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Table of Contents
Year : 2022  |  Volume : 6  |  Issue : 2  |  Page : 80-86

Characterization of nano-hydroxyapatite–collagen and epigallocatechin-3-gallate (EGCG) composites by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy

Department of Conservative Dentistry, Faculty of Dentistry, Trisakti University, Jakarta, Indonesia

Date of Submission31-Jan-2022
Date of Decision01-Apr-2022
Date of Acceptance28-Apr-2022
Date of Web Publication12-Jul-2022

Correspondence Address:
Department of Conservative Dentistry, Faculty of Dentistry, Trisakti University, Jakarta
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/SDJ.SDJ_4_22

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Background: Calcium hydroxide (Ca(OH)2) is most commonly used in vital pulp therapy, and it is the gold standard of pulp capping materials; however, it shows several limitations, including facile dissolution, stimulation of reparative dentin with tunnel defects, and inflammation. A previous study reported that nano-hydroxyapatite (nHA) might induce reparative dentin with no tunnel defects much better than Ca(OH)2. Another study reported that the addition of epigallocatechin-3-gallate (EGCG) to collagen (Col) gel can increase pulp cell proliferation and differentiation via the change in its mechanical properties. Objective: In this study, nHA–Col and EGCG composites were characterized by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Methods: Each material (i.e., nHA, Col type 1, and EGCG) was dissolved in 2 mL of deionized water. Three groups with varied nHA:Col ratios were prepared: 40:60 (group 1), 50:50 (group 2), and 60:40 (group 3). Each solution was mixed together using a magnetic stirrer at 40°C, followed by the addition of 2% hydroxypropyl methylcellulose (HPMC) into the mixture. Morphology observation, Ca/P ratio, crystallographic phase analysis, and functional group analysis were investigated by SEM-EDS, XRD, and FTIR. Results: SEM-EDS analysis revealed irregular agglomerated nHA between Col fibrils and a non-stoichiometric Ca/P ratio (>1.67). XRD analysis revealed hexagonal-phase nHA. FTIR analysis revealed chemical interaction between nHA, Col, and EGCG. Conclusions: SEM-EDS and XRD analysis confirmed that nHA does not change when it is mixed with Col and EGCG, and FTIR analysis revealed hydrogen bonding among materials.

Keywords: Collagen, composite, EGCG, nano-hydroxyapatite

How to cite this article:
Elline, Fibryanto E, Amanda HG. Characterization of nano-hydroxyapatite–collagen and epigallocatechin-3-gallate (EGCG) composites by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Sci Dent J 2022;6:80-6

How to cite this URL:
Elline, Fibryanto E, Amanda HG. Characterization of nano-hydroxyapatite–collagen and epigallocatechin-3-gallate (EGCG) composites by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Sci Dent J [serial online] 2022 [cited 2023 Jun 4];6:80-6. Available from: https://www.scidentj.com/text.asp?2022/6/2/80/350760

  Background Top

According to data reported by the World Health Organization (WHO) in 2019, caries or tooth decay is one of the most common oral health problems worldwide.[1] Moreover, this condition is prevalent in Indonesia, where caries accounts for 45.3% of the total proportion of oral health problems.[2] Calcium hydroxide (Ca(OH)2) is one of the materials that is typically used in vital pulp therapy, and it is the gold standard for pulp capping therapy owing to its ability to induce reparative dentin as well as its antibacterial properties. However, it shows several disadvantages, including facile dissolution, inadequate ability to bond with dentin, induction of pulp inflammation and necrosis, and stimulation of a dentinal bridge with tunnel defects and pores.[3],[4],[5]

Hydroxyapatite (Ca10(PO4)6(OH)2) is an inorganic material mainly containing calcium and phosphate in a Ca/P ratio of 1.67 (stoichiometric).[6] It has been used as a ceramic biomaterial in orthopedics and dentistry because of its several advantages, including osteoconductive properties, nontoxicity, bioactivity, good biocompatibility, and low solubility; however, it shows unsatisfactory mechanical properties.[6],[7] Previous studies reported that nano-sized hydroxyapatite can improve hydroxyapatite mechanical properties, solubility, and bioactivity.[6],[8] The improvement is attributed to the higher surface area of nano-hydroxyapatite (nHA) than that of conventional hydroxyapatite.[8] Hydroxyapatite can be synthesized by chemical methods or by using natural sources.[9] Chicken eggshells constitute one of the sources as it contains 94% of the calcium carbonate mineral (CaCO3). Therefore, chicken eggshells can be used as the calcium source.[10]

