oral Investigations → Version 1 posted 9 You are reading this latest preprint version Abstract Objectives to carry out physicomechanical characterization of the HA/DCPA/TiO 2 and to evaluate the tissue repair in rat calvaria. Methods Two bone defects were made in the calvaria of 36 Wistar rats, divided into groups: HA/DCPA, HA/DCPA/TiO 2 and sham (blood clot). The animals were euthanized at 30, 60 and 90 days and calvaria slides processed with hematoxylin/eosin. The newly formed bone, connective tissue, biomaterial remnant and total tissue repair percentages were calculated in relation to the total defect area. The HA/DCPA/TiO 2 was characterized structurally by scanning electron microscopy (SEM), and chemically by energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD). It was submitted to apparent density (AD), apparent porosity (AP), water absorption (WA) and compressive strength (CS) physical tests. The ANOVA test was applied, followed by Turkey’s test and Student’s t test (p ≤ 0,05). Results The SEM showed biomaterials inside the bone defects and newly formed bone. EDS identified oxygen, calcium, phosphorus and titanium in the sample. The HA/DCPA/TiO 2 and HA/DCPA groups presented a total tissue repair area was larger than the sham group (p < 0.001). Conclusions The physical-mechanical assays showed that HA/DCPA/TiO 2 has AD and CS properties within the limits of trabecular bone and with values higher than HA/DCPA.HA/DCPA/TiO 2 presented higher densification and compressive strength rates than HA/DCPA. Clinical Relevance : Both biomaterials are promising as bone defect fillers. The HA/DCPA/TiO 2 has potential as a scaffold for bone to application in areas subject to load. Chicken Egg Bone Cements Bone Substitutes Skull Bone Regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION With the progress of clinical applications of bioceramics, the limitations of their mechanical properties are becoming evident for areas subject to high loads. Hydroxyapatite (HA) is one of the most studied bioceramics and is widely used as a bone substitute [ 1 , 2 ], but it has low levels of bioactivity and solubility in physiological pH, slow degradation and low mechanical resistance [ 3 ]. These characteristics limit its clinical indications and stimulate the development of other calcium phosphate derivatives to obtain a bioceramic with improved mechanical properties and adequate reabsorption [ 4 ]. Dicalcium phosphate anhydrous (DCPA - CaHPO 4 ) is a relatively recent and still poorly researched material as a bone substitute [ 1 , 5 ]. It is biocompatible, bioactive, stable, has in vivo degradation and resorption rates superior to HA and better bone remodeling [ 6 ]. Zanelato et al. [ 7 ] developed and characterized an HA/DCPA cement, obtained from chicken eggshell, an inexhaustible source of calcium carbonate, a raw material for calcium phosphate synthesis. The HA/DCPA association results in a more soluble and stable graft, because DCPA increases HA reabsorption [ 2 ], but its mechanical resistance still needs improvement. Structural bioceramics, such as titanium dioxide (TiO 2 ), or titania, have high mechanical resistance under compression when compared to metals and polymers [ 8 ]. This ceramic is versatile in terms of applicability, it is bioinert and biocompatible, has high reactivity, electrochemical properties, and safe production [ 9 ]. It is a photocatalytic, antibacterial, low price and high stability biomaterial [ 10 ]. The addition of TiO 2 nanoparticles as a reinforcing agent during the development of hybrid materials can enhance the physicochemical properties of bioceramics. Thus, this study associated TiO 2 with HA/DCPA as produced by Zanelato et al. [ 7 ] developed the HA/DCPA/TiO 2 compound, as a biomaterial alternative for the anatomical and functional reconstruction of regions subjected to load [ 9 ] and aimed to perform the physical-mechanical and structural characterization of HA/DCPA/TiO 2 and evaluate the in vivo effect of this composite in the tissue repair process. MATERIALS AND METHODS 2.1 BIOMATERIALS The HA/DCPA was obtained by the precipitation method through the route of Oliveira's patent [ 11 ], at the Laboratory of Mechanical Tests of the Federal Institute of Espírito Santo (IFES – Vitória-ES). It consists of a biphasic calcium phosphate (BCP) cement composed of of a solid phase, in powder form, made of 84.5% HA,15.5% DCPA and the additive carboxymethylcellulose (CMC); and a liquid phase containing disodium hydrogen phosphate decahydrate diluted in distilled water as a reaction accelerator (RA). This biomaterial comes from chicken eggshells as a raw material for calcium phosphates synthesis [ 7 ]. To obtain the HA/DCPA/TiO 2 powder, TiO 2 (Sigma-Aldrich® (Barueri/SP, Brazil) was incorporated into HA/DCPA by means of sintering in a muffle furnace (Fortlab, ME1800, Industria de Fornos Elétricos LTDA, São Carlos/SP). 2.2 CHARACTERIZATION 2.2.1 Physical tests For HA/DCPA/TiO2 physical tests, 11 cylindrical specimens (12 mm diameter X 13 mm height) were made from HA/DCPA (50%), TiO2 (50%) (in equal mass proportions), and sunflower oil, mixed manually for 2 min. Sintering occurred by heating the sample from 20 to 1250°C for 246 min, using a 5°C/min ramp, remaining at this temperature for 2h, followed by subsequent cooling inside the furnace until reaching room temperature. To perform apparent porosity (AP), water absorption (WA) and apparent density (AD) physical tests, it was necessary to obtain the dry (W d ), wet (W w ) and immersed (W i ) weights of each specimen. Using Archimedes' principle, equations 1 to 3 were applied, respectively: $$\:PA\left(\%\right)=\left(\frac{{P}_{u}-{P}_{s}}{{P}_{u}-{P}_{i}}\right)\times\:100\left(1\right)$$ $$\:AA\left(\%\right)=\frac{{P}_{u}-{P}_{s}}{{P}_{s}}\times\:100\left(2\right)$$ $$\:DA\left(\frac{g}{{cm}^{3}}\right)=\frac{{P}_{s}}{{P}_{u}-{P}_{i}}\left(3\right)$$ The specimens were submitted to the compressive strength (CS) test, in accordance with ASTM F451-95 [ 12 ],, using a universal compression machine (Emic Instron 10000). CS values were obtained with Eq. 4, in which F represents the force applied on the specimen in kilonewtons, A indicates the cross-sectional area of the specimen and \(\:\sigma\:\) the maximum compressive strength: $$\:\sigma\:=\frac{F}{A}\left[\frac{kN}{{mm}^{2}}\right]\left(4\right)$$ 2.2.2 Chemical tests Energy-Dispersive X-Ray Spectroscopy (EDS) A sample of HA/DCPA/TiO2 powder and an operative specimen containing the critical-sized bone defects (CSDs) with the graft were submitted to microanalysis by EDS using an Energy Dispersive X-ray Detector (XFlash® Detector 6|10, Bruker, Billerica, USA), coupled to the scanning electron microscope (SEM), with a focal length of 25mm and voltage above 20Kv. The different energy levels of X-rays were analyzed in the software of the device itself. X-ray Diffractometry (XRD) The HA/DCPA/TiO2 powder was examined by XRD with an XRD-6000 diffractometer (Shimadzu), using copper Kɑ radiation with a wavelength of 1.5418 Å, at a voltage of 40 kV and current of 30 mA. A continuous scan was performed at a speed of 2°/min, going from 20° to 80°. 2.3 IN VIVO STUDY This research was approved by the local Animal Use Ethics Commission (number. 26/2021). The experiments were carried out following the ARRIVE guidelines [ 13 ]. We used 36 adult male Wistar rats with body weight ranging from 250g to 300g. This is a split-body study, in which two CSDs were performed in each animal, totaling 72 cavities distributed in three groups according to the filling or control material, observed in the periods of 30, 60 and 90 days each: HA/DCPA group, HA/DCPA/TiO2 group and sham group (control): CSDs filled by blood clot from surgical beds. During the experimental period, the animals were kept at a controlled-temperature (22 ± 2°C) and 12-hour light/dark cycle (7 a.m.-7 p.m.), with access to filtered water and food ad libitum . 2.3.1 Surgical Procedure The animals were anesthetized with 10% Ketamine Hydrochloride (100 mg/kg − 0.1 mL/100g, Ketamine Agener®, União Química Farmacêutica Nacional S/A. Embu-Guaçu /SP, Brazil), and 2% Xylazine Hydrochloride (10mg/kg − 0.05 mL/100 g, Anasedan®, Sespo Indústria e Comércio Ltda., Paulínia/SP, Brazil), intraperitoneally. Antibiotic prophylaxis was performed with Enrofloxacin 2.5% (Flotril® 2.5%, Intervet Schering-Plough, Rio de Janeiro/RJ, Brazil), at 10mg/weight of the animal in kg, subcutaneously, 1h before the surgical procedure. After anesthetic infiltration of 2% lidocaine hydrochloride with norepinephrine 1:100,000 (DFL Indústria e Comércio S/A, Rio de Janeiro, Brazil), a linear coronal incision of about 1.5 cm was made in the region between the ears. Periosteum skin was detached with the aid of a Molt detacher. Next, CSDs of 5mm diameters were made in the parietal bones, one to the right and another to the left of the median sagittal suture, with the aid of an electric motor (1500 rpm) and a pear-shaped multilaminate with 5 mm external diameter, maintaining the integrity of the meninges. The HA/DCPA or HA/DCPA/TiO2 biomaterials in the form of cement, with added CMC and RA, were inserted into the CSDs according to the experimental groups. In the sham group, no biomaterial was inserted, only a blood clot from the surgical bed (Fig. 1 A). The flap was repositioned and the suture was performed in a single plane with simple interrupted stitches, using a 5 − 0 nylon thread. After 30, 60 and 90 days, the animals were euthanized with a lethal intraperitoneal dose of 10% ketamine hydrochloride (300 mg/kg − 0.3 mL/100g) and 2% xylazine hydrochloride (30mg/kg − 0.15 mL/100g). The calvaria were dissected and blocks containing the parietal bones with the critical-sized bone deffects (Fig. 1 B) were stored in 10% buffered formalin for histological processing. 2.3.2 Histological Processing and Histomorphometric Analysis The surgical specimens were decalcified in ethylenediamine tetraacetic acid (EDTA) and included in paraffin to obtain 07µm sections stained with hematoxylin and eosin. The slides were analyzed under light microscopy (Leica DM500 trinocular optical microscope), with the ICC50 HD photodocumentation system of the UFES Multiuser Histotechnical Laboratory, in 4X and 10X objectives. The reading was performed by the same observer, previously calibrated by an experienced professional. For histological analysis, inter- and intra-examiner reproducibility was calculated by the Kappa coefficient, following the criteria of Landis and Koch (1977) [ 14 ]: weak (0–0.2), reasonable (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80), almost perfect (0.81–1.0). The histomorphometric analysis was performed using ImageJ1.50i® software (Madison, USA). The measurement was performed in µm 2 with the aid of the Set Measurements function of the Analyze tool to obtain measurements of total bone defect area, newly formed bone area, connective tissue area (including fibrous tissue, blood vessels, adipose tissue), remaining biomaterial area and total tissue repair area (sum of areas of newly formed bone, connective tissue and biomaterial remnant in the test groups), in relation to the total area of the originally created defect. The delimitation of the total area of the defect was obtained from the total extent of the defect (5mm) and using as limits the thickness of the calvaria for each photomicrograph (Fig. 2 ). For the researcher’s calibration for the histomorphometric analysis, an intraclass correlation coefficient (ICC) intra-examiner test-retest was performed using IBM SPSS® Statistics version 24 software, with a significance level of 5% (p < 0.05). The interpretation of the obtained values followed the criteria of Cicchetti and Sparrow (1981) [ 15 ]. 