Bioactive Biogenous Mineral for Bone Bonding Applications [311030]

Bioactive Biogenous Mineral for Bone Bonding Applications

Georgiana Dana DUMITRESCU1,*, Andrada SERAFIM1, Horia IOVU1, Izabela Cristina STANCU1

1[anonimizat], 011061, Bucharest, [anonimizat], porosity, compressive and flexural strength [1]. [anonimizat] [2]. We investigated the potential of this biogenous mineral to be bioactive with respect to bone integration when used as bone defects filler. To prove this hypothesis we verify if it is able to induce biomimetic mineralization when incorporated in inert poly(2-hydroxyethyl methacrylate(pHEMA)) hydrogels.

Keywords: [anonimizat], biodynamic test instrument

1. Introduction

The biomaterials used as fillers in bone regeneration and reconstruction should have a strong binding ability in order to prevent displacement. It has been demonstrated that biomaterials containing hydroxyapatite ([anonimizat]) have an enhanced ability to bind with living bone due to its similarity with host [1].

[anonimizat] [2]. As an example, a search using the term “cuttlefish bone” on ScienceDirect database showed that only ten research papers were published in the year 2000 and over sixty in 2018 [3]. This natural material is appealing as bioactive bone substitute due to its composition (a crystallized form of calcium carbonate and chitin [2]) [anonimizat] (porosity, light-weight, compressive and flexural strength) and in addition it presents the advantages of high availability and cost efficiency [4],[5]. [anonimizat]: a large biomineralization surface and an abundant organic matrix [3].

The use of cuttlefish bone as a filler in acrylic bone cements was investigated and found enhanced osseointegration and no evidence of secondary infection during in vivo testing on rabbits. The mechanical properties of the bone cement with up to 30% [anonimizat] [6], [7].

Another approach for using cuttlefish bone in tissue scaffolding applications revolves around the hydrothermal transformation of aragonite into hydroxyapatite, a ceramic which is already regarded as appropriate for bone tissue engineering applications. [anonimizat]. A [anonimizat] [6],[7].

The ability of a biomaterial to integrate with bone tissue can be evaluated using the simulated body fluid (SBF) test to study in vitro formation of Ca/P mineral phase at the surface of a material when immersed in SBF. The SBF solution (originally named simulated physiological solution, SPS [1],[8]) has similar ion concentration to human blood plasma and the test is carried out by maintaining solution pH and temperature to match that of blood plasma, as it has been found that this is required for formation of an apatite mineral [1],[8]. Thus, the ability of a scaffold to replace or repair natural bone tissue may be evaluated based on its ability to form bone-like hydroxyapatite. The test can be performed through immersion in a certain volume of SBF, for a pre-established period of time, as described by Kokubo in [2].

The present study aims the evaluation of the potential of the mineral phase of cuttlefish bone to act as biomimetic mineralization initiator when embedded in an inert polymeric matrix in order to be subsequently used in the field of hard tissue regeneration or repair. In this respect, cuttlefish bone fragments were immobilized into poly(2-hydroxyethyl methacrylate (samples further referred to as CB-pHEMA) and their mineralization was further investigated using two experimental conditions: uniaxial compression under continuous flow of SBF and conventional static incubation in SBF. The formation of biomimetic apatite into the pHEMA matrix was investigated through microcomputed tomography (microCT), scanning electron microscopy and Fourier transform infrared (FTIR) spectroscopy.

2. Materials and methods

2.1. Materials

2-hydroxyethyl methacrylate (HEMA), and silver nitrate, were purchased from Sigma Aldrich. Ammonium persulfate (APS), also purchased form Sigma Aldrich was used as initiator in the polymerization reaction. Ethylene glycol dimethacrylate (EGDMA) was purchased from Fluka and used as crosslinker. Cuttlefish bone (CB) was purchased from pet shops in Romania where it is sold as calcium supplement for birds.

For the preparation of SBF I used: sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO3), potassium chloride (KCl), di-potassium hydrogen phosphate trihydrate (K2HPO4·3H2O), magnesium chloride hexahydrate (MgCl2·6H2O), calcium chloride (CaCl2), sodium sulfate (Na2SO4), Tris-hydroxymethyl aminomethane: ((HOCH2)3CNH2) (Tris), 1 M (mol/l) Hydrochloric Acid, 1M-HCl, using the protocol described in [9].

2.2. Methods

2.2.1. Synthesis of CB – pHEMA materials

The CB powder was prepared as described in [5]. Briefly, the CB was cut in small fragments and extensively washed with double distilled water for 3 days at room temperature (RT), then and subsequently milled to a powder.

Cuttlefish bone (CB) fragments were embedded in pHEMA polymers (as described in Table 1) through the free radical bulk polymerization of HEMA in the presence of bone powder, using EGDMA as crosslinker (HEMA:EGDMA = 100:1 molar ratio) and APS as initiator (HEMA:APS = 100:1 molar ratio), at 60°C. pHEMA was also synthesized and used as a control matrix.

