
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12524-8
10.1016/j.heliyon.2024.e36493
e36493
Research Article
Eggshell derived scaffold of hydroxyapatite-ammonium bicarbonate nano-composite: Bioactivity and cytotoxicity studies
Oladipupo Oladoyinbo Fatai a
Adekola Adesokan Hameed a
Ofudje Edwin Andrew ofudjeandrew4real@yahoo.com
b⁎
Al-Ahmary Khairia Mohammed c
Al-Mhyawi Saedah R. c
Alshdoukhi Ibtehaj F. d
Alrahili Mazen R. e
Alsaiari Ahad Amer f
a Department of Chemistry, College of Physical Sciences, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria
b Department of Chemical Sciences, Mountain Top University, Ogun State, Nigeria
c Department of Chemistry, College of Science, University of Jeddah, Jeddah, Saudi Arabia
d Department of Basic Sciences, College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Science, King Abdullah International Medical Research Center, Riyadh, Saudi Arabia
e Physics Department, School of Science, Taibah University, Medina, 42353, Saudi Arabia
f Department of Clinical Laboratory Science, College of Applied Medical Science, Taif University, Taif, Saudi Arabia
⁎ Corresponding author. ofudjeandrew4real@yahoo.com
25 8 2024
15 9 2024
25 8 2024
10 17 e364931 6 2024
15 8 2024
16 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
This work investigated the facile synthesis of porous scaffold eggshell derived hydroxylapatite (ESHAp) as a composite with ammonium bicarbonate (AMB) for potential biomaterial in tissue engineering application. The phase purity, composition, size, functional groups and morphology of the apatite were elucidated using high resolution transmission electron microscopy (HTEM), X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FT-IR) and scanning electron microscopy (SEM). The results showed that hydroxylapatite (HAp) nanoparticles have round morphologies with average diameters between 20 nm and 80 nm, FT-IR analysis confirmed significant hydroxylapatite functional groups like carbonate, phosphate, and hydroxyl groups, while XRD analysis revealed a well crystalline monophasic HAp powder. The scaffold samples containing 10, 20, 25 and 30 % of AMB withstood a compressive stress up to 5, 20, 30 and 42 N/mm2 respectively which indicates that the compressive stress increased with the AMB content introduced as the pore forming agent. MTT assay performed using MG63 osteosarcoma cell lines showed that on comparing the sample of ESTHAp which contained 0 % AMB with other samples in the range of 0.01–1 mM, viability of above 85 % MG63 cells was achieved except for ESTHAp with 40 % AMB, which showed some level of toxicity. The cell adhesion studies of sintered ESTHAp porous scaffold with different weight percent of the pore forming agents using inverted microscopic images of MG 63 cells incubated with ESTHAp samples and treated with heat at 1000 °C appeared to be unstable in the media used with particle leaching observed, and no cells observed near to the samples.

Keywords

Bioactivity
Compressive stress
Biomedical engineering
Eggshell
Hydroxyapatite
==== Body
pmc1 Introduction

Conversion of waste such as Eggshell to resourceful biomaterial like Hydroxyapatite is a led-light to the realization of Sustainable Development Goal (SDG) 6 (Clean environment) and 11 (sustainable cities and communities), United Nation vision 2030. Hydroxyapatite which is normally abbreviated as HAp is the chief mineral constituent of human hard tissues has a molecular formula of Ca10(PO4)(OH)2 [1,2]. Synthetic hydroxyapatite is widely utilized as effective material for repairing damaged teeth and bones, drug delivery agent and dental applications as a result of its resemblance with key mineral constituent of hard tissue in human body which is as a result of its excellent osteoconductivity, biocompatibility and bioactivity [[1], [2], [3], [4]]. Moreso, variety of chemical reactions utilized fabricated hydroxyapatite (HAp) as a possible adsorbent, catalyst or as a catalyst support [2,5]. Besides, HAp powder can as well be used as an excellent adsorbent for the remediation of metals and organic contaminants due to its strong adsorptive property [6]. The synthesis of non-stoichiometric apatite for biomedical applications is on the increase since pure apatite only co-exists with its derivatives in any biological system [7,8]. Substituted hydroxyapatite are formed via chemical substitution of calcium ions (Ca2+), phosphate (PO43−) or hydroxyl (OH−) with other ions such as F−, Na+, Mg2+, K+, Cl−, and CO32−.

