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Published in final edited form as: Colloids Surf B Biointerfaces. 2018 Feb 15;165:158–164. doi: 10.1016/j.colsurfb.2018.02.034

Nano-scale Characterization of Nano-Hydroxyapatite Incorporated Chitosan Particles for Bone Repair

Bipin Gaihre 1, Suren Uswatta 1, Ambalangodage C Jayasuriya 1,2,*
PMCID: PMC5987766  NIHMSID: NIHMS945704  PMID: 29477936

Abstract

In this study, injectable porous spherical particles were fabricated using chitosan (CS) biopolymer, sodium tripolyphosphate (TPP), and nano-hydroxyapatite (nHA). TPP was primarily used as an ionic crosslinker to crosslink 2% (w/v) CS droplets. 2% (w/v) nHA was used to prepare nHA incorporated particles. The surface morphological properties and nanomechanical properties such as topography, deformation, adhesion, and dissipation of CS particles with and without nHA were studied using contact mode and peakforce quantitative nanomechanical property mapping mode in atomic force microscopy. The nHA spots have higher density than CS which leads to higher forces acting on the probe tip and higher energy dissipation to lift the tip from nHA areas. The cumulative release data showed that about 87% of total BMP-2 encapsulated within the particles was released by third week of experiment period. Degradation study was conducted to understand how the particles degradation occurs in the presence of phosphate buffered saline with continues shaking in an incubator at 37°C. In addition, BMP-2 release from the 2% nHA/CS particles was studied over a three weeks period and found that BMP-2 release was governed by the simple diffusion rather than the degradation of particles.

Keywords: Nano-hydroxyapatite, Atomic Force Microscopy, Chitosan, Particles, Topography, Bone Morphogenetic Protein-2

Graphical abstract

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INTRODUCTION

Development of injectable scaffolds for bone tissue regeneration is important since these scaffolds can be implanted using minimally invasive surgeries (MIS) avoiding the problematic and expensive open surgeries [1–2]. Different forms of injectable scaffolds have been developed including microparticles, hydrogel, nano-composite films and nanoparticles using variety of biomaterials such as natural and synthetic polymers, biocomposites and bioceramics to apply in bone repair [3–7].

Chitosan (CS) is a semi crystalline polysaccharide derived from chitin which is the second most abundant natural biopolymer commonly found in the shells of marine crustaceans and cell walls of fungi [8–9]. CS can be ionically cross-linked through its cationic amine group with anionic phosphate group in sodium tripolyphosphate (TPP) to obtain rigid CS scaffolds [10–11]. The resultant cross-linked CS scaffolds exhibited higher structural integrity and provided controllability of release of encapsulated growth factors [10,12–14]. Hydroxyapatite (HA) resembles to apatite in bone mineral. Therefore, HA is incorporated with CS and HA-CS scaffolds have shown to increase the mechanical properties of CS alone [15–16]. HA-CS scaffolds have shown good cytocompatibilty with osteoblasts, which is an indication for HA to be used in bone tissue engineering applications [17–18]. In this study, we developed nHA incorporated CS particles, which are more versatile than the other form of scaffolds. These particles can be used as carrier for the drugs and growth factors that can release these agents in a controlled manner at the defect site. They can also be used to provide suitable porous surface and sufficient mechanical support for the ingrowth of cells and tissue formation.

Atomic force microscopy (AFM) can be used to collect detailed information about surface and interface properties in micro and sub-micron level [19–20]. Peak force quantitative nanomechanical property mapping (PeakForce QNM) is a recently developed imaging technique. In PeakForce QNM, each tip-sample interaction is analyzed to extract quantitative nanomechanical properties including adhesion, deformation, and dissipation. PeakForce QNM allows each of these properties to be mapped quantitatively at high resolution while still collecting standard topography images at normal imaging rates. PeakForce QNM works well on a wide range of sample types from soft delicate materials with modulus < 1 MPa all the way up to materials with modulus > 50 MPa [21].

Bone morphogenetic protein-2 (BMP-2) is a member of the BMP subfamily of the transforming growth factor-β super family [22–23]. BMP-2 is FDA approved to use for a wide range of bone defects repair including sinus augmentations, localized alveolar ridge augmentations, spine fusion and tibia fractures. It is important to develop controlled release delivery system for BMP-2 to maintain the efficacy and safety.

In this paper, CS particles were prepared using a simple coacervation method and those particles were ionically cross-linked using TPP solution. The surface morphological properties and nanomechanical properties such as topography, deformation, adhesion, and dissipation of the CS only and nHA/CS particles were studied using AFM. In addition, BMP-2 release from the 2% nHA/CS particles was studied over a three weeks period.

MATERIALS AND METHODS

Materials

Medium molecular weight CS (85% deacetylation), hydroxyapatite nanopowder (nHA, < 200 nm particle size), sodium tripolyphosphate (TPP) and acetic acid were purchased from Sigma-Aldrich (USA). BMP-2 and relevant ELISA kit were purchased from R&D SYSTEMS (USA).