3 SCANNING ELECTRON MICROSCOPY (SEM) The microstructural analysis was performed by SEM in one operative specimen of each test group, in each observation period, and compared with the powdered biomaterial. The samples were metallized with a thin gold layer (Desk V, Denton Vacuum, NJ, USA) for scanning, made with the SEM (JSM-6610LV, JEOL Ltda., Tokyo, Japan). 2.4 STATISTICAL ANALYSIS The independent samples ANOVA followed by Tukey's test was used to compare the percentages of newly formed bone, connective tissue and tissue repair area of HA/DCPA/TiO 2 , HA/DCPA and sham intergroups, at each observation time. To compare the percentage of remaining biomaterial of HA/DCPA/TiO 2 and HA/DCPA intergroups, Student's t-test for paired measurements was used. To compare the studied intragroup variables over the observation periods, ANOVA was used followed by the Bonferroni test. The significance level was 5% in all analyses. The program used in the analyses was IBM SPSS Statistics version 24. RESULTS 3.1 HA/DCPA/TiO 2 COMPOUND CHARACTERIZATION 3.1.1 Analysis of chemical composition and XRD The chemical composition of titania is shown in Table 1 . It can be observed that the titania is almost pure (over 99%). Table 1 Chemical composition of TiO 2 in the anatase phase. Chemical compound Chemical Composition (%) TiO 2 99,03 Al 2 O 3 0,41 SiO 2 0,28 P 2 O 5 0,28 Figure 3 shows the XRD for the ceramic compound 50% HA/DCPA + 50% TiO 2 , by mass, sintered at 1250°C. The main peaks of HA/DCPA and TiO 2 can be observed, as well as the chemical reaction between them, which produced perovskite (CaTiO 3 ). TiO 2 is represented in two polymorphs: rutile and anatase. Two phases related to calcium phosphate were also identified: its beta phase (β-TCP) and calcium pyrophosphate (Ca 2 O 7 P 2 ). 3.1.2 Physical and mechanical tests 3.1.2.1 Apparent porosity, bulk density, water absorption and compressive strength Table 2 shows the results obtained for the proposed physical assays of AP, AD and WA through dry, wet and immersed weight data and through the application of equations 1 to 3, as well as the CS values for 50% HA/DCPA + 50% TiO2. These results can be compared with other findings in the literature for pure HA [ 3 ], HATiO 2 compounds [ 3 , 16 ] and trabecular and cortical human bone [ 17 – 20 ]. Table 2 Physical and mechanical properties of the HA/DCPA/TiO 2 compound, compared with other bioceramics reinforced with titania and human trabecular and cortical bone described in the literature. Author/Year Bioceramic Physical and mechanical properties DA (g/cm 3 ) AA (%) PA (%) RC (MPa) HA/DCPA Oliveira et al. (2023) This study 84,5% HA/ + 15,5% DCPA 1,10 ± 0,03 84,54 ± 3,41 56,69 ± 1,83 6,09 ± 1,64 HA/DCPA/TiO 2 This study 50% HA/DCPA + 50%TiO 2 1,42 ± 0,04 78,36 ± 2,38 56,81 ± 0,65 8,44 ± 1,10 César et al. (2019) 50%HA + 50%TiO 2 1,66 ± 0,05 17,7 ± 2,9 15,5 ± 2,0 3,5 ± 0,3 Galdino and Zavaglia (2012a) 100% HA - - - 2,4 ± 0,8 50%HA + 50%TiO 2 1,51 ± 0,05 33,6 ± 2,3 50,7 ± 2,0 5,5 ± 1,2 Morgan et al., 2018 Trabecular human bone - - 40–95 - Human cortical bone 2,0 - 5–15 - Reina et al., 2021 Trabecular human bone 1,18 - - - Human cortical bone 1,85 − 2,0 - - - Matsunake and Frantz, 2021 Trabecular human bone - - - 2–12 Human cortical bone - - - 100–150 3.2 HISTOLOGICAL ANALYSIS At 30 days, in the HA/DCPA/TiO 2 group, there were conglomerates of biomaterial granules inside the CSDs, which made the external surface more irregular, surrounded mainly by loose connective tissue. There was, predominantly, a formation of medullary centripetal bone from the margins and the internal limit of the critical-sized bone defect facing the meninges (Fig. 4 A). In the HA/DCPA group, multiple graft granules are evenly distributed in the defect area and surrounded predominantly by cellular connective tissue. In the central region of the defect image, newly formed bone areas can be observed in the middle of the granules (Fig. 4 B). At 60 days, in the HA/DCPA/TiO 2 group, there were clusters of biomaterial with different dimensions surrounded by newly formed bone tissue, predominantly medullary, in the central region of the defect, surrounded by loose connective tissue on the surface of the CSDs (Fig. 4 D). In the HA/DCPA group, the central area of the defect was filled with highly cellular connective tissue and with biomaterial granules distributed within it. There was newly formed bone from the surrounding regions all the way to the central area of the defect, especially at its internal limit (Fig. 4 E). At 90 days, the HA/DCPA/TiO 2 group presented a loose connective tissue band above the newly formed bone, with small biomaterial granules dispersed inside it. The centripetal bone grew and more bone filled the defect, in relation to the other observation periods (Fig. 4 G). In the HA/DCPA group, connective tissue covered the upper limit of the defect (Fig. 4 H). There were graft granules distributed throughout the tissue repair area, surrounded by connective tissue in the most superficial portion of the defect and by bone tissue in the innermost areas. The graft granules were more evenly distributed throughout the defect than in the HA/DCPA/TiO 2 group. In the three experimental periods, the sham group showed newly formed bone areas distributed regularly and covered by cellular, fibrous connective tissue with a mild depression on top (Fig. 4 C, 4 F and 4 I). In general, the total tissue repair volume was maintained inside the defects in all experimental groups. In all periods, the HA/DCPA group had a more regular repair surface than the HA/DCPA/TiO 2 group. At the end of the experiment, it was possible to observe a greater volume of newly formed bone in the central region of the defect, in all three groups. 3.3 HISTOMORPHOMETRIC ANALYSIS Figure 5 shows the graphical representation of the histomorphometric analysis results. At 30 days, the groups were similar to each other in relation to the variables studied (p > 0.05). At 60 days, there was a higher percentage of HA/DCPA connective tissue area (69.58 ± 10.82%) compared to that of HA/DCPA/TiO 2 (47.52 ± 12.88%), and the difference was statistically significant (p = 0.027). The same was observed at 90 days, with a higher percentage of HA/DCPA connective tissue area (57.54 ± 16.37%) than that of HA/DCPA/TiO 2 (37.06 ± 5.55%) (p = 0.001). In summary, at the end of the experiment, there was a difference in the connective tissue variables of the HA/DCPA and HA/DCPA/TiO 2 groups. It is noteworthy that the total tissue repair areas in both experimental groups completely filled the CSDs, with percentage means higher than 100% (HA/DCPA/TiO 2 = 103.88 ± 21.02% and HA/DCPA = 120.35 ± 17.51%) and significantly higher than that of the sham group (p < 0.001). 3.4 SCANNING ELECTRON MICROSCOPY AND ENERGY DISPERSIVE X-RAY SPECTROSCOPY In the images obtained by SEM, it Is possible to observe an irregularly shaped and porous biomaterial with granular morphology, similar in all groups (Fig. 6 ). In greater increase, the result obtained on the surface of the granulated biomaterial can be observed, revealing the interconnected microporous microstructure formed by fine grains. There is also the presence of newly formed bone and biomaterial remnants in the critical-sized bone defect area. After selecting a random location in the SEM images, EDS was used to identify the chemical composition of the samples at 30, 60 and 90 days, revealing oxygen, calcium, phosphorus and titanium as the main chemical elements of hydroxyapatite, monetite and titania (Fig. 6 ). The results suggest a heterogeneous percentage distribution of these chemical elements, with evidence of their concentration in certain regions. DISCUSSION The calcium phosphate obtained from chicken eggshells is an innovative alternative and comes from an inexhaustible source of calcium carbonate with low cost and a safe production process, since it does not require chemicals used in the production of other phosphates, considered dangerous and corrosive in their raw states [ 7 , 20 ]. The addition of TiO 2 particles to calcium phosphates has attracted the attention of researchers, since TiO 2 can increase osteoblast adhesion and induce cell growth [ 21 – 23 ]. Considering that although the HA/DCPA compound presents better reabsorption and stability than does pure HA, its low mechanical resistance still limits its clinical application. Thus, TiO 2 addition can contribute to improve the mechanical characteristics of the biomaterial and bone tissue regeneration, by presenting values of resistance strength, elastic modulus and porosity close to those of trabecular bone [ 2 , 24 ]. Galdino and Zavaglia [ 3 ] observed that the addition and sintering of TiO 2 to pure HA results in increased mechanical compressive strength. The ideal biomaterial should have dimensional accuracy, adequate CS and not deform easily under compression. The 8.44 ± 1.10 MPa CS of HA/DCPA/TiO 2 met the criteria for trabecular bone compression [ 18 – 20 ] to receive masticatory pressure [ 18 ]. However, it would not be indicated for applications in cortical bones, such as the femur, because it does not reach the values between 100 MPa and 150 MPa [ 19 ]. According to our findings, with the addition of TiO 2, there was a 38.6% increase in the CS of HA/DCPA, in addition to the increase in AD, maintenance of AP and reduction of WA. These data are corroborated by Galdino and Zavaglia [ 3 ] and César et al , who observed that the sintering of TiO2 to pure HA increased the CS of the biomaterial. It is worth mentioning that the CS values of HA/DCPA/TiO2 were higher than those achieved by César et al. [ 16 ] for HA/TiO2 although the specimens presented close AD values and were more porous (> AP). The different TiO2 phases used in this study, rutile and anatase, have different crystalline characteristics, which interfere with the properties of TiO2 and probably guarantee superior results. Because it is a metastable polymorph at high temperatures (approximate range between 400°C and 1200°C), anatase irreversibly transforms into rutile, a thermodynamically stable polymorph [ 25 ]. As this transformation is a function of time and temperature, the sintering process was not long enough to transform the entire anatase titania into rutile titania. The mixture of rutile and anatase phases possibly contributed to a CS increase and helped to obtain different calcium phosphates: the beta phase of tricalcium phosphate (β-TCP) and calcium pyrophosphate (Ca 2 O 7 P 2 ). These phases occur due to the calcium phosphate synthesis method, in which crystalline β-TCP forms above 1125°C and is widely used as a ceramic biocomposite. Considering that this composite is