Collagen (Col) is a protein formed by amino acids in a triple helix structure.[11] The human body contains 28 types of Cols, where Col types I–V are the most common. Col type I is the main component of dentin, and it is typically used in tissue engineering, the food industry, and cosmetics.[11],[12],[13] Col shows several benefits such as good compatibility, absorbance, biodegradability, and stimulation of tissue regeneration; however, it shows low mechanical properties and thermal stability.[14],[15]

Green tea (Camellia sinensis) contains catechin, which is one of the main polyphenols. Catechin is predominantly composed of epigallocatechin-3-gallate (EGCG, 59%), followed by epigallocatechin (EGC, 19%), epicatechin-3-gallate (ECG, 13,6%), and epicatechin (EC, 6.4%).[16] The polyphenol component of green tea renders several benefits to green tea, including antibacterial, anti-inflammation, antioxidant, antimutagenic, and anticancer effects.[3],[16]

Previous studies reported that nHA might induce a dentinal bridge with no tunnel defects better than Ca(OH)2.[17] Another study revealed that dental human pulp cell can be cultured in Col scaffolds and shows odontogenic differentiation with high vascularization.[3] Widjiastuti et al.[4] reported that EGCG gel can reduce inflammation response via binding to nitrate oxide (NO) and increase the number of fibroblast cells proliferation from dental pulp; hence, it can be concluded that EGCG gel might have a role in the pulp healing process. Kwon et al.[18] reported that the addition of EGCG to Col gel increases the proliferation and differentiation of dental pulp. Their study also concluded that the use of gel in pulp therapy is more preferable because it can be easily applied and can adapt to the complex anatomy of tooth root.

This study was based on a previous study in which a mixture of nHA-gelatin-streptomycin was synthesized, and several ratios of nHA and Col were used.[19] Hydroxypropyl methylcellulose (HPMC) also has been used as the suspension agent.[19] HPMC is a semi-synthesized polymer and a cellulose derivative, which is commonly used as a gelling agent, suspension agent, and drug delivery system matrix, as well as in film making owing to its biocompatibility and biodegradability.[13] In this study, nHA–Col and EGCG composite are characterized by SEM-EDS, XRD, and FTIR.

  Materials and Methods Top

nHA from chicken eggshells, hydrolyzed Col type I, EGCG, 2% HPMC, and deionized water were used in this study. The particle size of nHA (PT. Alesha Berkah Utama (ProDB), Indonesia) was less than 60 nm, and the Ca/P ratio was 1,9. Hydrolyzed Col type I (Wuhan Healthdream Biological Technology, China) was derived from bovine. Other materials included EGCG (Sigma Aldrich, USA) and HPMC (Ashland Benecel, Belgium).

A Barnstead Cimarec digital hot plate, freeze dryer, a Thermo Scientific Quanta USA scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), a PANalytical Aeris Suite X-ray Diffraction (XRD), and a Thermo Scientific Nicolet iS-10 Fourier transform infrared (FTIR) Spectroscopy equipment were used.

Synthesis of the nHA–Col–EGCG composite

The nHA–Col–EGCG composite was synthesized by dissolving each material, i.e., nHA, 20% w/v Col, and 0,45 mg/mL of EGCG in 2 mL of deionized water. Three groups with varied ratios of nHA and Col were prepared: 40:60 (group 1), 50:50 (group 2), and 60:40 (group 3). All solutions were mixed together using a magnetic stirrer at 40°C until homogenized. Then, 0.07 g/mL of 2% HPMC was added into the mixture and stirred at 40°C until homogenized.

Sample characterization

The morphology of the nHA–Col–EGCG composite was observed by SEM; calcium and phosphate compositions (Ca/P ratio) were analyzed by EDS; crystallographic phase analysis was conducted by XRD; and functional group analysis was performed by FTIR. All samples were freeze-dried at −80°C for 24 h. SEM-EDS analysis (Thermo Scientific Quanta, USA) was conducted at 1000× magnification for each sample. The crystallographic structure and composition phase of nHA in the composite were analyzed by XRD (PANalytical Aeris Suite). The freeze-dried samples were irradiated by monochromatic Cu/Kα radiation at 2θ angles ranging from 20° to 100°. FTIR analysis was employed to identify the functional groups present in the materials and observe whether chemical interactions occurred among the materials. The freeze-dried samples were mixed with KBr into a pellet and then analyzed by FTIR (Thermo Scientific Nicolet iS-10) at wavelengths of 400–4000 cm−1. All data were presented in a descriptive manner.