Several synthetic routes for the preparation of HAp has been discussed among which include sol–gel [9], aqueous precipitation [10,11], hydrothermal technique [12] and solid-state reactions [13,14]. However, commercially available HAp is very expensive as a result of the high purity of chemicals involves in the fabrication of HAp and also the possibility of toxicity to cell arising from the incorporation of desired ions. Recently, biological derived HAp from the components of natural materials such as bone, shell and plants have been documented in literature [2,8,[15], [16], [17]]. For instance, Shih-Ching et al. [2] reported the formation of HAp phase via ball milling followed by calcinations to form a biphasic calcium phosphate after 5 h of ball milling and 1 h of heat treatment at 1000 °C. Also, Aseel and Nihad [18] used calcium oxide from eggshell which was calcined at 1000 °C and reacted with phosphoric acid in ratio 1:1 to produced HAp through mechanical milling. Leyla et al. [19] adsorbed heavy metal with HAp with the same precursor in ratio 1:2 via precipitation method. Higher crystallinity and smaller particles size with HAp from eggshell were found by Sandile et al. [20] when compared with HAp extracted from fish scales. Though, there have been reports on the synthesis of HAp powder from waste ago-materials as mentioned above, the fabrication of porous scaffold nano-composite to enhance the cell diffusion during implant and the detailed study of their toxicity has not been well explored. Thus, this work tends to utilize agro-waste of eggshell for the production of porous scaffold hydroxyapatite-ammonium bicarbonate nano-composite.

To reduce and utilize these wastes, Nongnuch et al. [21] reported the use of eggshell wastes for the production of low- or high-purity calcium carbonate grades via washing, crushing, and after drying, was utilized as precursor for the fabrication of calcium phosphate. The waste eggshell was used to fabricate dicalcium phosphate dihydrate, monocalcium phosphate monohydrate, and tricalcium phosphate respectively. Eggshells are waste materials which are readily available in larger amounts in Nigeria and their presence in the environmental often constitute nuisance which could cause the growth and spread of microorganisms because they contain organic content which could leads to other environmental challenges. Herein, eggshells waste which is often discarded as useless materials will be utilized as source of calcium in the production of hydroxyapatite biomaterial that can be deployed in biomedical application, thus, adding values to agricultural wastes. Since the use of chemicals for the synthesis of HAp could pose environmental challenges and also very expensive, the use of readily available waste agricultural materials like eggshells could serves as better alternatives.

In this present study, our aim was to fabricate HAp powder from waste eggshells which serve as calcium source and reacted with ammonium dihydrogen phosphate {(NH4)H2(PO4)} solution via wet chemical method to form a scaffold nano-composite. The structure of the fabricated HAp-scaffold nano-composite was investigated with various analytical tools like XRD, SEM, TGA/DTA, EDX, FT-IR and TEM. Ammonium bicarbonate was used as pore forming agent in the production of porous scaffolds HAp to demonstrate its usefulness in tissue engineering and biological applications. The bioactivity and biocompatibility assessment of the HAp-scaffold nano-composite powder was also demonstrated.

2 Materials and methods

2.1 Chemicals and materials

The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, Dimethyl sulfoxide (DMSO), Phosphate-buffered saline (PBS), Ca(OH)2, Ammonia, ammonium bicarbonate, simulated body fluid, (NH4)H2PO4, and ELISA plate used were purchased from Sigma, India which are of analytical grade and used without further purification.

2.2 Synthesis of calcium source

Uncrushed eggshells were sourced locally from Abeokuta, Ogun State of Nigeria and boiled for 30 min with distilled water and filtered. After hot water treatment, the shell was oven dried at 100 °C for 8 h. The dried samples were crushed into smaller pieces and calcined in a box furnace at a temperature of 800 °C at 5 °C/min in three stages of calcination. The removal of organic matrix as well as the decomposition of the calcium carbonate of the eggshell into calcium oxide was achieved by maintaining the temperature for 3 h. The CaO powder produced was cooled to room temperature and kept for the synthesis of hydroxyapatite.