intended for use in tissue bioengineering, pores with adequate dimensions, shapes, quantity and interconnectivity are needed to favor tissue growth, intertwining the newly formed bone with the scaffold, increasing the CS of the in vivo biomaterial [ 26 ]. The HA/DCPA granules are composed of finely distributed spherical particles of less than 30 µm. Powdered biomaterials containing round particles with an average size of 2–20 µm are ideal for use in the form of cement [ 7 , 27 ]. The HA/DCPA presented 56.69 ± 1.83 AP and the addition of TiO2 maintained the porosity of the HA/DCPA/TiO2 compound, which remained within the 45–95% range of trabecular bone [ 17 ]. It is important to highlight that although porosity is similar for the two composites, the HA/DCPA/TiO2 presented higher AD and CS values due to the presence of TiO2. The HA/DCPA/TiO2 also presented higher AP values in relation to the HA/TiO2 of Galdino and Zavaglia’s studies [ 3 ], and even higher values in comparison to the findings of César et al. [ 16 ]. It is noteworthy that the manufacturing processes of the specimens were different, since Galdino and Zavaglia [ 3 ] used the polymeric sponge method and César et al. [ 16 ] used polymeric wax to generate porosity during the sintering of specimens manufactured by uniaxial pressing. In our study, the specimens were manufactured by uniaxial pressing, but with a low compaction load. As for the chemical analysis, the formation of perovskite was also observed by Galdino and Zavaglia [ 28 ] and Assmar et al. [ 29 ], who developed porous ceramic HA/TiO 2 compounds by different methods, formed in all compounds sintered at 1250°C, 1300°C and 1350°C. These phases occur due to the calcium phosphate synthesis method, in which crystalline β-TCP forms above 1125°C and is widely used as a ceramic biocomposite, since it promotes bone growth. The formation of calcium pyrophosphate by the wet method used in the present study was also observed in other studies [ 30 – 32 ]. EDS provided information on the oxygen, calcium, phosphorus and titanium present in HA/DCPA/TiO 2 granules and adjacent tissues. The energy dispersion generated by the titanium suggests the successful incorporation of this element into the biomaterial [ 33 ]. Although EDS identified the presence of Ca and P, these elements can be both from biomaterial and newly formed bone [ 34 ], requiring in vivo histological and histomorphometric analysis to identify and quantify them. The photomicrographs demonstrated that the biomaterial is safe for clinical use due to the absence of significant adverse effects, such as osteolytic reactions or persistent inflammatory processes, which evidences good biocompatibility and interaction with the native calvarial bone. During bone repair, an initial inflammatory process followed by new bone formation and a subsequent bone matrix remodeling process is a normal occurrence [ 35 ]. With the histological analysis, newly formed bone with an immature appearance could be observed, accompanied by intense vascular formation and irregularly organized collagen fibers, without the presence of Havers channels with well-defined morphology. Over the observed periods, the bone acquired a mature appearance, making it difficult to identify the line of intersection between it and the native bone and possible to identify the presence of osteocytes. HA presents slow biodegradation and can be gradually reabsorbed 4 to 5 years after implantation [ 1 ]. Since the resorption of the material and its replacement by bone tissue are desired characteristics, the association of HA with DCPA aims to increase reabsorption. Tamimi et al. [ 6 ] showed that DCPA shows signs of graft resorption as the newly formed bone tissue grows, 8 weeks after its implantation, surrounding and penetrating DCPA granules. Comparing the resorption process of the material over the three observation periods, we noticed a slight reduction of the HA/DCPA biomaterial and a similar resorption pattern in the intergroup comparison. However, even without differences, it should be considered that the remaining HA/DCPA and HA/DCPA/TiO 2 granules influence the greater volume and maintenance of the bone defect contour when compared to the sham, because the granules are surrounded by connective tissue and newly formed bone [ 36 ], which may explain why the total tissue repair area in these groups was larger than that of the sham group (p < 0.001). Both biomaterials in the form of cement have the advantage of ease of handling and insertion in the experimental cavities, which allowed the biomaterial to adapt and helped to control the volume of the grafted material. In general, calcium phosphate cement paste allows injection, solidification and in situ modeling of complex and vertical bone cavities, besides being a great option for minimally invasive surgery [ 2 ]. The possibility of using a porous biomaterial in the form of cement paste brings together two configurations that can contribute to tissue repair. The granule porosity of between 40% and 60% can promote rapid diffusion or flow of nutrients and cell-biomaterial interactions [ 37 , 38 ]. The cement in paste form has biomaterial clusters, which were histologically observed, in its morphology, and this configuration could both stimulate collagen fibers and release calcium ions for new bone formation [ 38 ]. There are no previous studies in the literature that have evaluated the effects of HA/DCPA/TiO 2 on bone regeneration. We believe that future research to evaluate the use of TiO 2 particles at different mass percentages and in other bone sites is necessary for our findings to be confirmed. Thus, there will be greater safety and efficacy in its application in clinical practice. It is worth mentioning that the combination of TiO 2 with other types of phosphate provides better quality in the bone regeneration process, impacts clinical management and provides new treatments for acquired or congenital bone defects. According to the results obtained, both biomaterials are promising as bone defect fillers. Nevertheless, the scaffolds used in bone tissue engineering must be able to provide mechanical strength similar to natural bone tissue [ 39 ], when in function. Regarding their application in areas subject to load, despite the CS increase obtained with the addition of TiO 2 to HA/DCPA, the tests were performed in vitro on specimens, and future studies may investigate the mechanical strength of these biomaterials after in vivo implantation. CONCLUSIONS The HA/DCPA/TiO 2 presented higher densification and compressive strength rates than the HA/DCPA. Biocompatibility and bone conductivity denote the potential of this bioceramic as a framework for bone engineering, with in vitro mechanical resistance similar to that of trabecular bone. Declarations Conflicts of interest : The authors have no conflicts of interest to declare that are relevant to the content of this article. Nom-financial interests All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Author Contribution L.K.R.O: Methodology- Development of all stages of the experiment; Collection, interpretation and analysis of data; Writing, Review and Edition; Approval of the final version and submission of the article.C.D.N.N: Methodology- Surgical stage of the animals experiment, approval of the final version of the article.A.B.C.S: Methodology- Surgical stage of the animals experiment , approval of the final version of the article.S.M.W.R: Conceptualization; Design of methodology, definition of the theme; Critical analysis and approval of the final version of the article.P.R.B: Methodology - Surgical stage of the animals experiment; Critical analysis and approval of the final version of the article.A.G.S.G: Conceptualization; Design of methodology; Definition of the theme/subject; Critical analysis and approval of the final version of the article.D.N.S: Conceptualization; Design of methodology ; Supervision; Project administration; Interpretation and analysis of data; Critical analysis and approval of the final version of the article. Acknowledgement Laboratories: Carbonaceous and Ceramic Materials/UFES Thermal Plasma Laboratory; Mechanical Tests at the Federal Institute of Espírito Santo (IFES – Vitória-ES); Cellular Ultrastructure Carlos Alberto Redins; Multiuser of Histotechnics at UFES; and cardiac electromechanics and vascular reactivity of UFES References Guastaldi AC, Aparecida AH (2010) Fosfatos de cálcio de interesse biológico: importância como biomateriais, propriedades e métodos de obtenção de recobrimentos. Quím. https://doi.org/10.1590/S0100-40422010000600025 Jeong J, Kim JH, Shim JH, Hwang NS, Heo CY (2019) Bioactive calcium phosphate materials and applications in bone regeneration. Biomater Res. https://doi.org/10.1186/s40824-018-0149-3 Galdino AGS, Zavaglia CAC (2012a) Caracterização físico-mecânica de compósitos porosos de hidroxiapatita-titânia confeccionados pelo método da esponja polimérica. https://doi.org/10.1590/S0366-69132012000300017 . 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[Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica] 126. https://repositorio.unicamp.br/acervo/detalhe/805519 Anaya-Esparza LM, Villagrán-de la Mora Z, Ruvalcaba-Gómez JM et al (2020) Use of Titanium Dioxide (TiO 2 ) Nanoparticles as Reinforcement Agent of Polysaccharide-Based Materials. https://doi.org/10.3390/pr8111395 . Process Chen J, Shan M, Shi X, Zhang S, Li J, Luan J et al (2022) BiSnSbO6–TiO 2 composites enhance LED light-driven photocatalytic antibacterial activity. Ceram Int. https://doi.org/10.1016/j.ceramint.2022.03.192 Oliveira LTD Production of calcium phosphate compositions https://patents.google.com/patent/BR112018005871A2/en . Acessed 2023 May 12 ASTM F451-16 Standard Specification for Acrylic Bone Cement. https://www.astm.org/f0451-16.html Percie du Sert N, Hurst V, Ahluwalia A et al (2020) The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. https://doi.org/10.1371/journal.pbio.3000410 Landis JR, Koch GG (1977) The measurement of observer agreement for categorical data. Biometrics.PMID: 843571 Cicchetti DV, Sparrow SA (1981) Developing criteria for establishing interrater reliability of specific items: applications to assessment of adaptive behavior. Am J Ment Defic. https://pubmed.ncbi.nlm.nih.gov/7315877/ César FSC, Júnior CP, Rocha RC, Galdino AGS (2019) Assessing the Physical-Mechanical Characteristics of Hydroxyapatite-Titanium Oxide Biocomposites Produced by the Polymeric Wax Addition Method. Mater Sci Forum. https://doi.org/10.4028/www.scientific.net/MSF.958.87 Morgan EF, Unnikrisnan GU, Hussein AI (2018) Bone Mechanical Properties in Healthy and Diseased States. Annu Rev Biomed Eng. https://doi.org/10.1146/annurev-bioeng-062117-121139 Reina SA, Tito BJE, Malini MH, Iqrimatien FG, Sa’diyah E, Aminatun (2021) Porosity and compressive strength of PLA-based scaffold coated with hydroxyapatite-gelatin to reconstruct mandibula: a literature review. NASA ADS. https://ui.adsabs.harvard.edu/abs/2021JPhCS1816a2085R/abstract Matsunaka LEZ, Frantz JC (2021) Analysis of the compressive strength of scaffolds manufactured by additive manufacturing applied in bone regeneration https//. doi.org/10.34117/bjdv7n9-461 