For simplicity, in the denomination of the samples with the highest CB loading incubated in dynamic conditions the letter D will be added at the end, and the letter S will be added to those incubated in static conditions (i.e. pHEMA_S and pHEMA_D and pHEMA-CB10_S and pHEMA-CB10_D, respectively).

Table 1. Compositions of the synthesized materials

2.2.2. Characterization of hybrid materials

Mineralization testing

The potential of the CB to act as promoter of a biomimetic mineralization was evaluated in both static and dynamic conditions.

Static tests were conducted using samples of each composition immersed in simulated body fluid (SBF) following the protocol described in [9].

For dynamic tests, samples were compressed up to two weeks using an ElectroForce 5210 biodynamic test instrument, at a frequency of 1 Hz, and a displacement of ± 2 mm in continuous flow of SBF at a flow rate of 0.2 ml/min and a temperature of 37°C (Fig.1.). At the end of the incubation the samples were removed and gently washed with distilled water to remove residual salts. They were dried at drying stove.

Fig.1. Representative experimental: a) Conventional vial with incubated sample,

b) ElectroForce 5210 biodynamic test instrument

The biodynamic test instrument helps characterize implant materials by imitating natural conditions in the human body: compression or traction, temperature, dynamic flow of a simulated fluid, such as SBF.

Von Kossa staining

To visually monitor the formation of calcium salts into the pHEMA matrix after SBF incubation, the samples were immersed into 3 ml silver nitrate solution (1% wt/v), for 60 minutes and then exposed to strong light as described in [10].

Fourier transform infrared (FTIR) analysis

FTIR spectra were recorded using a JASCO 4200 spectrometer equipped with a Specac Golden Gate attenuated total reflectance (ATR) device in the 4.000-600 cm-1 wavenumber region with a resolution of 4 cm-1 and an accumulation of 200 spectra.

Micro computed tomography (microCT)

The microCT investigation allowed the comparison of the formation of new mineral phase, in dynamic versus static experimental set up. Cylindrical samples dried after the incubation in SBF were scanned.

A Sky Scan 1272 microCT (Bruker) was used to visualize mineral deposits generated in the polymeric matrix. The equipment uses an X-Ray source with peak energies ranging from 20-100 kV and a 6-position automatic filter changer. The samples were fixed on the sample holder using modeling clay. All samples were scanned without filter at a voltage of 50 kV and an emission current of 175 µA. The images were registered at a resolution of 2452 x 1640 and a pixel size of 7.000 µm, with a rotation step of 0.4 degrees. All images were processed using CT NRecon software and reconstructed as a 3D object using CTVox. DataViewer was used to visualize 2D slices of the samples and CT Analyser (Version 1.17.7.2) software was used for quantitative data regarding the samples’ opacity.

Morpho-structural characterization

Morphological and microstructural characterization of the hydrogels was performed through scanning electron microscopy (SEM) using QUANTA INSPECT F SEM device equipped with a field emission gun with 1.2 nm resolution and a and with an X-ray energy dispersive spectrometer (EDAX). The samples were coated with a thin layer of gold prior to analysis.

3. Results and discussions

The present study aims the evaluation of the potential of the mineral phase of cuttlefish bone to act as biomimetic mineralization initiator when embedded in an inert polymeric matrix in order to be subsequently used in the field of hard tissue regeneration or repair. In this respect, cuttlefish bone fragments were immobilized into poly(2-hydroxyethyl methacrylate (samples further referred to as CB-pHEMA) and their mineralization was further investigated using two experimental conditions: uniaxial compression under continuous flow of SBF and conventional static incubation in SBF.

Layered scaffolds were obtained, containing a pHEMA hydrogel upper layer and a composite bottom layer following mineral sedimentation.

Fig.2. Representative SEM micrograph of a longitudinal section of sample:

a – pHEMA matrix, b – composite region containing CB.

The scaffolds were subjected to uniaxial compression under continuous flow of SBF and the formation of a new biomimetic mineral phase was investigated through microCT, SEM and FTIR spectroscopy. The properties of the synthesized materials were correlated with the biogenous mineral content.

Micro computed tomography (microCT)

Micro-computed tomography (microCT) was used to visualize mineral deposits generated in the polymeric matrix. MicroCT images of the samples prior to incubation showed that the mineral deposited during the synthesis generated non-homogeneous specimens.

MicroCT images provided microstructural details of the layered samples confirming the precipitation of CB fragments in the pHEMA matrix. The analysis of the samples incubated in SBF revealed different responses in terms of mineralization. When comparing the two types of mineralization tests – static vs. dynamic – the microCT investigation showed that the mineralization was more efficient in dynamic conditions (Fig.3.). The images revealed clusters of newly formed mineral phase in the inert pHEMA hydrogel only in the samples submitted to the biodynamic testing.