2.3 Synthesis of hydroxyapatite from calcined eggshells

0.75 M solution of Ca(OH)2 was made and placed in a 500 mL beaker on a hot plate and stirred for 30 min with a magnetic stirrer. This was followed by stepwise addition of 0.45 M solution of (NH4)H2PO4 and stirring for 40 min. Ammonia solution was used for the adjustment of the solution pH after which the mixture was left on hot plate with continuous stirring for 24 h at room temperature for the development of apatite. The product formed was centrifuged, while excess ammonia solution was removed by washing severally with distilled water. The hydroxyapatite formed was oven dried for 8 h at a temperature of 150 °C followed by pulverization to formed amorphous HAp powder. Fig. 1 shows the schematic synthesis procedure of HAp-nano composite powder from eggshells. The expressions for the synthesis are shown in equations 1 to 3 follows [22].(1) Eggshells(CaCO3)→CalcinationCaO+CO2

(2) CaO + H2O → Ca(OH)2

(3) 10Ca(OH)2 + 6(NH4)2HPO4 → Ca10(PO4)6(OH)2+6H2O+12NH4OH

2.4 Scaffold preparation and bioactivity study

Scaffolds hydroxyapatites were prepared by adjusting different weight percent of ammonium bicarbonate (AMB) which serves as the pore forming agent. The AMB were added in the ratio of 0, 10, 20, 30 40 and 50 wt % to 100, 90, 80, 70, 60 and 50 wt % of HAp. The mixture was then subjected to sintering at 600, 800, 1000, and 1200 °C and thereafter characterized using scanning electron microscope, while the pore diameters were evaluated using mercury porosimeter. After sintering, the bulk density of the sintered pellet was calculated using equation (4) below [23]:(4) B.D=mV

where, B.D is the bulk density in g/cm3, m is the mass in g and V is the volume in cm3. The volume V of the lattice was estimated using equation (5) [23]:(5) V=πr2h

Where r is the radius of the sintered pellet and h is the height (thickness). The densification of the synthesized scaffold was evaluated using equation (6) below [23]:(6) Densification=B.DT.D×100

where theoretical density of HAp is (3.15 g/cm3).

The porosity was evaluated by adopting equation (7) below [23]:(7) Porosity=(1−B.DT.D)×100

2.5 Characterization

Structural investigation of treated eggshells and as-prepared HAp powder were performed with several analytical tools. The XRD patterns of all the samples were measured using Bruker D8 Advance X-ray Diffractometer with Cu Kɑ (λ = 1.5405 Å) radiation in the 2θ range from 20 to 60°. To identify the functional groups found in ESTHAp, a Bruker spectrometer (Model no. TENSOR 27, Germany) of Fourier Transform infrared (FT-IR) was utilized. After mixing the sample with potassium bromide (KBr) to create a pellet, the 400–4000 cm−1 spectrum was recorded. To determine the structure and morphology of samples, field emission scanning electron microscopy (FE-SEM) characterization was done. For this, a Carl Zeiss Supra 55VP microscope with an accelerating voltage range of 0.1–30 kV was utilized. A FEI Tecnai G220 (Netherland) with an acceleration voltage of 200 kV was used to study the internal morphology and structural characteristics of the samples using Transmission electron microscopy (TEM) and Scanning TEM (STEM-EDX) with elemental mapping. Thermogravimetric analysis (TGA) was performed using SDT Q600 V8.3 Build 101simultaneous DSC-TGA instrument to measure the thermal stability of the biomaterial prepared.

Evaluation of average crystallite size of the prepared apatite powder was done using the Scherrer's formula as given in equation (8) below [24]:(8) XC=0.9λFWHMcosθ

where, Xc denotes the crystallite size in nm, λ denote the diffraction wavelength (0.154 nm) and the diffraction angle is given as θ. Since hydroxyapatite is hexagonal crystal, the crystal parameters were evaluated using the equation below [24]:(9) 1d(hkl)={4(h2+k2+kh)3a2+l2c2}1/2

where the distance between adjacent planes of the Miller indices (h k l) is given as d, and the plane at (002) was used for this purpose and a, b and c measure the dimensions of the unit volume and denote the lattice parameters.

2.6 In vitro bioactivity study

In order to create the simulated body fluid (SBF), which had ion concentrations almost identical to those of human blood plasma as reported by Kokubo and Takadama [25], reagent-grade chemicals were dissolved in de-ionized water and buffered to pH 7.4 at 37 °C using tris(hydoxylmethyl) aminomethane and 1mol HCl. In SBF, scaffolds pellets were put in an abrade bottle filled with 150 ml SBF. A shaking water bath was used to maintain the SBF temperature at 37 °C. Following a week-long submersion, the samples were taken out of the SBF, carefully cleaned with deionized water, and allowed to air dry. The surface morphologies of pellets were examined using SEM, while the changing in the functional groups was examined with FT-IR.Fig. 1 Flow chart for the synthesis of ESTHAp from eggshell.

