Synthesis and Characterization of Naringin Functionalized Nano-Hydroxyapatite for Bone Tissue Engineering

ABSTRACT

Bone is a unique nanocomposite tissue composed of organic and inorganic materials. Bone grafting is a common surgical method used to improve bone regeneration in dentistry and orthopedic surgery. Because standard therapies have substantial drawbacks, nanomaterials provide alternative options for bone repair. Owing to its high bioactivity, osteoconductivity, biocompatibility, and topography that matches the architecture of real bone, hydroxyapatite nanoparticles (n-HA) are commonly used in bone treatment. We report here the synthesis and characterization of Naringin (NA) functionalized n-HA using HRTEM, FTIR, XRD, and UV-visible spectroscopy. The obtained results indicated that the n-HA can be functionalized with Naringin and they might be used as a bone regenerative material in medical and dental fields.

INTRODUCTION

Bones are a unique type of organ due to their innate ability to regenerate in response to injuries. However, the regenerative potential of the bone is impaired in conditions such as osteoporosis, osteoarthritis, osteomyelitis, and osteogenesis imperfecta. Furthermore, in orthopedic and dental surgery, considerable amounts of bone regeneration are required for skeletal restoration of massive bony defects caused by trauma, infection, and tumour excision.[1]

Autologous bone grafting has long been accepted as the “gold standard” technique. Autograft harvesting is traumatic, expensive, and linked with donor-site morbidity.[2] These drawbacks prompted the invention of allografts and xenografts. However, these transplant materials carry the danger of rejection and disease transmission.[3] To tackle these issues, several synthetic bone grafts are currently available on the market. These materials only have osteoconductive properties. As a result, there is a strong drive to create innovative synthetic materials to repair segmental defects and enhance the bone healing response.

The inorganic component of the bone matrix is largely comprised of hydroxyapatite (HA).[4] Due to this similarity, substantial research is underway on the utilization of HA as a bone transplant. Technological breakthroughs in nanoscience and nanotechnology have paved the way for the synthesis of nano-sized bonegrafts. Nanohydroxyapatite (n-HA) crystals have higher biocompatibility, better bioactivity, and osseointegration ability than porous hydroxyapatite and a lower inflammatory response.[5]

Herbal medicines have recently seen a resurgence of attention as an alternate treatment option for intractable conditions like rheumatoid arthritis. A herbal medicine known as “Rhizoma drynariae” is extensively used in China to treat orthopedic problems. The flavanone glycoside “Naringin” (NA) is the main active natural element contained in several Chinese herbal preparations and citrus fruits.[6,7] As per a comprehensive literature research, Naringin exhibits antioxidant, antibacterial, anti-inflammatory, antiosteoporotic, and anticarcinogenic properties.[7]

Naringin was found to stimulate the osteoblastic cell proliferation and differentiation[8] while suppressing osteoclasts formation.[9-11] It also inhibits the HMG-CoA reductase and activates the promotor region of bone morphogenic protein-2 (BMP-2) resulting in enhanced bone formation.[12] In a work done by Chen et al.,[10] Naringin was found to decrease the number of osteoclasts while increasing the alkaline phosphatase activity, osteocalcin levels, and bone mineral density.

In this study, we explored the feasibility of functionalizing nano-hydroxyapatite with Naringin to develop novel bone repair materials by local administration of the flavonoid.

MATERIALS AND METHODS

Materials

Naringin (≥ 95% HPLC) was purchased from Sigma-Aldrich. Commercially available analytical grade reagents like orthophosphoric acid (H₃PO₄ 85%), calcium hydroxide (Ca(OH)₂ 96%), sodium hydroxide (NaOH), Phosphate Buffer Solution (PBS), and ethanol were purchased from Sisco Research Laboratories Pvt. Ltd (India).

Synthesis of Nano-hydroxyapatite (n-HA)

The wet-chemical precipitation technique was employed for the production of nano-hydroxyapatite (n-HA) powder.[13] In this procedure, 250 mL of an aqueous solution of H₃PO₄ (0.6 M) was slowly added to 250 mL of an aqueous suspension of Ca(OH)₂ (1 M) and agitated with a magnetic stirrer for two hours at room temperature. The pH was then adjusted with concentrated NaOH until it reached 11. The white suspension was rinsed in deionized water and dried for 24 hours in an oven at 80°C.

Synthesis of n-HA/NA Composite

To load the Naringin molecules into the hydroxyapatite nanoparticles, the Naringin and nano-hydroxyapatite powders (in the same weight ratio) are taken in a separate beaker. Both powders were dissolved using ethanol under constant stirring at 350 rpm for 40 minutes. The Naringin solution was then added using a dropper into n-HA solution under constant stirring for another 40 minutes. The suspension was then centrifuged for 5 minutes at 2,000 rpm to separate the supernatant from the precipitate and dried.

Characterisation of n-HA and n-HA/NA composite

A high resolution transmission electron microscope (HRTEM, JEOL Japan, JEM-2100 Plus) was used to analyse the crystal morphological characteristics, microstructure, and architecture of n-HA and n-HA/NA nanoparticles. Powder X-ray diffraction with 2q angle ranging from 20 to 80 was used to determine the crystalline phase composition (XRD, BRUKER USA D8 Advance, Davinci). A FTIR spectrometer was used to analyse the FTIR spectra of functional groups found in n-HA and n-HA/NA particles (SHIMADZU, IRTRACER 100). For this experiment, 1 gram of dry powder material was mixed thoroughly with 100 mg of potassium bromide at a 1:100 ratio (sample: KBr) and pelleted. The IR spectra of the pellets were then determined with an FTIR spectrometer functioning in the 400–4,000 cm-1 range. The molecular composition and absorbance of the sample were determined using UV-visible spectroscopy (UV-Vis, SHIMADZU, UV 3600 PLUS).