Agbeboh NI, Oladele IO, Daramola OO, Adediran AA, Olasukanmi OO, Tanimola MO (2020) Environmentally sustainable processes for the synthesis of hydroxyapatite. Heliyon https://doi.org/10.1016/j.heliyon.2020.e03765 Galdino AGS, Oliveira EM, Filippin-Monteiro FB, Zavaglia CAC, Cerâmica (2014) https://doi.org/10.1590/S0366-69132014000400019 Webster TJ, Siegel RW, Bizios R (1999) Osteoblast adhesion on nanophase ceramics. Biomaterials. https://doi.org/10.1016/s0142-9612(99)00020-4 Pourmollaabbassi B, Karbasi S, Hashemibeni B (2016) Evaluate the growth and adhesion of osteoblast cells on nanocomposite scaffold of hydroxyapatite/titania coated with poly hydroxybutyrate. Adv Biomedical Res. https://doi.org/10.4103/2277-9175.188486 Osório GC, Valencia AMJ, Agualimpia CM, Sierra DME (2018) Manufacture of titanium dioxide scaffolds for medical applications. https://doi.org/10.19053/01211129.v27.n48.2018.8017 . Revista Facultad de Ingeniería Hanaor DAH, Sorrell CC (2010) Review of the anatase to rutile phase transformation. J Mater Sci https://doi. org/10.1007/s10853-010-5113-0 Bertoni PMV (2014) Obtenção de corpos cerâmicos porosos de TiO2 para o emprego como biomateriais.Dissertation, Federal University de Alfenas https://bdtd.unifal-mg.edu.br:8443/handle/tede/638 Dorozhkin SV Calcium orthophosphates., Biomatter (2011) https://doi.org/10.4161%2Fbiom.18790 Galdino AGS, Zavaglia CAC (2012b) Characterization of Hydroxyapatite-Titanium Oxide Scaffolds Made by the Polymeric Sponge Method. Materials Science Forum https://doi.org/ 10.4028/www.scientific.net/MSF.727-728.1113 Assmar DC, Boldrini RS, Rocha RC, Galdino AGS (2017) Avaliação das características estruturais de arcabouços de hidroxiapatita-titânia pelo método da adição de cera polimérica para uso em engenharia tecidual. Revista IFES Ciência file:///C:/Users/Administrador/Downloads/294-Texto%20do%20Artigo-1118-1-10-20191121.pdf Torres PMC, Vieira SI, Cerqueira S, Pina da Cruz Silva OAB, Abrantes JCC et al (2014) Effects of Mn-doping on the structure and biological properties of β-tricalcium phosphate. J Inorg Biochemistr. http://dx.doi.org/10.1016/j.jinorgbio.2014.03.013 Torres PMC, Marote A, Cerqueira AR, Calado AJ, Abrantes JCC, Olhero S et al (2017) Injectable MnSr-doped brushite bone cements with improved biological performance. J Mater Chem B. https://doi.org/10.1039/c6tb03119f Sinusaite L, Renner AM, Schütz MB, Antuzevics A, Rogulis U, Grigoraviciute-Puroniene I et al (2019) Effect of Mn doping on the low-temperature synthesis of tricalcium phosphate (TCP) polymorphs. J Eur Ceram Soc. https://doi.org/10.1016/j.jeurceramsoc.2019.03.057 Qiang T, Xia Y, Zhao J (2019) Homogeneous Zr and Ti co-doped SBA-15 with high specific surface area: preparation, characterization and application. J Leather Sci Eng. https://doi.org/10.1186/s42825-019-0004-x Lozano-Carrascal N, Satorres-Nieto M, Delgado-Ruiz R, Maté-Sánchez de Val JE, Gehrke SA, Gargallo-Albiol J et al (2017) Scanning electron microscopy study of new bone formation following small and large defects preserved with xenografts supplemented with pamidronate-A pilot study in Fox-Hound dogs at 4 and 8 weeks. https://doi.org/10.1016/j.aanat.2016.09.009 . Ann Anat Lin CY, Chang YH, Sung LY, Chen CL, Lin SY, Li KC et al (2014) Long-Term Tracking of Segmental Bone Healing Mediated by Genetically Engineered Adipose-Derived Stem Cells: Focuses on Bone Remodeling and Potential Side Effects. https://doi.org/10.1089/ten.TEA.2013.0314 . Tissue Engineering Part A Grandi G, Heitz C, Santos LA, Silva ML, Sant’Ana Filho M, Pagnocelli RM et al (2011) Comparative histomorphometric analysis between α-Tcp cement and β-Tcp/Ha granules in the bone repair of rat calvaria. Mater Res https://www.scielo.br/j/mr/a/hLyFnqswPgcjC3M4m 3Fbz3R/?lang=en#:~:text=Alpha%2DTCP%20cement%20is%20more Li X, Wang L, Fan Y, Feng Q, Cui FZ, Watari F (2013) Nanostructured scaffolds for bone tissue engineering. J Biomedical Mater Res – Part A. 10.1002/jbm.a.34539 Pintor AVB et al (2018) In Vitro and In Vivo Biocompatibility Of ReOss® in Powder and Putty Configurations. Braz Dent J. 10.1590/0103-6440201802017 Teimouri T, Abnous K, Taghdisi SM et al (2024) Protein-Based Hybrid Scaffolds: Application in Bone Tissue Engineering. J Polym Environ. https://doi.org/10.1007/s10924-024-03264-y Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Clinical Oral Investigations → Version 1 posted Editorial decision: Revision requested 06 Nov, 2024 Reviews received at journal 06 Nov, 2024 Reviewers agreed at journal 06 Nov, 2024 Reviews received at journal 22 Oct, 2024 Reviewers agreed at journal 08 Oct, 2024 Reviewers invited by journal 08 Aug, 2024 Editor assigned by journal 30 Jul, 2024 Submission checks completed at journal 30 Jul, 2024 First submitted to journal 26 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4807871","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":339588870,"identity":"b406e6c6-923f-4537-8da4-68cc913a4fba","order_by":0,"name":"Laisa Kindely Ramos Oliveira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACPh7GBgYeBgsGfhAvoYAILWwQLRIMkg0gLQZEaQESIC0GB0Bc4rQcbnvwpkIicfP51YkfHhgwyPOLHSCghbex3XDOGYnEbTfebpYAOsxw5uwEAlr4GdukedtAWs5uAGlJMLhNlJZ/QIfNOLv5B3FaeBuBWhokEjfw924j0haeg22Sc45JGM+4wbvNIsFAgrBf+HnSn0m8qbGR7e8/u/nmjwobeX5pAlpgwLFBAqxSgjjlIGDPwH+AeNWjYBSMglEwsgAAlmpBFDsRjJcAAAAASUVORK5CYII=","orcid":"","institution":"Federal University of Espírito Santo","correspondingAuthor":true,"prefix":"","firstName":"Laisa","middleName":"Kindely Ramos","lastName":"Oliveira","suffix":""},{"id":339588871,"identity":"91d7ec9c-9070-4898-be79-48fcd787627a","order_by":1,"name":"Conrado Dias do Nascimento Neto","email":"","orcid":"","institution":"Federal University of Espírito Santo","correspondingAuthor":false,"prefix":"","firstName":"Conrado","middleName":"Dias do Nascimento","lastName":"Neto","suffix":""},{"id":339588872,"identity":"e29675ee-658f-4134-b02f-24f736d8de8a","order_by":2,"name":"Amy Brian Costa e Silva","email":"","orcid":"","institution":"Federal University of Espírito Santo","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"Brian Costa e","lastName":"Silva","suffix":""},{"id":339588873,"identity":"83ae0315-f80c-41da-aaea-326d790537a1","order_by":3,"name":"Stela Maris Wanderley Rocha","email":"","orcid":"","institution":"Federal University of Alagoas","correspondingAuthor":false,"prefix":"","firstName":"Stela","middleName":"Maris Wanderley","lastName":"Rocha","suffix":""},{"id":339588874,"identity":"6fb9f44b-3f12-4f20-aa4c-ba173e521f0d","order_by":4,"name":"Patrícia Roccon Bianchi","email":"","orcid":"","institution":"Brazilian Dental Association","correspondingAuthor":false,"prefix":"","firstName":"Patrícia","middleName":"Roccon","lastName":"Bianchi","suffix":""},{"id":339588875,"identity":"cdee1503-5354-4f03-a68d-29a49da9a393","order_by":5,"name":"André Gustavo de Sousa Galdino","email":"","orcid":"","institution":"Federal Institute of Espírito Santo","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"Gustavo de Sousa","lastName":"Galdino","suffix":""},{"id":339588876,"identity":"55f6be9c-ea41-4f2d-8e6e-ae7a8a358537","order_by":6,"name":"Daniela Nascimento Silva","email":"","orcid":"","institution":"Federal University of Espírito Santo","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"Nascimento","lastName":"Silva","suffix":""}],"badges":[],"createdAt":"2024-07-26 12:00:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4807871/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4807871/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00784-025-06195-7","type":"published","date":"2025-02-04T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63341771,"identity":"7233e28f-65f1-421f-9a41-148d3039988b","added_by":"auto","created_at":"2024-08-27 06:46:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eBilateral bone defects made in parietal bones with 5 mm in diameter\u003cstrong\u003e; \u003c/strong\u003eleft side filled with graft and right-side sham group (blood clot). \u003cstrong\u003eB: \u003c/strong\u003eOperative specimen of the parietal bones containing the regenerating bone areas after the animal was euthanized.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4807871/v1/a2a68bfcddca66e6ce2a28e0.jpg"},{"id":63341769,"identity":"13e62aa6-d596-442f-b75a-21b579bd79ad","added_by":"auto","created_at":"2024-08-27 06:46:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70725,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of histomorphometry using the ImageJ® software and the Analyze\u0026gt;Set Measurements tools.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4807871/v1/7097d4117d0953e21dc99b9b.jpg"},{"id":63341772,"identity":"c0faff09-3263-4985-96b5-46a8a17c6046","added_by":"auto","created_at":"2024-08-27 06:46:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38064,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray diffractogram of the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e sample.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4807871/v1/2ab332a7230c79666f7d5fda.jpg"},{"id":63341770,"identity":"869c027a-f8c9-4b35-9e38-c8d67ac9795a","added_by":"auto","created_at":"2024-08-27 06:46:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127344,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs in hematoxylin-eosin of the repair area and bone defect fillings. The animals were evaluated 30 (A, B, C), 60 (D, E, F) and 90 (G, H, I) days after the procedures. C and I: areas of highly cellular connective tissue (CT) above the trabeculae of newly formed bone tissue (NB). F: Immature NB, surrounded by fibrous CT. A, B and E: Biomaterial granules (arrow) inside the CT. D and G: NB and CT surrounding numerous biomaterial granules in the region of the medullary bone in the defect. H: Biomaterial granules juxtaposed to NB with extensive CT area inside the bone defect and inside the CT.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4807871/v1/a24e8b62fd2cd68c121becf8.jpg"},{"id":63341768,"identity":"1fee148b-b262-47a3-8bee-9b8107816f2a","added_by":"auto","created_at":"2024-08-27 06:46:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63704,"visible":true,"origin":"","legend":"\u003cp\u003eHistomorphometric analysis – newly formed bone areas, biomaterial remnant, connective tissue, and total tissue repair (%) in the three experimental periods.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4807871/v1/a01142141d5b417129caba82.jpg"},{"id":63341774,"identity":"ccb94fc0-28fd-462d-8b34-880bafb8ca7d","added_by":"auto","created_at":"2024-08-27 06:46:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105802,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of EDS and BCP samples surface micrographs at 30, 60 and 90 days. Figures A, B, C and D show areas of newly formed bone tissue (NB) and biomaterial remnant (BCP) in the critical-sized bone defect. In greater increase, the morphology and microstructure of microporous granulated HA/DCPA/TiO\u003csub\u003e2 \u003c/sub\u003ebiomaterials can be noted. Figures E, F, G and H represent the normalized mass percentage distributions of the chemical elements present in the pure BCP sample, and in the\u003cem\u003e in vivo\u003c/em\u003e samples of the area of defects filled with BCP at 30, 60 and 90 days.