  Results Top

SEM images were recorded to observe the morphology of the nHA–Col–EGCG composite. [Figure 1] shows the SEM images of group 1 (40:60), group 2 (50:50), and group 3 (60:40). The SEM image of group 1 revealed spherical nHA between Col fibers and irregular agglomeration of nHA particles. In group 3 (60:40), nHA and Col fibers, as well as agglomerated nHA, were visible, albeit in a non-homogeneous manner. However, the SEM image of group 2 (50:50) revealed only large agglomerated nHA with no Col fibers. EDS analysis revealed that the calcium and phosphate compositions (Ca/P ratio) of the three groups are 1.96, 1.98, and 1.82 [Figure 2], respectively; all of these values are greater than the stoichiometric hydroxyapatite Ca/P ratio (1.67).
Figure 1: SEM images of nHA:Col 40:60 (A), 50:50 (B), and 60:40 (C)

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Figure 2: EDS elemental maps of nHA:Col 40:60 (A), 50:50 (B), and 60:40 (C)

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The XRD patterns of pure nHA in [Figure 3] revealed 2θ diffraction peaks at 25°, 31°, 32°, 33°, 34°, 39°, 40°, 46°, 49°, and 53°, corresponding to the (002), (211), (112), (300), (202), (310), (310), (222), (213), and (004) crystallographic plans of the apatite structure, respectively. The XRD analysis of each group revealed the same 2θ diffraction peaks as those present in pure nHA. The XRD patterns of Group 1 (40:60) revealed diffraction peaks at 25°, 31°, 34°, and 40°; the XRD pattern of group 2 (50:50) revealed diffraction peaks at 25° and 40°; and the XRD pattern of group 3 (60:40) revealed diffraction peaks at 25°, 31°, and 40°. Crystallographic structure analysis confirmed the inorganic phase of hydroxyapatite with lattice crystals of a = b ≠ c.
Figure 3: XRD patterns of pure nHA (A), and nHA:Col 40:60 (B), 50:50 (C), and 60:40 (D)

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FTIR analysis revealed the presence of several absorbance peaks related to some specific functional groups, including a peak at 3390–3250 cm−1 corresponding to the stretching vibration of the hydroxyl group (O–H), a peak at 2875–2937,5 cm−1 corresponding to the stretching vibration of C–H, a peak at 1625–1655 cm−1 corresponding to the stretching vibration of the carbonyl group (C = O), a peak at 1562–1548 cm−1 corresponding to N–H bending and C–N stretching, and a peak at 1250 cm−1 corresponding to C–N stretching and N–H bending. In addition, a carbonate (CO32−) peak was observed at 875 cm−1, and a stretching and bending peak of phosphate (PO43−) at 1000 cm−1 and 500–625 cm−1 [Figure 4].
Figure 4: FTIR spectra of nHA:Col 40:60 (A), 50:50 (B), and 60:40 (C)

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  Discussion Top

A previous study reported that nHA can induce dentinal bridge formation with no tunnel defects owing to its osteoconductive ability to increase osteoblast cells and produce an osteodentin structure.[17] nHA has been reported to also induce an acute inflammation reaction owing to its high pH (11) and calcium release, but it is resolved within 2 weeks, much better and faster than Ca(OH)2.[17] EGCG can be used as a crosslinking agent for Col gel to improve its mechanical properties and thermal stability. By improving its mechanical ability, the biological properties of Col gel are changed, thereby increasing dental pulp proliferation and differentiation. EGCG also can be used in pulp therapy because of its antibacterial activity against oral pathogens, such as Streptococcus mutans and Enterococcus faecalis.[18]

SEM analysis revealed agglomerated nHA in all samples. This result is similar to that reported in previous studies, where nHA tends to agglomerate.[20] This also might be caused by the direct stirring of nHA, which renders temporary distribution effects of the particles and causing rebonding of particles.[21]

