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

doi:10.21203/rs.3.rs-4807871/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-4807871/v1

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Objectives

to carry out physicomechanical characterization of the HA/DCPA/TiO₂ 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/TiO₂ 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/TiO₂ 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/TiO₂ 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/TiO₂ has AD and CS properties within the limits of trabecular bone and with values higher than HA/DCPA.HA/DCPA/TiO₂ presented higher densification and compressive strength rates than HA/DCPA.

Clinical Relevance: Both biomaterials are promising as bone defect fillers. The HA/DCPA/TiO₂ has potential as a scaffold for bone to application in areas subject to load.

Chicken Egg

Bone Cements

Bone Substitutes

Skull

Bone Regeneration

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 - CaHPO₄) 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 (TiO₂), 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 TiO₂ nanoparticles as a reinforcing agent during the development of hybrid materials can enhance the physicochemical properties of bioceramics. Thus, this study associated TiO₂ with HA/DCPA as produced by Zanelato et al. [7] developed the HA/DCPA/TiO₂ 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/TiO₂ and evaluate the in vivo effect of this composite in the tissue repair process.

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/TiO₂ powder, TiO₂ (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. 1A). 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. 1B) 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 µm² 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/TiO₂, HA/DCPA and sham intergroups, at each observation time. To compare the percentage of remaining biomaterial of HA/DCPA/TiO₂ 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.

3.1 HA/DCPA/TiO₂ 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 TiO₂ in the anatase phase.
Chemical compound	Chemical Composition (%)
TiO₂	99,03
Al₂O₃	0,41
SiO₂	0,28
P₂O₅	0,28

Figure 3 shows the XRD for the ceramic compound 50% HA/DCPA + 50% TiO₂, by mass, sintered at 1250°C. The main peaks of HA/DCPA and TiO₂ can be observed, as well as the chemical reaction between them, which produced perovskite (CaTiO₃). TiO₂ 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 (Ca₂O₇P₂).

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], HATiO₂ compounds [3, 16] and trabecular and cortical human bone [17–20].

Table 2

Physical and mechanical properties of the HA/DCPA/TiO₂ 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
Author/Year	Bioceramic	DA (g/cm³)	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/TiO₂ This study	50% HA/DCPA + 50%TiO₂	1,42 ± 0,04	78,36 ± 2,38	56,81 ± 0,65		8,44 ± 1,10
César et al. (2019)	50%HA + 50%TiO₂	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
Galdino and Zavaglia (2012a)	50%HA + 50%TiO₂	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		-
Morgan et al., 2018	Human cortical bone	2,0	-	5–15		-
Reina et al., 2021	Trabecular human bone	1,18	-	-		-
Reina et al., 2021	Human cortical bone	1,85 − 2,0	-	-		-
Matsunake and Frantz, 2021	Trabecular human bone	-	-	-		2–12
Matsunake and Frantz, 2021	Human cortical bone	-	-	-		100–150

3.2 HISTOLOGICAL ANALYSIS

At 30 days, in the HA/DCPA/TiO₂ 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. 4A). 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. 4B).

At 60 days, in the HA/DCPA/TiO₂ 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. 4D). 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. 4E).

At 90 days, the HA/DCPA/TiO₂ 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. 4G). In the HA/DCPA group, connective tissue covered the upper limit of the defect (Fig. 4H). 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/TiO₂ 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. 4C, 4F and 4I).

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/TiO₂ 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/TiO₂ (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/TiO₂ (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/TiO₂ 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/TiO₂ = 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.

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 TiO₂ particles to calcium phosphates has attracted the attention of researchers, since TiO₂ 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, TiO₂ 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 TiO₂ 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/TiO₂ 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 (Ca₂O₇P₂). 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/TiO₂ 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/TiO₂ 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/TiO₂ 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/TiO₂ on bone regeneration. We believe that future research to evaluate the use of TiO₂ 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 TiO₂ 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 TiO₂ 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.

The HA/DCPA/TiO₂ 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.

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

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

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

2.1 BIOMATERIALS

2.2 CHARACTERIZATION

2.2.1 Physical tests

2.2.2 Chemical tests

Energy-Dispersive X-Ray Spectroscopy (EDS)

X-ray Diffractometry (XRD)

2.3.1 Surgical Procedure

2.3.2 Histological Processing and Histomorphometric Analysis

3 SCANNING ELECTRON MICROSCOPY (SEM)

2.4 STATISTICAL ANALYSIS

RESULTS

3.1.1 Analysis of chemical composition and XRD

3.1.2 Physical and mechanical tests

3.1.2.1 Apparent porosity, bulk density, water absorption and compressive strength

3.2 HISTOLOGICAL ANALYSIS

3.3 HISTOMORPHOMETRIC ANALYSIS

3.4 SCANNING ELECTRON MICROSCOPY AND ENERGY DISPERSIVE X-RAY SPECTROSCOPY

DISCUSSION

CONCLUSIONS

Declarations

Conflicts of interest:

Author Contribution

Acknowledgement

References

Additional Declarations

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