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4807871/v1/a4dc890a25e4324d6b153566.jpg"},{"id":75929954,"identity":"5c27ea9d-dc55-45da-837f-484bf9c1baf7","added_by":"auto","created_at":"2025-02-10 16:07:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1441783,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4807871/v1/9bcdfe78-dab9-42fa-b193-33ded94f30b2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003ePhysicochemical Characterization and Effects of Monetite Obtained from Titania-Reinforced Eggshell on Bone Repair: A New Possibility for Tissue Bioengineering?\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eWith the progress of clinical applications of bioceramics, the limitations of their mechanical properties are becoming evident for areas subject to high loads. Hydroxyapatite (HA) is one of the most studied bioceramics and is widely used as a bone substitute [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], but it has low levels of bioactivity and solubility in physiological pH, slow degradation and low mechanical resistance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These characteristics limit its clinical indications and stimulate the development of other calcium phosphate derivatives to obtain a bioceramic with improved mechanical properties and adequate reabsorption [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDicalcium phosphate anhydrous (DCPA - CaHPO\u003csub\u003e4\u003c/sub\u003e) is a relatively recent and still poorly researched material as a bone substitute [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is biocompatible, bioactive, stable, has \u003cem\u003ein vivo\u003c/em\u003e degradation and resorption rates superior to HA and better bone remodeling [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Zanelato et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] developed and characterized an HA/DCPA cement, obtained from chicken eggshell, an inexhaustible source of calcium carbonate, a raw material for calcium phosphate synthesis. The HA/DCPA association results in a more soluble and stable graft, because DCPA increases HA reabsorption [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], but its mechanical resistance still needs improvement.\u003c/p\u003e \u003cp\u003eStructural bioceramics, such as titanium dioxide (TiO\u003csub\u003e2\u003c/sub\u003e), or titania, have high mechanical resistance under compression when compared to metals and polymers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This ceramic is versatile in terms of applicability, it is bioinert and biocompatible, has high reactivity, electrochemical properties, and safe production [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is a photocatalytic, antibacterial, low price and high stability biomaterial [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe addition of TiO\u003csub\u003e2\u003c/sub\u003e nanoparticles as a reinforcing agent during the development of hybrid materials can enhance the physicochemical properties of bioceramics. Thus, this study associated TiO\u003csub\u003e2\u003c/sub\u003e with HA/DCPA as produced by Zanelato et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] developed the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e compound, as a biomaterial alternative for the anatomical and functional reconstruction of regions subjected to load [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and aimed to perform the physical-mechanical and structural characterization of HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e and evaluate the \u003cem\u003ein vivo\u003c/em\u003e effect of this composite in the tissue repair process.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 BIOMATERIALS\u003c/h2\u003e \u003cp\u003eThe HA/DCPA was obtained by the precipitation method through the route of Oliveira's patent [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], at the Laboratory of Mechanical Tests of the Federal Institute of Espírito Santo (IFES – Vitória-ES). It consists of a biphasic calcium phosphate (BCP) cement composed of of a solid phase, in powder form, made of 84.5% HA,15.5% DCPA and the additive carboxymethylcellulose (CMC); and a liquid phase containing disodium hydrogen phosphate decahydrate diluted in distilled water as a reaction accelerator (RA). This biomaterial comes from chicken eggshells as a raw material for calcium phosphates synthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo obtain the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e powder, TiO\u003csub\u003e2\u003c/sub\u003e (Sigma-Aldrich® (Barueri/SP, Brazil) was incorporated into HA/DCPA by means of sintering in a muffle furnace (Fortlab, ME1800, Industria de Fornos Elétricos LTDA, São Carlos/SP).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 CHARACTERIZATION\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Physical tests\u003c/h2\u003e \u003cp\u003eFor HA/DCPA/TiO2 physical tests, 11 cylindrical specimens (12 mm diameter \u003csub\u003eX\u003c/sub\u003e 13 mm height) were made from HA/DCPA (50%), TiO2 (50%) (in equal mass proportions), and sunflower oil, mixed manually for 2 min. Sintering occurred by heating the sample from 20 to 1250°C for 246 min, using a 5°C/min ramp, remaining at this temperature for 2h, followed by subsequent cooling inside the furnace until reaching room temperature.\u003c/p\u003e \u003cp\u003eTo perform apparent porosity (AP), water absorption (WA) and apparent density (AD) physical tests, it was necessary to obtain the dry (W\u003csub\u003ed\u003c/sub\u003e), wet (W\u003csub\u003ew\u003c/sub\u003e) and immersed (W\u003csub\u003ei\u003c/sub\u003e) weights of each specimen. Using Archimedes' principle, equations 1 to 3 were applied, respectively:\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:PA\\left(\\%\\right)=\\left(\\frac{{P}_{u}-{P}_{s}}{{P}_{u}-{P}_{i}}\\right)\\times\\:100\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:AA\\left(\\%\\right)=\\frac{{P}_{u}-{P}_{s}}{{P}_{s}}\\times\\:100\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:DA\\left(\\frac{g}{{cm}^{3}}\\right)=\\frac{{P}_{s}}{{P}_{u}-{P}_{i}}\\left(3\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003cp\u003eThe specimens were submitted to the compressive strength (CS) test, in accordance with ASTM F451-95 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e],, using a universal compression machine (Emic Instron 10000). CS values were obtained with Eq.\u0026nbsp;4, in which F represents the force applied on the specimen in kilonewtons, A indicates the cross-sectional area of the specimen and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sigma\\:\\)\u003c/span\u003e\u003c/span\u003ethe maximum compressive strength:\u003c/p\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:\\sigma\\:=\\frac{F}{A}\\left[\\frac{kN}{{mm}^{2}}\\right]\\left(4\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2.2 Chemical tests\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003eEnergy-Dispersive X-Ray Spectroscopy (EDS)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA sample of HA/DCPA/TiO2 powder and an operative specimen containing the critical-sized bone defects (CSDs) with the graft were submitted to microanalysis by EDS using an Energy Dispersive X-ray Detector (XFlash® Detector 6|10, Bruker, Billerica, USA), coupled to the scanning electron microscope (SEM), with a focal length of 25mm and voltage above 20Kv. The different energy levels of X-rays were analyzed in the software of the device itself.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eX-ray Diffractometry (XRD)\u003c/h2\u003e \u003cp\u003eThe HA/DCPA/TiO2 powder was examined by XRD with an XRD-6000 diffractometer (Shimadzu), using copper Kɑ radiation with a wavelength of 1.5418 Å, at a voltage of 40 kV and current of 30 mA. A continuous scan was performed at a speed of 2°/min, going from 20° to 80°.\u003c/p\u003e \u003cp\u003e2.3 \u003cem\u003eIN VIVO\u003c/em\u003e STUDY\u003c/p\u003e \u003cp\u003e This research was approved by the local Animal Use Ethics Commission (number. 26/2021). The experiments were carried out following the ARRIVE guidelines [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We used 36 adult male Wistar rats with body weight ranging from 250g to 300g. This is a split-body study, in which two CSDs were performed in each animal, totaling 72 cavities distributed in three groups according to the filling or control material, observed in the periods of 30, 60 and 90 days each: HA/DCPA group, HA/DCPA/TiO2 group and sham group (control): CSDs filled by blood clot from surgical beds. During the experimental period, the animals were kept at a controlled-temperature (22 ± 2°C) and 12-hour light/dark cycle (7 a.m.-7 p.m.), with access to filtered water and food \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3.1 Surgical Procedure\u003c/h2\u003e \u003cp\u003eThe animals were anesthetized with 10% Ketamine Hydrochloride (100 mg/kg − 0.1 mL/100g, Ketamine Agener®, União Química Farmacêutica Nacional S/A. Embu-Guaçu /SP, Brazil), and 2% Xylazine Hydrochloride (10mg/kg − 0.05 mL/100 g, Anasedan®, Sespo Indústria e Comércio Ltda., Paulínia/SP, Brazil), intraperitoneally. Antibiotic prophylaxis was performed with Enrofloxacin 2.5% (Flotril® 2.5%, Intervet Schering-Plough, Rio de Janeiro/RJ, Brazil), at 10mg/weight of the animal in kg, subcutaneously, 1h before the surgical procedure. After anesthetic infiltration of 2% lidocaine hydrochloride with norepinephrine 1:100,000 (DFL Indústria e Comércio S/A, Rio de Janeiro, Brazil), a linear coronal incision of about 1.5 cm was made in the region between the ears. Periosteum skin was detached with the aid of a Molt detacher. Next, CSDs of 5mm diameters were made in the parietal bones, one to the right and another to the left of the median sagittal suture, with the aid of an electric motor (1500 rpm) and a pear-shaped multilaminate with 5 mm external diameter, maintaining the integrity of the meninges. The HA/DCPA or HA/DCPA/TiO2 biomaterials in the form of cement, with added CMC and RA, were inserted into the CSDs according to the experimental groups. In the sham group, no biomaterial was inserted, only a blood clot from the surgical bed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The flap was repositioned and the suture was performed in a single plane with simple interrupted stitches, using a 5 − 0 nylon thread.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 30, 60 and 90 days, the animals were euthanized with a lethal intraperitoneal dose of 10% ketamine hydrochloride (300 mg/kg − 0.3 mL/100g) and 2% xylazine hydrochloride (30mg/kg − 0.15 mL/100g). The calvaria were dissected and blocks containing the parietal bones with the critical-sized bone deffects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) were stored in 10% buffered formalin for histological processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.3.2 Histological Processing and Histomorphometric Analysis\u003c/h2\u003e \u003cp\u003eThe surgical specimens were decalcified in ethylenediamine tetraacetic acid (EDTA) and included in paraffin to obtain 07µm sections stained with hematoxylin and eosin. The slides were analyzed under light microscopy (Leica DM500 trinocular optical microscope), with the ICC50 HD photodocumentation system of the UFES Multiuser Histotechnical Laboratory, in 4X and 10X objectives. The reading was performed by the same observer, previously calibrated by an experienced professional. For histological analysis, inter- and intra-examiner reproducibility was calculated by the Kappa coefficient, following the criteria of Landis and Koch (1977) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]: weak (0–0.2), reasonable (0.21–0.40), moderate (0.41–0.60), substantial (0.61–0.80), almost perfect (0.81–1.0).