EDS analysis revealed a Ca/P ratio of 1.9 for all samples, which is greater than the stoichiometric ratio of hydroxyapatite (1.67). This result is related to the use of chicken eggshells as a calcium source of hydroxyapatite.[22] Hydroxyapatite derived from natural sources is usually non-stoichiometric because it contains other ions such as Mg2+, Fe2+, Na+, Cl, and F.[23]

XRD analysis of all samples confirmed the inorganic phase of hydroxyapatite, which can be observed at the same 2θ diffraction peaks as those observed for pure nHA (25° (002), 31° (211), 33° (300), and 40° (310)).[20],[21] Crystallographic structure analysis revealed a parameter crystal lattice of a = b ≠ c, which confirmed the hexagonal-phase of hydroxyapatite. Hexagonal crystals of hydroxyapatite are typically observed in non-stoichiometric hydroxyapatite, corresponding to the EDS analysis (Ca/P ratio of 1,9). Therefore, based on SEM-EDS and XRD analyses, when nHA is mixed with Col and EGCG, nHA does not show changes.

FTIR analysis of all samples confirmed the presence of nHA, Col, EGCG, and HPMC based on their functional groups. The hydroxyl group (O–H) peak at 3600–3150 cm−1 confirmed the presence of nHA, EGCG, and HPMC.[13],[15],[20],[21] Phosphate (PO43−) stretching and bending at 1000 cm−1 and 500–600 cm−1 also confirmed the presence of nHA.[21],[24] The C–H stretching vibration at 2900 cm−1 confirmed the presence of the methyl and hydroxypropyl groups of HPMC.[19] The Col structure peaks represented by amide I (C = O), amide II (N–H bending, C–N stretching), and amide III (C–N stretching, N–H bending) at 1600–1690 cm−1, 1480–1575 cm−1, and 1229–1301 cm−1 were observed in all samples.[25] In this study, peak absorbance shifts, especially for the O–H vibration at 3200–3300 cm−1, between samples were observed, indicative of a possible chemical interaction between the materials. This is confirmed by a previous study that revealed that shifts in absorbance peaks of O–H stretching, amide I, and amide II indicate hydrogen-bond interactions between molecules. The broadening of the O–H stretching absorbance peak also indicated hydrogen bond among materials.[13]

  Conclusions Top

SEM imaging revealed agglomerated nHA between Col fibrils. EDS analysis revealed a non-stoichiometric Ca/P ratio (1.9) for all samples. XRD analysis confirmed a hexagonal crystal structure and phase of nHA. SEM-EDS and XRD analysis revealed neither degradation nor changes in nHA when it is mixed with Col and EGCG. FTIR analysis revealed possible hydrogen bonding among materials.


The authors would like to appreciate Puspitek LAPTIAB-BPPT for providing help and facility in completing this research.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