Fig. 1

2.7 Pellet density determination

Density for all the successfully pelletized sampled at 50 kg/cm pressure was measured using Schimadzu balance density measurement kit. By employing Archimedes's principle, the density of each material was extrapolated using equation (10) below [22]:(10) Density=W1W1−W2

Where W1 = Weight of pellet in air.

W2 = Weight of pellet in water.

2.8 Assessment of biocompatibility and cell growth

To assess the biocompatibility of porous ESTHAp scaffolds with different percentage of pore forming agent, an MTT assay was carried out using the conventional protocol. ESTHAp pellet samples (with 0–40 %) of ammonium bicarbonate (AMB) pore forming agent were placed into compatible size tissue culture dish and cells were seeded on them at a density of 1 × 104 cells/well. The cells were kept alive in a CO2 incubator with the previously mentioned medium for 72 h. After that, the medium was changed, and each well was incubated for an extra 4 h with the addition of 100 μL of MTT (0.5 mg/mL). The resultant purple formazan crystals were dissolved in DMSO, and an ELISA plate reader (BIORAD) was used to quantify the absorbance at 570 nm. The vitality percentage of cells cultured with 0 % AMB ESTHAp pellets and various AMB modified scaffolds was determined by comparing its values to 100 % of control cells cultured without ESTHAp pellets.

2.9 Cell adhesion studies

Following the protocol as described [26], immunostaining with an antibody against the cytoskeletal marker αtubulin (Sigma) was done to further confirm cell adhesion onto the surface of the 0 % AMB ESTHAp pellets and it's various AMB modified scaffolds, as well as to determine whether they displayed normal growth characteristics. In short, the cell-filled ESTHAp pellets were blocked with 5 % fetal bovine serum (FBS) for 1 h at room temperature, rinsed with PBS, and fixed with 4 % paraformaldehyde for 20 min. Using the specified antibody, the cells were incubated for two to 3 h at room temperature in order to carry out the immunostaining process. After that, the pellets underwent three PBS rinses before being incubated with the suitable secondary antibody coupled with Cy3 and 4,6-diamidino-2-phenylindole (DAPI), which serves as the nuclear stain. A second PBS rinse was performed following a 1-h incubation period. After mounting the cell-filled pellets on a cover glass, photos were taken at 63 × magnification using a laser scanning spectral confocal microscope (Leica TCS SP2).

2.10 Characterization of the cells

A variety of distinct cluster-of-differentiation (CD) markers that are expressed on the surface of MSCs are typically used to identify them. Flow cytometry detection and quantification of the same were done to confirm if cells cultivated in the presence of 0 % AMB ESTHAp pellets and its various AMB modified scaffolds would keep their intrinsic antigen expression feature. At the prescribed doses from the manufacturer, the cells were stained with antihuman CD29-PE, CD73-PE, and CD44-FITC antibodies (all from BD Pharmingen) or their corresponding isotype-matched controls (Biolegend). Summarily, the cells were taken out, tallied, and then incubated for 1 h at 4 °C in the dark with sporadic mixing in the presence of the appropriate antibodies. Subsequently, the cells were cleaned three times with PBS, collected by centrifugation, and taken for flow cytometry after resuspending the pellet in PBS. About 10 000 events were acquired using a BD FACS Calibur (BD Biosciences). The Becton Dickinson CellquestPro software was used to evaluate the data.

2.11 Scaffold compressive strength determination

In order to investigate the mechanical properties of the 1400 °C calcined ESTHAp scaffold samples with diﬀerent percentage of pore forming agents. Sample with porosity in close proximity with the theoretical (50–60 %) porosity of fabricated tissue was used for the test. In a typically test, samples with 55 % porosity was uniaxially compressed (PCi Hydraulic press, CAP.- 15T) into cylindrical specimen with 13.17 mm diameter and a h/d ratio of 1.2 in a stainless steel die applying a pressure of 50 kgcm−2 for 60s. The prepared scaffolds were sintered at 1000, 1200 and 1400 °C for 3h (holding time) with a heating and cooling rate of 5 °C/min. These molded cylindrical samples were brought forward for a compression test, which was conducted on a minimum of five samples per group using a universal testing machine (Shimadzu (Asia Pacific) Pvt. Ltd., Singapore) with a 5 kN load cell and a cross-head speed of 5.0 mm/min to ascertain the impact of sintering temperature, amount of added AMB, and calcinations on the final products' compressive strength.