RESULTS AND DISCUSSION

Characterisation of n-HA and n-HA/NA

The wet chemical precipitation approach was used to effectively synthesize pure n-HA and n-HA/NA composites. The XRD pattern of pure hydroxyapatite nanoparticles is shown in Figure 1. At 25.88, 31.78, 32.18, and 32.98 2 theta values, the spectrum matches JCPDS values (09-0432) such as (002), (211), (211), and (300). It reveals the presence of highly pure, single-phase nano hydroxyapatite particles and the spectrum matches JCPDS values (09-0432) such as (002), (210), (211), (300), (202), (310), (311), (213), and (211) at 25.58, 28.57, 31.69, 32.85, 34.15, 39.04, 41.07, 49.24, 52.05, and 53.09 2θ. There were no additional phases found. Furthermore, the findings confirmed the presence of sharper diffraction peaks indicating sample’s crystalline nature. The results matched those of previous investigations by Sebastiammal et al.[14] and Poinern et al.[15]

Figure 1.

XRD pattern of n-HA synthesized by wet chemical precipitation

The structure and morphology of the samples were further confirmed by the HRTEM. The HRTEM images of the prepared HA nanoparticles and the n-HA/NA composites were depicted in Figure 2a and 2b. The image depicts the presence of nanocrystalline hydroxyapatite as a rod-shaped structure. Discrete and slightly agglomerated particles of nanoscale were clearly evident. There were no noticeable morphological changes between the n-HA nanoparticles and the n-HA/NA composites. The SAED pattern obtained [Figure 2a and 2b] showed the crystalline nature with the formation of bright spot and well defined bands confirming the attributes of nanorange particles.[16] Also, the addition of Naringin did not alter the crystalline nature of HA nanoparticles.

Figure 2.

(a) HRTEM images of n-HA at 50 nm and the SAED pattern. (b) n-HA/NA at 50 nm and the SAED pattern

The functional groups of the synthesized nanocomposites were analysed using FTIR spectroscopy with wavenumbers from 4,000 to 500 cm^-1 and the results were depicted in Figure 3. The PO₄ group showed prominent bands at 527 cm-1, 558 cm-1, 891 cm-1, and 1027 cm-1 in Figures 3a and 3c. The bands at 3,569 cm-1 confirm the presence of hydroxyapatite’s OH group and the bands at 3,415 cm-1 and 1,642 cm-1 indicate the presence of adsorbed water. The carbonate group detected at 1,407 cm^-1 and 891 cm^-1 might be the consequence of atmospheric CO₂ being dissolved in solution at the time of n-HA synthesis.[17] The FT-IR spectra of Naringin is characterized by vibrational bands which are mostly related to the functional groups of Naringin such as OH, C-H, C = O, and C = C [Figure 3b].[18] The findings revealed the characteristic peaks of the functional groups OH at 3,358.12cm^-1 and 1,038.68 cm^-1, C = O at 1,637.59 cm^-1, and C = C at 1,584.54 cm^-1, respectively.[19] The aromatic is seen at 1,207.45 cm^-1, which is one of Naringin’s distinctive bands. The majority of the characteristic bands of n-HA and Naringin were found in the n-HA/NA composite spectra. Some extremely weak bands were not discernible due to the presence of both n-HA and Naringin, while some bands were broadened or slightly shifted due to overlap. In addition, there is minimal variation between the pure n-HA and n-HA/NA composite, indicating that the drug has little impact on the structure. The well-crystallized apatite structure is confirmed by the appearance of prominent peaks in the FTIR spectrum associated with the hydroxyl and phosphate groups.[20]

Figure 3.

FTIR spectra of (a) pure n-HA, (b) pure Naringin, and (c) n-HA functionalized with Naringin

The spectrum of flavonoids is often represented by two distinct bands with maximum absorption, band II in the 240-295 nm range and band I in the 300-400 nm range, corresponding to ring A and ring B, respectively.[21] The UV-Vis spectra of n-HA, NA, and n-HA/NA composites are shown in Figure 4. The findings indicate that the n-HA/NA composites had two prominent UV absorption peaks, one at 282 nm (band II) connected to ring A absorption and the other at 336 nm (band I) indicating ring B absorption, which are typical for flavones and flavonoids.[19] It is also obvious that the absorbance of Naringin was maximum at 282 nm as indicated by the peak suggesting that the absorption of Naringin into HA nanoparticles.

Figure 4.

UV-vis spectra of n-HA, NA, and n-HA/NA composites

CONCLUSIONS

The global need for biomaterials is constantly increasing and there is a huge demand for the synthetic bone substitute materials. Therefore, the focus of our work was on creating a biocomposite scaffold made of ceramic nano-hydroxyapatite that was combined with Naringin using a wet chemical method. The synthesized biomaterials, n-HA and n-HA/NA, were characterized by HRTEM, FTIR, XRD, and UV-visible spectroscopy studies. However, further research is necessary to determine the bone regeneration potential and the safety profile of these scaffolds.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.