World Health Organization. Oral Health. Geneva: World Health Organization; 2021.  Back to cited text no. 1
Kementerian Kesehatan RI. Infodatin: Pusat Data dan Informasi Kementerian Kesehatan RI. Jakarta: Kementerian Kesehatan Republik Indonesia; 2019. p. 1-6.  Back to cited text no. 2
Hanna SN, Perez Alfayate R, Prichard J. Vital pulp therapy an insight over the available literature and future expectations. Eur Endod J 2020;5:46-53.  Back to cited text no. 3
Widjiastuti I, Setyabudi , Ismiyanti K, Purwanto DA, Sukmawati T. Effect of hydrogel epigallocatechin-3-gallate (EGCG) to the number of fibroblast cell proliferation in the perforation of Wistar rat tooth pulp. Conserv Dent J 2019;9:93-6.  Back to cited text no. 4
Akhlaghi N, Khademi A. Outcomes of vital pulp therapy in permanent teeth with different medicaments based on review of the literature. Dent Res J (Isfahan) 2015;12:406-17.  Back to cited text no. 5
Gshalaev VS, Demirchan AC. Hydroxyapatite: Synthesis, Properties, and Applications. New York: Nova Science Publisher; 2012. p. 17.  Back to cited text no. 6
Milovac D, Gallego Ferrer G, Ivankovic M, Ivankovic H. PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: Morphology, mechanical properties and bioactivity. Mater Sci Eng C Mater Biol Appl 2014;34:437-45.  Back to cited text no. 7
Pelpa E, Besherat LK, Palaia G, Tenore G, Migliau G. Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: A review of literature. Ann Stomatol (Roma) 2014;5:108-14.  Back to cited text no. 8
Mohd Pu’ad NAS, Koshy P, Abdullah HZ, Idris MI, Lee TC. Syntheses of hydroxyapatite from natural sources. Heliyon 2019;5:e01588.  Back to cited text no. 9
Tijani HI, Mohammed BA, Saidu H, Yusuf H, Jibrin MN, Mohammed S. From garbage to biomaterials: An overview on egg shell based hydroxyapatite. J Mater 2014;2014:1-6.  Back to cited text no. 10
Leon-Lopez A, Morales-Penaloza A, Matinez-Juarez VM, Vargas-Torres A, Zeugolis DI, Aguirre-Alvarez G. Hydrolyzed collagen: Source and applications. Mol 2019;24: 4031.  Back to cited text no. 11
Fratzl P. Collagen Structure and Mechanics. New York: Springer; 2008. p. 1-15.  Back to cited text no. 12
Ding C, Zhang M, Li G. Preparation and characterization of collagen/hydroxypropyl methylcellulose (HPMC) blend film. Carbohydr Polym 2015;119:194-201.  Back to cited text no. 13
Wu M, Cronin K, Crane JS. Biochemistry, collagen synthesis. In: StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing; 2022. Available from: https://www.ncbi.nlm.nih.gov/books/NBK507709/. [Last accessed on 2021 Aug 19].  Back to cited text no. 14
Chu C, Deng J, Xiang L, Wu Y, Wei X, Qu Y, et al. Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts. Mater Sci Eng C Mater Biol Appl 2016;67:386-94.  Back to cited text no. 15
Narotzki B, Reznick AZ, Aizenbud D, Levy Y. Green tea: A promising natural product in oral health. Arch Oral Biol 2012;57:429-35.  Back to cited text no. 16
Swarup SJ, Rao A, Boaz K, Srikant N, Shenoy R. Pulpal response to nano hydroxyapatite, mineral trioxide aggregate and calcium hydroxide when used as a direct pulp capping agent: An in vivo study. J Clin Pediatr Dent 2014;38: 201-6.  Back to cited text no. 17
Kwon YS, Kim HJ, Hwang YC, Rosa V, Yu MK, Min KS. Effects of epigallocatechin gallate, an antibacterial cross-linking agent, on proliferation and differentiation of human dental pulp cells cultured in collagen scaffolds. J Endod 2017;43:289-96.  Back to cited text no. 18
Hikmawati D, Maulida HN, Budiatin AS. Synthesis and characterization of nanohydroxyapatite-gelatin composite with streptomycin as tuberculosis injectable bone substitute. Inter J Biomater 2019;2019:7179243.  Back to cited text no. 19
Rogina A, Sandrk N, Teruel-Biosca L, Antunovic M, Ivankovic M. Bone-mimicking injectable gelation/hydroxyapatite hydrogels. Chem Biochem Eng Q 2019;33: 325-35.  Back to cited text no. 20
Takallu S, Mirzaei E, Azadi A, Karimizade A, Tavakol S. Plate-shape carbonated hydroxyapatite/collagen nanocomposite hydrogel via in situ mineralization of hydroxyapatite concurrent with gelation of collagen at ph = 7.4 and 37°C. J Biomed Mater Res B Appl Biomater 2019;107:1920-9.  Back to cited text no. 21
Yousefi AM, Oudadesse H, Akbarzadeh R, Wers E, Lucas-Girot A. Physical and biological characteristics of nanohydroxyapatite and bioactive glasses used for bone tissue engineering. Nanotechnol Rev 2014;3:527-52.  Back to cited text no. 22
Akram M, Ahmed R, Shakir I, Ibrahim WA, Hussain R. Extracting hydroxyapatite and its precursors from natural resources. J Mater Sci 2014;49:1461-75.  Back to cited text no. 23
Moreno-Vásquez MJ, Valenzuela-Buitimea EL, Plascencia-Jatomea M, Encinas-Encinas JC, Rodríguez-Félix F, Sánchez-Valdes S, et al. Functionalization of chitosan by a free radical reaction: Characterization, antioxidant and antibacterial potential. Carbohydr Polym 2017;155:117-27.  Back to cited text no. 24
Liu J, Yong H, Yao X, Hu H, Yun D, Xiao L. Recent advances in phenolic-protein conjugates: Synthesis, characterization, biological activities and potential applications. RSC Adv 2019;9:35825-40.  Back to cited text no. 25


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