3 Results and discussions

3.1 Crystallography evaluation

The XRD evaluation of calcium oxide which is the major phase composition of the CaCO3 in the eggshell was investigated and is as presented in Fig. 2a which exhibits the XRD pattern of calcium oxide with major characteristic peaks as detected by JCPDS file no 28–0775. This inferred that calcite phase (CaCO3) is the major composition of the microstructure of the eggshell having rhombohedral crystal system [24]. A typical XRD diffraction of hydroxyapatite as-synthesized from eggshell and with commercial HAp as obtained from Sigma Aldrich is equally presented in Fig. 2a, while those obtained after heat treatments are listed in Fig. 2b. The most important peaks noticed were evaluated using the standard JCPDS file (Ref. code: 09–0432) for HAp powder. Particularly the strong diffraction peaks attributed to hydroxyapatite are (002), (112), (211), (213) and (300) in addition to other planes were identified with broad peaks. The manifestation of a lot of sharp peaks with increased intensity after calcinations at higher temperature suggests the development of polycrystalline hydroxylapatite.Fig. 2 XRD diffractograms of: (a) Raw/treated eggshell (Raw-EST), calcined eggshell (Cal- EST), As-synthesized Hap (ESTHAp) and commercially available HAp (Comm. HAp) (b) Synthesized Hap from Tamil Nadu eggshell at different temperature conditions (600, 800 and 1000 °C).

Fig. 2

The average crystallite size is denoted as Xc (nm), X-ray wavelength is represented as λ (1.54178 °A) and the full width at half maximum is given as FWHM as obtained from the diffraction peak. For this purpose, the diffraction peak of (211) was selected to compute the crystallite size. The calculated crystallite sizes as well as lattice parameters are as shown in Table 1. The size of the crystallites ranged from 21.22 nm–81.61 nm. This is an indication of crystallite grain growth of the apatite on increasing the temperature owning to the removal of water molecules and other organic matrix. The lattice parameters calculated were very close to those obtained from standard JCPDS data contained in file no. 09–432. It was observed that lattice parameter ‘a’ decreases as temperature increases, while that of parameter ‘c’ rises with temperature. This might be due to the incorporation of carbonate group into the apatite which substitutes the phosphate group. The replacement of the phosphate anion with carbonate anion can cause an increase along the c-axis of the unit cell length and a reduction alongside the a-axis [6,27]. This is in accordant with the observations from this current study because the FT-IR and EDX analyses confirmed the presence of carbonate ions in the structure of the hydroxyapatite powder.Table 1 Crystallite size and lattice parameters of synthesized HAp from eggshell at different calcinations temperature.

Table 1Samples	FWHM (°)	Parameter a (nm)	Parameter c (nm)	c/a	Parameter V (À3)	Crystallite size (nm)	
HAp (JCPDF no. 09–432)	–	0.9418	0.6884	0.73094		–	
As-prepared ESTHAP	0.42030	0.9423	0.6860	0.7289	531.22	21.22	
ESTHAp-600	0.25910	0.9415	0.6870	0.7290	530.15	32.13	
ESTHAp-800	0.19324	0.9413	0.6881	0.7310	529.20	56.32	
ESTHAp-1000	0.10138	0.9414	0.6895	0.7324	529.20	81.61	

3.2 FT-IR analysis

The observations described by FT-IR analysis of treated eggshell, calcium oxide derived from eggshell and commercial calcium oxide obtained from Sigma Aldrich is presented in the spectra shown in Fig. 3a, while the various bands assignment are represented in Table 2. The spectra demonstrated the major important diffraction peaks intensity corresponding to eggshell powder at 1054–1424 cm−1 which is due to carbonate components present in the structure of the eggshell [6,27,28]. The out-plane deformation modes of calcium carbonate peaks were observable in the spectrum range of 874 cm−1 to 876 cm−1 [21]. The presences of Ca-O peaks were noticed at 439, 505 and 572 cm−1 of the spectrum. Vibration bands between 3667 and 3639 cm−1 corresponding to O–H band which is due to humidity absorption [27]. The total disappearance of the broad band peak of the OH group at higher calcinations temperature could be due to the rupture of the hydrogen bond in the structure and also dehydroxylation of the OH group [6].Fig. 3 FT-IR spectra of (a) Commercial CaO, Treated and Calcined eggshell at 1000 °C and (b) As-synthesized ESTHAp from eggshell at different temperature conditions.