\u003c/p\u003e \u003cp\u003eThe histomorphometric analysis was performed using ImageJ1.50i® software (Madison, USA). The measurement was performed in µm\u003csup\u003e2\u003c/sup\u003e with the aid of the \u003cem\u003eSet Measurements\u003c/em\u003e function of the \u003cem\u003eAnalyze\u003c/em\u003e tool to obtain measurements of total bone defect area, newly formed bone area, connective tissue area (including fibrous tissue, blood vessels, adipose tissue), remaining biomaterial area and total tissue repair area (sum of areas of newly formed bone, connective tissue and biomaterial remnant in the test groups), in relation to the total area of the originally created defect. The delimitation of the total area of the defect was obtained from the total extent of the defect (5mm) and using as limits the thickness of the calvaria for each photomicrograph (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the researcher’s calibration for the histomorphometric analysis, an intraclass correlation coefficient (ICC) intra-examiner test-retest was performed using IBM SPSS® Statistics version 24 software, with a significance level of 5% (p \u0026lt; 0.05). The interpretation of the obtained values followed the criteria of Cicchetti and Sparrow (1981) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3 SCANNING ELECTRON MICROSCOPY (SEM)\u003c/h2\u003e \u003cp\u003eThe microstructural analysis was performed by SEM in one operative specimen of each test group, in each observation period, and compared with the powdered biomaterial. The samples were metallized with a thin gold layer (Desk V, Denton Vacuum, NJ, USA) for scanning, made with the SEM (JSM-6610LV, JEOL Ltda., Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.4 STATISTICAL ANALYSIS\u003c/h2\u003e \u003cp\u003eThe independent samples ANOVA followed by Tukey's test was used to compare the percentages of newly formed bone, connective tissue and tissue repair area of HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e, HA/DCPA and sham intergroups, at each observation time. To compare the percentage of remaining biomaterial of HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e and HA/DCPA intergroups, Student's t-test for paired measurements was used. To compare the studied intragroup variables over the observation periods, ANOVA was used followed by the Bonferroni test. The significance level was 5% in all analyses. The program used in the analyses was IBM SPSS Statistics version 24.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e3.1 HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e COMPOUND CHARACTERIZATION\u003c/p\u003e\u003ch2\u003e3.1.1 Analysis of chemical composition and XRD\u003c/h2\u003e\u003cp\u003eThe chemical composition of titania is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It can be observed that the titania is almost pure (over 99%).\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical composition of TiO\u003csub\u003e2\u003c/sub\u003e in the anatase phase.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical compound\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChemical Composition (%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99,03\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAl\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,41\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,28\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,28\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the XRD for the ceramic compound 50% HA/DCPA + 50% TiO\u003csub\u003e2\u003c/sub\u003e, by mass, sintered at 1250°C. The main peaks of HA/DCPA and TiO\u003csub\u003e2\u003c/sub\u003e can be observed, as well as the chemical reaction between them, which produced perovskite (CaTiO\u003csub\u003e3\u003c/sub\u003e). TiO\u003csub\u003e2\u003c/sub\u003e is represented in two polymorphs: rutile and anatase. Two phases related to calcium phosphate were also identified: its beta phase (β-TCP) and calcium pyrophosphate (Ca\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\u003ch2\u003e3.1.2 Physical and mechanical tests\u003c/h2\u003e\u003ch2\u003e3.1.2.1 Apparent porosity, bulk density, water absorption and compressive strength\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results obtained for the proposed physical assays of AP, AD and WA through dry, wet and immersed weight data and through the application of equations 1 to 3, as well as the CS values for 50% HA/DCPA + 50% TiO2. These results can be compared with other findings in the literature for pure HA [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], HATiO\u003csub\u003e2\u003c/sub\u003e compounds [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and trabecular and cortical human bone [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e–\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysical and mechanical properties of the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e compound, compared with other bioceramics reinforced with titania and human trabecular and cortical bone described in the literature.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAuthor/Year\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBioceramic\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003ePhysical and mechanical properties\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDA (g/cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAA (%)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003ePA (%)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRC (MPa)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA/DCPA \u003cb\u003eOliveira et al. (2023)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eThis study\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84,5%\u003cb\u003eHA/ +\u003c/b\u003e15,5% \u003cb\u003eDCPA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,10 ± 0,03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e84,54 ± 3,41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56,69 ± 1,83\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6,09 ± 1,64\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e \u003cb\u003eThis study\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e50% HA/DCPA + 50%TiO\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,42 ± 0,04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78,36 ± 2,38\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e56,81 ± 0,65\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8,44 ± 1,10\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCésar et al. (2019)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50%HA + 50%TiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,66 ± 0,05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17,7 ± 2,9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e15,5 ± 2,0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3,5 ± 0,3\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGaldino and Zavaglia (2012a)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100% HA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2,4 ± 0,8\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50%HA + 50%TiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,51 ± 0,05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33,6 ± 2,3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e50,7 ± 2,0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5,5 ± 1,2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMorgan et al., 2018\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrabecular human bone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e40–95\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman cortical bone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e5–15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eReina et al., 2021\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrabecular human bone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman cortical bone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,85 − 2,0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMatsunake and Frantz, 2021\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrabecular human bone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2–12\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman cortical bone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100–150\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003e3.2 HISTOLOGICAL ANALYSIS\u003c/h2\u003e\u003cp\u003eAt 30 days, in the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e group, there were conglomerates of biomaterial granules inside the CSDs, which made the external surface more irregular, surrounded mainly by loose connective tissue. There was, predominantly, a formation of medullary centripetal bone from the margins and the internal limit of the critical-sized bone defect facing the meninges (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In the HA/DCPA group, multiple graft granules are evenly distributed in the defect area and surrounded predominantly by cellular connective tissue. In the central region of the defect image, newly formed bone areas can be observed in the middle of the granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eAt 60 days, in the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e group, there were clusters of biomaterial with different dimensions surrounded by newly formed bone tissue, predominantly medullary, in the central region of the defect, surrounded by loose connective tissue on the surface of the CSDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In the HA/DCPA group, the central area of the defect was filled with highly cellular connective tissue and with biomaterial granules distributed within it. There was newly formed bone from the surrounding regions all the way to the central area of the defect, especially at its internal limit (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eAt 90 days, the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e group presented a loose connective tissue band above the newly formed bone, with small biomaterial granules dispersed inside it. The centripetal bone grew and more bone filled the defect, in relation to the other observation periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In the HA/DCPA group, connective tissue covered the upper limit of the defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). There were graft granules distributed throughout the tissue repair area, surrounded by connective tissue in the most superficial portion of the defect and by bone tissue in the innermost areas. The graft granules were more evenly distributed throughout the defect than in the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e group.