Fig.3. Representative microCT images revealing the formation of new mineral phase after

2 weeks incubation in SBF

Staining of the mineral phase

The samples were subjected to Von Kossa assay before and after incubation in SBF. The control sample showed only slight traces of mineral, probably due to the physical attachment of salt on the polymeric surface (Fig.4., upper row). After two weeks of incubation of the pHEMA-CB10 series, in both static and dynamic set-up, the samples showed a drastic change of colour when compared to the un-incubated pHEMA-CB10 sample. As shown in fig.4., lower row, the un-incubated sample has a brownish aspect, while pHEMA-CB10_S and pHEMA-CB10_D are almost black in the mineral region. Moreover, the digital images registered for these samples are in good agreement with the microCT images, showing that testing in dynamic conditions led to the formation of mineral in the middle region of the specimen.

Fig.4. Digital images of the Von Kossa stained samples

Fourier transform infrared (FTIR) analysis

FTIR was used in order to evaluate the formation of new mineral into the incubated samples. To this end, spectra were registered before and after incubation in SBF. The samples in the FTIR analysis were analyzed in the area where the CB was loaded where the mineralization formed.

Fig.5. FTIR spectra for pHEMA, pHEMA_S and pHEMA_D

The spectrum of pHEMA displayed typical vibrations at 1.716, 1.450 and 1.072 cm-1, assigned to O-H stretching, C-H symmetric and asymmetric stretching, as well as to C=O stretching, respectively, of mixed ester and ether origin, but vibrations at 1.268 and 1.165 cm-1 assigned to C-O stretching. The three spectra of pHEMA, pHEMA_S and pHEMA_D, respectively (Fig.5.) don’t show any significant differences, stating for the inertness of the un-loaded polymer at mineralization.

Fig.6. FTIR spectra for pHEMA-CB10, pHEMA-CB10_S, pHEMA-CB10_D and CB

Aragonite and chitin were detected by FTIR analysis of the cuttlefish bone. Besides the bands characteristic for the aragonite: 1.080 cm-1, 711 cm-1 and 852 cm-1 for C-O in plane band, but there are bands derived from interval vibrations of CO3-ions at 1.442 cm-1. A weak contribution to the cuttlefish bone spectrum from chitin is observed in the range 1.080 cm-1 (C-O stretching).

All CB-loaded samples presented typical O-H vibrations at 1.716 cm-1 (specific for pHEMA spectrum) and the peak characteristic for aragonite at 852 cm-1; the peak specific for chitin, present in the CB at 1.080 cm-1 is also present in the pHEMA-CB10 samples at 1.072 cm-1. All the peaks are visible for samples pHEMA-CB10, pHEMA-CB10_S and pHEMA-CB10_D, where CB is incorporated in the polymeric matrix (Fig.6.).

Morpho-structural characterization

SEM was used to evaluate the morphology and microstructure of the materials. New mineral was only detected on the samples incubated under dynamic conditions (Fig.7.). The microstructure suggests formation of small deposits of nanoapatite during testing.

Fig.7. SEM micrographs revealing the influence of the testing conditions (dynamic – D versus static – S) on the mineralization potential of pHEMA-CB10 samples (longitudinal sections); pHEMA-CB10 was used as a control: a – general appearance with two layers (bottom layer (*): pHEMA hydrogel and top layer (**): pHEMA-CB10 composite); b-e – morpho-and microstructural details of the top composite layer in ETD mode – b,e and BSED mode – c,d; white arrow – newly formed mineral ( ) during incubation in SBF;

white circle – nanostructured newly formed mineral phase

In the SEM analysis, samples incubated under dynamic conditions can be observed newly formed mineral during incubation in SBF, but also nanostructured newly structured mineral phase, and samples incubated under static conditions can be observed areas with newly formed mineral, but much less.

Fig.8. EDX spectra of the testing conditions on the mineralization potential of pHEMA-CB10 samples (longitudinal sections): a) dynamic – D versus b) static – S; m1 – pHEMA matrix, m2 – newly formed mineral

The potential of CB to be used as promoter of biomimetic mineralization was confirmed with SEM-EDX mapping. The dynamic testing stimulates more efficient mineralization when compared to static experiments.

After incubation it is observed in two samples in EDAX peak intense Ca but also the appearance of a characteristic peak P, it is more intense in the sample incubated under dynamic conditions because P formed Ca.

4. Conclusions

The present study aims the evaluation of the potential of the mineral phase of cuttlefish bone to act as biomimetic mineralization initiator when embedded in an inert polymeric matrix in order to be subsequently used in the field of hard tissue regeneration or repair. In this respect, cuttlefish bone fragments were immobilized into poly(2-hydroxyethyl methacrylate (samples further referred to as CB-pHEMA) and their mineralization was further investigated using two experimental conditions: uniaxial compression under continuous flow of SBF and conventional static incubation in SBF.

Layered scaffolds were obtained, containing a pHEMA hydrogel upper layer and a composite bottom layer following mineral sedimentation.

This study shows the potential of cuttlefish bone to be used as a biogenous mineral. The properties of an inert matrix based on pHEMA loaded with CB were compared.

The potential of CB to be used as promoter of biomimetic mineralization was confirmed with SEM-EDX mapping. The dynamic testing stimulates more efficient mineralization when compared to static experiments. An improvement of the mechanical properties of the matrix loaded with CB can be noticed, the dispersion of the biogenous mineral is best observed at SEM.

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