Fig. 3

Table 2 Functional groups assignment of treated eggshell, calcined eggshell and commercial CaO.

Table 2Treated eggshell	Calcined eggshell	Commercial eggshell	Functional group assignment (cm−1)	
572	439, 505	443, 556	Ca-O band	
708	687	706	Ca-O band	
874	876	877	CO32-	
1066	1054	1124	CO32-	
1424	1411	1426	CO32-	
1799	–	–	C=O	
2515	–	–	Adsorbed CO2	
2875	–	–	Adsorbed CO2	
3344	–	–	Adsorbed H2O	
3521	–	3530	O-H stretching	
3667	3639	3690	O-H stretching	

Fig. 3b shows the FT-IR spectra of the as-synthesized HAp from eggshells as well as those obtained at different temperature with various peaks assignment as listed in Table 3. The FT-IR spectra of the entire synthesized nano particle showed distinct bands observed at 470–472 cm−1, 561–607 cm−1 and 1024–1094 cm−1 which are characteristic of crystallized hydroxyapatite powder [6,[27], [28], [29]]. The bands which were observed between 1024 and 1094 cm−1 were assigned to asymmetric vibration (v3) corresponding to the functional groups of phosphate. The sharp peaks in the range of 561–607 cm−1 are associated with the bending mode (V4) of O-P-O. Likewise, the presence of adsorbed H2O bands was confirmed with peaks appearing between 1639 and 1644 cm−1 for the prepared HAp powders with the band intensity disappearing at higher calcinations temperature which indicates the loss of H2O (dehydroxylation) molecules during calcinations [30,31]. However, the appearance of the bands in the range of 3452–3572 cm−1 evidently confirmed the presence of hydroxyl group of the hydroxyapatite [32,33]. The presence of B-type carbonate functional group is observed at 873, 876, 14211–1456 cm−1. The gradual decrease in peak intensity of the carbonate bands at higher calcinations temperature inferred decarboxylation since CO2 is driven out [30].Table 3 Functional groups assignment ESTHAp at different temperature conditions.

Table 3ESHAp	ESHAp+ 600 °C	ESHAp+ 800 °C	ESHAp+ 1000 °C	Functional group assignment (cm−1)	
470	471	472	472	PO43-	
566, 602	568, 601	585, 604	561, 607	PO43-	
873	876	873	–	CO32-	
1053	1024	1030	1094	PO43-	
1420, 1456	1417, 1456	1420	1411	CO32-	
1639	1644	–	–	Adsorbed H2O	
3452	3480, 3573	3572, 3655	3540, 3571	O-H stretching	

3.3 TGA analysis

In order to evaluate the calcination temperature, the eggshell was studied by thermogravimetric analysis as shown in Fig. 4. The TGA of the eggshell showed weight loss in three categorized regions. The first region is from 25 °C to 200 °C with a total weight loss of 2.19 % corresponding to physically adsorbed water. The weight loss of about 6.38 % around 340 to 520 °C corresponds to the breakdown of MgCO3 in addition to the burning of hydrocarbons. Recall that the presence of Mg was observed in the EDX measurement. The weight loss of about 24.77 % at 696 °C in TGA indicates the decomposition of CaCO3 to form calcium oxide and carbon (IV) oxide respectively. No further weight loss was observed at higher calcinations temperature, thus suggesting the complete transformation of eggshell into calcium oxide. Similar reports have been documented in literature [6,[32], [33], [34], [35]].Fig. 4 (a) TGA thermogram of as-prepared ESTHAp.