\u003c/p\u003e\u003cp\u003eIn the three experimental periods, the sham group showed newly formed bone areas distributed regularly and covered by cellular, fibrous connective tissue with a mild depression on top (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eIn general, the total tissue repair volume was maintained inside the defects in all experimental groups. In all periods, the HA/DCPA group had a more regular repair surface than the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e group. At the end of the experiment, it was possible to observe a greater volume of newly formed bone in the central region of the defect, in all three groups.\u003c/p\u003e\u003ch2\u003e3.3 HISTOMORPHOMETRIC ANALYSIS\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the graphical representation of the histomorphometric analysis results. At 30 days, the groups were similar to each other in relation to the variables studied (p \u0026gt; 0.05). At 60 days, there was a higher percentage of HA/DCPA connective tissue area (69.58 ± 10.82%) compared to that of HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e (47.52 ± 12.88%), and the difference was statistically significant (p = 0.027). The same was observed at 90 days, with a higher percentage of HA/DCPA connective tissue area (57.54 ± 16.37%) than that of HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e (37.06 ± 5.55%) (p = 0.001). In summary, at the end of the experiment, there was a difference in the connective tissue variables of the HA/DCPA and HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e groups. It is noteworthy that the total tissue repair areas in both experimental groups completely filled the CSDs, with percentage means higher than 100% (HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e = 103.88 ± 21.02% and HA/DCPA = 120.35 ± 17.51%) and significantly higher than that of the sham group (p \u0026lt; 0.001).\u003c/p\u003e\u003ch2\u003e3.4 SCANNING ELECTRON MICROSCOPY AND ENERGY DISPERSIVE X-RAY SPECTROSCOPY\u003c/h2\u003e\u003cp\u003eIn the images obtained by SEM, it Is possible to observe an irregularly shaped and porous biomaterial with granular morphology, similar in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In greater increase, the result obtained on the surface of the granulated biomaterial can be observed, revealing the interconnected microporous microstructure formed by fine grains. There is also the presence of newly formed bone and biomaterial remnants in the critical-sized bone defect area. After selecting a random location in the SEM images, EDS was used to identify the chemical composition of the samples at 30, 60 and 90 days, revealing oxygen, calcium, phosphorus and titanium as the main chemical elements of hydroxyapatite, monetite and titania (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results suggest a heterogeneous percentage distribution of these chemical elements, with evidence of their concentration in certain regions.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe calcium phosphate obtained from chicken eggshells is an innovative alternative and comes from an inexhaustible source of calcium carbonate with low cost and a safe production process, since it does not require chemicals used in the production of other phosphates, considered dangerous and corrosive in their raw states [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The addition of TiO\u003csub\u003e2\u003c/sub\u003e particles to calcium phosphates has attracted the attention of researchers, since TiO\u003csub\u003e2\u003c/sub\u003e can increase osteoblast adhesion and induce cell growth [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Considering that although the HA/DCPA compound presents better reabsorption and stability than does pure HA, its low mechanical resistance still limits its clinical application. Thus, TiO\u003csub\u003e2\u003c/sub\u003e addition can contribute to improve the mechanical characteristics of the biomaterial and bone tissue regeneration, by presenting values of resistance strength, elastic modulus and porosity close to those of trabecular bone [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Galdino and Zavaglia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] observed that the addition and sintering of TiO\u003csub\u003e2\u003c/sub\u003e to pure HA results in increased mechanical compressive strength.\u003c/p\u003e \u003cp\u003eThe ideal biomaterial should have dimensional accuracy, adequate CS and not deform easily under compression. The 8.44 ± 1.10 MPa CS of HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e met the criteria for trabecular bone compression [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e–\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] to receive masticatory pressure [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, it would not be indicated for applications in cortical bones, such as the femur, because it does not reach the values between 100 MPa and 150 MPa [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to our findings, with the addition of TiO\u003csub\u003e2,\u003c/sub\u003e there was a 38.6% increase in the CS of HA/DCPA, in addition to the increase in AD, maintenance of AP and reduction of WA. These data are corroborated by Galdino and Zavaglia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and César \u003cem\u003eet al\u003c/em\u003e, who observed that the sintering of TiO2 to pure HA increased the CS of the biomaterial. It is worth mentioning that the CS values of HA/DCPA/TiO2 were higher than those achieved by César et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] for HA/TiO2 although the specimens presented close AD values and were more porous (\u0026gt; AP). The different TiO2 phases used in this study, rutile and anatase, have different crystalline characteristics, which interfere with the properties of TiO2 and probably guarantee superior results. Because it is a metastable polymorph at high temperatures (approximate range between 400°C and 1200°C), anatase irreversibly transforms into rutile, a thermodynamically stable polymorph [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. As this transformation is a function of time and temperature, the sintering process was not long enough to transform the entire anatase titania into rutile titania. The mixture of rutile and anatase phases possibly contributed to a CS increase and helped to obtain different calcium phosphates: the beta phase of tricalcium phosphate (β-TCP) and calcium pyrophosphate (Ca\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003eP\u003csub\u003e2\u003c/sub\u003e). These phases occur due to the calcium phosphate synthesis method, in which crystalline β-TCP forms above 1125°C and is widely used as a ceramic biocomposite.\u003c/p\u003e \u003cp\u003eConsidering that this composite is intended for use in tissue bioengineering, pores with adequate dimensions, shapes, quantity and interconnectivity are needed to favor tissue growth, intertwining the newly formed bone with the scaffold, increasing the CS of the \u003cem\u003ein vivo\u003c/em\u003e biomaterial [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The HA/DCPA granules are composed of finely distributed spherical particles of less than 30 µm. Powdered biomaterials containing round particles with an average size of 2–20 µm are ideal for use in the form of cement [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The HA/DCPA presented 56.69 ± 1.83 AP and the addition of TiO2 maintained the porosity of the HA/DCPA/TiO2 compound, which remained within the 45–95% range of trabecular bone [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is important to highlight that although porosity is similar for the two composites, the HA/DCPA/TiO2 presented higher AD and CS values due to the presence of TiO2. The HA/DCPA/TiO2 also presented higher AP values in relation to the HA/TiO2 of Galdino and Zavaglia’s studies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and even higher values in comparison to the findings of César et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It is noteworthy that the manufacturing processes of the specimens were different, since Galdino and Zavaglia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] used the polymeric sponge method and César et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] used polymeric wax to generate porosity during the sintering of specimens manufactured by uniaxial pressing. In our study, the specimens were manufactured by uniaxial pressing, but with a low compaction load.\u003c/p\u003e \u003cp\u003eAs for the chemical analysis, the formation of perovskite was also observed by Galdino and Zavaglia [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and Assmar et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], who developed porous ceramic HA/TiO\u003csub\u003e2\u003c/sub\u003e compounds by different methods, formed in all compounds sintered at 1250°C, 1300°C and 1350°C. These phases occur due to the calcium phosphate synthesis method, in which crystalline β-TCP forms above 1125°C and is widely used as a ceramic biocomposite, since it promotes bone growth. The formation of calcium pyrophosphate by the wet method used in the present study was also observed in other studies [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e–\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEDS provided information on the oxygen, calcium, phosphorus and titanium present in HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e granules and adjacent tissues. The energy dispersion generated by the titanium suggests the successful incorporation of this element into the biomaterial [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although EDS identified the presence of Ca and P, these elements can be both from biomaterial and newly formed bone [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], requiring \u003cem\u003ein vivo\u003c/em\u003e histological and histomorphometric analysis to identify and quantify them.\u003c/p\u003e \u003cp\u003eThe photomicrographs demonstrated that the biomaterial is safe for clinical use due to the absence of significant adverse effects, such as osteolytic reactions or persistent inflammatory processes, which evidences good biocompatibility and interaction with the native calvarial bone. During bone repair, an initial inflammatory process followed by new bone formation and a subsequent bone matrix remodeling process is a normal occurrence [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. With the histological analysis, newly formed bone with an immature appearance could be observed, accompanied by intense vascular formation and irregularly organized collagen fibers, without the presence of Havers channels with well-defined morphology. Over the observed periods, the bone acquired a mature appearance, making it difficult to identify the line of intersection between it and the native bone and possible to identify the presence of osteocytes.