Fig. 4

3.4 Morphological and elemental evaluations

The microscopic analysis of the as-prepared HAp and calcined HAp powders are as presented in Fig. 5. SEM images of the as-prepared HAp powder (Fig. 5a) showed agglomerated of rod like shapes. However, upon heat treatment, grain growth was observed as the particle accumulate to form little chains, with chains clustering to form apatite structure of a dimension in the range of 25–50 nm in width, while the length was observed to be in the range of 62–78 nm. It was discovered from the analysis of the scanning electron microscope that heat treatment has great impact on the HAp morphology. The HRTEM image of the HAp powder is shown in Fig. 5b which also corroborated the SEM results. The Micrograph showed that the ESTHAp have high surface area nanorod in which particles are more or less uniformed in size and round shape in all cases with size approximately in the range of 20–74 nm in diameter and about 35 nm in length. The SAED (Fig. 5c) studies indicated the formation of polycrystalline powders as a result of the continuous spotted ring structure observed. Interestingly, the fibre axis (fringes) observed in the agglomerated HAp has a d-spacing which is in good agreement with XRD crystallite size of the most intense peak. The HAADF (STEM) elemental mapping of the ESTHAp-1000 confirm the presence Ca, P and O in the proportions of 30.63 %, 18.02 % and 46.09 % respectively with Ca/P of 1.70 as shown in Fig. 6.Fig. 5 SEM (A1 &2), HRTEM (B1&2) and SAED (C1&2) micrographs of As-synthesized ESTHAp (Top1) and ESTHAp-1000 (Bottom2).

Fig. 5

Fig. 6 HRTEM (HAADF)-STEM elemental mapping images of (Ca) Calcium, (P) Phosphorus and (O) Oxygen for ESTHAp (Top) and ESTHAp (Below) with their corresponding EDX spectrum (inset show the weight and atomic percentage of elements).

Fig. 6

3.5 Scaffold HAp-AMB nano-composite

Fig. 7 shows the heating cycle for the fabrication of eggshell derived porous scaffold HAp, while the SEM images of the various HAp-AMB nano-composites formed with different weight percent of AMB are as listed in Fig. 8. The formation of interconnected porous structure was observed with the pore size increasing with the weight percent of AMB. Fig. 9a illustrates the variation in densification of sintered HAp-AMB nano-composite at different temperature with the weight percent of AMB. As depicted from the plots, the nano composite density was found to have decrease with a rise in the AMB weight percentage with the HAp-AMB nano-composite sintered at 1400 °C having the highest densification. Fig. 9b showed the effect of ammonium bicarbonate as the pore forming agent on the porosity of the eggshells derived HAp scaffold. Generally, it was observed that while the densification of the nano-composite apatite decreases with increase in weight percent of AMB, the porosities of the scaffold showed enormous increased. All the samples investigated showed considerable degree of porosities of 18 % for 10 wt% and 44 % for 40 wt% ammonium bicarbonate –hydroxyapatite (for consistency) composite respectively. Greater number of porosities coupled with interconnected porous structure will boost the transportation of metabolic products; improve ingrowth of cell in the porous space, and vascularization which are very crucial for biocompatibility [[36], [37], [38]].Fig. 7 Heating Cycle for the synthesis of ESTHAp-1000 °C porous pellet scaffold.

Fig. 7

Fig. 8 SEM micrographs of fractured surface of sintered ESTHAp-1000 °C porous scaffold with different weight percent of pore forming agents.

Fig. 8

Fig. 9 Comparative plot of (a) densification and (b) porosity of ESTHAp scaffold sintered at 1000, 1200 and 1400 °C against weight percent of pore forming agents.

Fig. 9

3.6 Compressive strength evaluation of the ESTHAP-1400 °C Scaffold

Fig. 10 Shows the results of the comparative stress versus compressive strain of compressed sintered ESTHAp with different weight percent of pore forming agents. It was found that the compressive strength rose with an increase in the content of the pore forming agent. The ESTHAp samples containing 10, 20, 25 and 30 % of AMB have withstood a compressive stress up to 5, 20, 30 and 42 N/mm2 respectively. The result clearly shows that the compressive stress increased with the AMB content introduce as the pore forming agent. The report of this work is similar to the work of Ahmed et al. [39] where it was reported that the compression stress of HAp mixed with different contents of titania and sintered at 1000C for 1 h increase with the titania contents and that the sample containing 20 % titania was able to withstood the highest compressive stress of 215 MPa. In a literature report by Esmaeilkhanian et al. [40], the compressive strength of natural nanohydroxyapatite obtained via calcination from raw turkey femurs bone after ball milling are 13.2, 24.23, 35.24, and 37.44 MPa at 850, 950, 1050, and 1150 °C, respectively as the results indicated that the strength and hardness of the nanohydroxyapatite is a function of the sintering temperature. In a similar way, Benbo et al. [41], showed that the compression properties of the two forms of double-crosslinked hydroxyapatite composite hydrogels increase with methacrylate anhydride substitution which improved the hydrogel strength.Fig. 10 Fig. 10: Comparative Stress Vs Strain plot of compression strength test of sintered ESTHAp porous scaffold with different weight percent of pore forming agents. Optical images of sintered ESTHAp porous scaffold with different weight percent of pore forming agents.

Fig. 10

3.7 Biocompatibility and cell adhesion studies

MTT assay was performed using MG63 osteosarcoma cell lines and the result is shown in Fig. 11a. ESTHAp with 0 % AMB was considered as the control sample, while cell without any samples which determines the viability of control cells, was set as 100 %. It was observed that on comparing the sample of ESTHAp containing 0 % AMB with other samples in the range of 0.01–1 mM, viability of above 85 % MG63 cells was achieved except for ESTHAp with 40 % AMB which showed some level of toxicity. This result showed that the sample of ESTHAp with higher degree of viability is biocompatible even up to a weight percent of AMB of 30 %. The confocal laser scanning microscopic images of MG 63 cells showed adhesion on different HAP samples as shown in Fig. 11b shows that cell morphology appears little distracted, while dead cells were also observed (round shaped). Fig. 12 shows the cell adhesion studies of sintered ESTHAp porous scaffold with different weight percent of pore forming agents. It was observed that the inverted microscopic images of MG 63 cells incubated with ESTHAp samples and treated with heat at 1000 °C appears to be unstable in the media used with particle leaching observed, and no cells observed near to the samples.Fig. 11 (a) MTT studies of sintered ESTHAp porous scaffold with different weight percent of pore forming agents.

Fig. 11b Confocal laser scanning microscopic images of MG 63 cells adhered on different HAP samples.

Fig. 11

Fig. 12 Cell adhesion studies of sintered ESTHAp porous scaffold with different weight percent of pore forming agents.

Fig. 12

3.8 Limitation of the study

There is one major limitation in this study that could be addressed in future research work. This study focused on the use of eggshell as the main source material for the synthesis of the prepared hydroxylapatite. Although, the prepared material was compared with standard reference material (JCPD file and commercial hydroxylapatite) to ascertain the authentication of the prepared bio-material, future study could focused on the use of some other biological origins as precursors for the synthesis of hydroxylapatite.

4 Conclusion

This work aimed at fabricating porous hydroxyapatite scaffold powders derived from eggshell via chemical precipitation. Structural investigations of the fabricated apatite were done with transmission electron microscopy, X-Ray Diffraction, Fourier Transform Infrared Spectroscopy and scanning electron microscopy. The results obtained revealed that the scaffold eggshell derived hydroxyapatite nanoparticles contain functional groups such as carbonate, phosphate, and hydroxyl groups with round morphologies and average diameters between 20 nm and 80 nm. The scaffold eggshell derived hydroxyapatite/ammonium bicarbonate nanocomposites with a highest compressive strength of 42 N/mm2 was obtained for the composite of 30 % ammonium bicarbonate. The (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay results showed that samples of scaffold eggshell derived hydroxyapatite showed higher degree of viability which suggests the biocompatibility of the samples even up to a weight percent of ammonium bicarbonate of 30 %.

Ethics statement

The cells used in this experiment were simulated cells to mimic those of living components which were obtained from Sigma, India. We didn't use cells extracted from either animal or human.

Funding

This research was funded by 10.13039/501100006261 Taif University , Saudi Arabia, project No.(TU-DSPP-2024-13 ).

Data and code availability

Data will be made available on request.

CRediT authorship contribution statement

Oladoyinbo Fatai Oladipupo: Writing – original draft, Supervision, Formal analysis, Data curation, Conceptualization. Adesokan Hameed Adekola: Resources, Methodology, Investigation. Edwin Andrew Ofudje: Writing – original draft, Supervision, Resources, Investigation, Conceptualization. Khairia Mohammed Al-Ahmary: Software, Resources, Funding acquisition. Saedah R. Al-Mhyawi: Writing – review & editing, Software, Formal analysis. Ibtehaj F. Alshdoukhi: Visualization, Validation, Resources. Mazen R. Alrahili: Visualization, Software, Data curation. Ahad Amer Alsaiari: Writing – review & editing, Resources, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

Authors extend their appreciation to 10.13039/501100006261 Taif University , Saudi Arabia, for supporting this work through project number (TU-DSPP-2024-13 )
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