\u003c/p\u003e \u003cp\u003eHA presents slow biodegradation and can be gradually reabsorbed 4 to 5 years after implantation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Since the resorption of the material and its replacement by bone tissue are desired characteristics, the association of HA with DCPA aims to increase reabsorption. Tamimi et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] showed that DCPA shows signs of graft resorption as the newly formed bone tissue grows, 8 weeks after its implantation, surrounding and penetrating DCPA granules. Comparing the resorption process of the material over the three observation periods, we noticed a slight reduction of the HA/DCPA biomaterial and a similar resorption pattern in the intergroup comparison. However, even without differences, it should be considered that the remaining HA/DCPA and HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e granules influence the greater volume and maintenance of the bone defect contour when compared to the sham, because the granules are surrounded by connective tissue and newly formed bone [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which may explain why the total tissue repair area in these groups was larger than that of the sham group (p \u0026lt; 0.001).\u003c/p\u003e \u003cp\u003eBoth biomaterials in the form of cement have the advantage of ease of handling and insertion in the experimental cavities, which allowed the biomaterial to adapt and helped to control the volume of the grafted material. In general, calcium phosphate cement paste allows injection, solidification and \u003cem\u003ein situ\u003c/em\u003e modeling of complex and vertical bone cavities, besides being a great option for minimally invasive surgery [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The possibility of using a porous biomaterial in the form of cement paste brings together two configurations that can contribute to tissue repair. The granule porosity of between 40% and 60% can promote rapid diffusion or flow of nutrients and cell-biomaterial interactions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The cement in paste form has biomaterial clusters, which were histologically observed, in its morphology, and this configuration could both stimulate collagen fibers and release calcium ions for new bone formation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are no previous studies in the literature that have evaluated the effects of HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e on bone regeneration. We believe that future research to evaluate the use of TiO\u003csub\u003e2\u003c/sub\u003e particles at different mass percentages and in other bone sites is necessary for our findings to be confirmed. Thus, there will be greater safety and efficacy in its application in clinical practice. It is worth mentioning that the combination of TiO\u003csub\u003e2\u003c/sub\u003e with other types of phosphate provides better quality in the bone regeneration process, impacts clinical management and provides new treatments for acquired or congenital bone defects.\u003c/p\u003e \u003cp\u003eAccording to the results obtained, both biomaterials are promising as bone defect fillers. Nevertheless, the scaffolds used in bone tissue engineering must be able to provide mechanical strength similar to natural bone tissue [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], when in function. Regarding their application in areas subject to load, despite the CS increase obtained with the addition of TiO\u003csub\u003e2\u003c/sub\u003e to HA/DCPA, the tests were performed \u003cem\u003ein vitro\u003c/em\u003e on specimens, and future studies may investigate the mechanical strength of these biomaterials after \u003cem\u003ein vivo\u003c/em\u003e implantation.\u003c/p\u003e "},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e presented higher densification and compressive strength rates than the HA/DCPA. Biocompatibility and bone conductivity denote the potential of this bioceramic as a framework for bone engineering, with \u003cem\u003ein vitro\u003c/em\u003e mechanical resistance similar to that of trabecular bone.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003e \u003cb\u003eConflicts of interest\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNom-financial interests\u003c/strong\u003e \u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL.K.R.O: Methodology- Development of all stages of the experiment; Collection, interpretation and analysis of data; Writing, Review and Edition; Approval of the final version and submission of the article.C.D.N.N: Methodology- Surgical stage of the animals experiment, approval of the final version of the article.A.B.C.S: Methodology- Surgical stage of the animals experiment , approval of the final version of the article.S.M.W.R: Conceptualization; Design of methodology, definition of the theme; Critical analysis and approval of the final version of the article.P.R.B: Methodology - Surgical stage of the animals experiment; Critical analysis and approval of the final version of the article.A.G.S.G: Conceptualization; Design of methodology; Definition of the theme/subject; Critical analysis and approval of the final version of the article.D.N.S: Conceptualization; Design of methodology ; Supervision; Project administration; Interpretation and analysis of data; Critical analysis and approval of the final version of the article.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eLaboratories: Carbonaceous and Ceramic Materials/UFES Thermal Plasma Laboratory; Mechanical Tests at the Federal Institute of Esp\u0026iacute;rito Santo (IFES \u0026ndash; Vit\u0026oacute;ria-ES); Cellular Ultrastructure Carlos Alberto Redins; Multiuser of Histotechnics at UFES; and cardiac electromechanics and vascular reactivity of UFES\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuastaldi AC, Aparecida AH (2010) Fosfatos de c\u0026aacute;lcio de interesse biol\u0026oacute;gico: import\u0026acirc;ncia como biomateriais, propriedades e m\u0026eacute;todos de obten\u0026ccedil;\u0026atilde;o de recobrimentos. 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Braz Dent J. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1590/0103-6440201802017\u003c/span\u003e\u003cspan address=\"10.1590/0103-6440201802017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeimouri T, Abnous K, Taghdisi SM et al (2024) Protein-Based Hybrid Scaffolds: Application in Bone Tissue Engineering. J Polym Environ. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10924-024-03264-y\u003c/span\u003e\u003cspan address=\"10.1007/s10924-024-03264-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chicken Egg, Bone Cements, Bone Substitutes, Skull, Bone Regeneration","lastPublishedDoi":"10.21203/rs.3.rs-4807871/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4807871/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eto carry out physicomechanical characterization of the HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e and to evaluate the tissue repair in rat calvaria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo bone defects were made in the calvaria of 36 Wistar rats, divided into groups: HA/DCPA, HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e and sham (blood clot). The animals were euthanized at 30, 60 and 90 days and calvaria slides processed with hematoxylin/eosin. The newly formed bone, connective tissue, biomaterial remnant and total tissue repair percentages were calculated in relation to the total defect area. The HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e was characterized structurally by scanning electron microscopy (SEM), and chemically by energy-dispersive X-ray spectroscopy (EDS) and \u003cem\u003eX-ray diffraction\u003c/em\u003e (XRD). It was submitted to apparent density (AD), apparent porosity (AP), water absorption (WA) and compressive strength (CS) physical tests. The ANOVA test was applied, followed by Turkey’s test and \u003cem\u003eStudent’s\u003c/em\u003e t test (p ≤ 0,05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SEM showed biomaterials inside the bone defects and newly formed bone. EDS identified oxygen, calcium, phosphorus and titanium in the sample. The HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e and HA/DCPA groups presented a total tissue repair area was larger than the sham group (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physical-mechanical assays showed that HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e has AD and CS properties within the limits of trabecular bone and with values higher than HA/DCPA.HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e presented higher densification and compressive strength rates than HA/DCPA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Relevance\u003c/strong\u003e: Both biomaterials are promising as bone defect fillers. The HA/DCPA/TiO\u003csub\u003e2\u003c/sub\u003e has potential as a scaffold for bone to application in areas subject to load.\u003c/p\u003e","manuscriptTitle":"Physicochemical Characterization and Effects of Monetite Obtained from Titania-Reinforced Eggshell on Bone Repair: A New Possibility for Tissue Bioengineering?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-27 06:46:39","doi":"10.21203/rs.3.rs-4807871/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-06T12:09:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-06T12:08:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75084652593237150120049355979361479737","date":"2024-11-06T12:07:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-22T11:05:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186096861931122401548275467524865429747","date":"2024-10-08T19:13:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-08T06:48:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-30T14:56:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-30T14:56:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Oral Investigations","date":"2024-07-26T11:59:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-oral-investigations","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cloi","sideBox":"Learn more about [Clinical Oral Investigations](http://link.springer.com/journal/784)","snPcode":"784","submissionUrl":"https://submission.nature.com/new-submission/784/3","title":"Clinical Oral Investigations","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"919dc695-a3a4-4464-b83e-f59da8d2ddb5","owner":[],"postedDate":"August 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T15:59:09+00:00","versionOfRecord":{"articleIdentity":"rs-4807871","link":"https://doi.org/10.1007/s00784-025-06195-7","journal":{"identity":"clinical-oral-investigations","isVorOnly":false,"title":"Clinical Oral Investigations"},"publishedOn":"2025-02-04 15:56:58","publishedOnDateReadable":"February 4th, 2025"},"versionCreatedAt":"2024-08-27 06:46:39","video":"","vorDoi":"10.1007/s00784-025-06195-7","vorDoiUrl":"https://doi.org/10.1007/s00784-025-06195-7","workflowStages":[]},"version":"v1","identity":"rs-4807871","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4807871","identity":"rs-4807871","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}