Particles Preparation

The particles were prepared by coacervation technique followed by lyophilizaton similar to our previous study [24]. Briefly, 200 mg of medium molecular weight CS was measured and added into 10 ml of 1% (v/v) acetic acid in a 50 ml beaker. Mixture was stirred using a magnetic stirrer at 300 rpm. The resultant mixture is 2% (w/v) CS solution. 2% (w/v) nHA powder was added to the CS solution and subjected to the vigorous mixing using magnetic stirrer. The nHA powder was further dispersed in the CS solution by sonication. Sonicated nHA/CS suspensions were filtered by 50 μm nylon mesh to remove nHA agglomerates thus to avoid clogging in the needle. The suspension was dripped into 27.18 mM TPP/deionized water solution (crosslinking medium) using 30 gauge needles and stirred at 600 rpm. After 30 min, TPP crosslinked nHA/CS beads were filtered out from the TPP/deionized water solutions and lyophilized at −52°C and 0.02 mbar pressure for 24 h. Portion of lyophilized particles from each sample were soaked in 300 ml distilled water and stirred at 300 rpm for an hour. Then particles were separated and dried inside the chemical hood for 24 h.

Atomic Force Microscopy

Atomic Force Microscopy (AFM) imaging was performed with Bruker MultiMode 8 with J scanner. The NanoScope software was used to adjust parameters for different scanning modes and NanoScope Analysis software was used to analyze the scanned images. The particles were scanned using contact mode and PeakForce Quantitative Nanomechanical property mapping (PeakForce QNM) mode.

Contact Mode

Particles were scanned using Bruker SNL-10 probes with Silicon tip over Nitride lever. SNL-10 probes have four cantilevers A, B, C and D with resonance frequency 65, 23, 56 and 18 kHz, and spring constant 0.35, 0.12, 0.24 and 0.06 N/m. In this experiment we have chosen the cantilever A as it has produced better quality images in our test experiments. The scanning parameters were set to 2.03 Hz for scan rate, 1.54 μm for scan size, 1024 for sample lines, 357.79 mV for amplitude set point and 122.38 for drive amplitude. Five particles from each group were scanned. The scanned images were retrieved using NanoScope Analysis software and were processed to determine the roughness (Rq) values and 3-D topography representation.

PeakForce QNM Mode

In this mode we have used SCANASYST-AIR probes (Bruker) with Silicon tip on Nitride lever. These probes have cantilever with a length of 115 μm, width of 25 μm, tip of 2 nm, resonance frequency of 70 kHz and 0.4 N/m spring constant. The scanning parameters were set to 1 Hz scan rate, 1 μm scan size, 512 scan lines, 1 nN peak force set point and 150 nm peak force amplitude. The sample was first scanned along 5 μm scan size to get a clean scan. Three different regions of 1 μm scan size along the 5 μm scan was then selected and collected for the analysis. Five particles from each group were taken in order to generate the quantitative data for adhesion, dissipation and deformation.

Degradation of Particles

Degradation study was conducted to understand how the particles surface degradation occurs in the presence of PBS with continues shaking in an incubator at 37°C. 20 mg of 2% nHA/CS particles were immersed in glass vials containing 1 ml of PBS and kept on a plate shaker at 150 rpm in an incubator. Experiment was conducted on 8 samples. PBS was removed from the vial after 30 days and remaining particles were lyophilized for 24 h at −52°C and 0.02 mbar pressure. After 24 h, weight of the samples was measured. Then again samples were immersed in 1 ml of PBS in glass vial and kept it in the shaker at 150 rpm. This step was repeated at every 30th day until total of 120 days.

Release Kinetics of BMP-2

1 μg of BMP-2 dissolved in 4mM HCl was added to 10 ml (BMP-2 concentration of 100 ng/ml) of 2% (w/v) nHA/CS solutions and mixed by stirring for 5 min at 0°C. Then nHA/CS/BMP-2 solutions were dripped into crosslinking medium and stirred at 300 rpm. After 4 h, nHA/CS/BMP-2 beads were separated and lyophilized using similar conditions as described earlier.

To study the release of BMP-2 from lyophilized particles, 10 mg of nHA/CS/BMP-2 particles were immersed in 2 ml of PBS in a glass vials and incubated at 37°C on an orbital shaker at 50 rpm. On day 1, 3, 5, 7, 10, 14 and 22, the PBS containing released BMP-2 was collected and replaced with fresh 2 ml of PBS. The collected samples were stored at −20°C before ELISA was conducted for the quantification of released BMP-2 from the particles (n=3). Optical density of wells in 96 well plate was measured using Molecular Devices Spectramax190 plate reader. In order to find the total BMP-2 encapsulated, the particles at the end of 22nd day, were transferred into 1% acetic acid and incubated on same conditions for 24 h, to dissolve those particles. ELISA was again conducted to quantify the BMP-2 remaining in the solution.

Statistical Analysis

IBM SPSS Statistics version 23 software was used to compare data using one-way analysis of variance (ANOVA) with 95% confidence interval and Tukey’s Post Hoc single-step multiple statistical comparison procedure.

RESULTS

Contact AFM

AFM contact mode is ideal for topographic imaging because probe tip is always in contact with the sample. 3-D topography image of the particles captured using contact mode is shown in the figure 1. It can be seen that surface of both of the particles was rough with many nano peaks and pores. The dark spots represent deeper areas on the surface while light spots represent peaks on the surface. The average Rq for the CS only particles was 41.1±2.61 nm and that for CS/nHA particles was 24.95±2.87 nm. In AFM contact mode, an initial probe contact point on surface is considered as the base height, and the peaks and pores height are measured from this initial contact point. The lower roughness value of CS/nHA particles compared to CS only particles might be due to the reduction of pores height caused by the deposition of nHA on pore walls.

Figure 1.

Figure 1

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3-D topography image of (A) only CS and (B) CS/nHA lyophilized particles captured using contact mode imaging.

PeakForce QNM

PeakForce QNM mode of AFM was used to capture and measure the surface properties of the particles. Figure 2 shows the images obtained from different channels during a single scan using PeakForce QNM for CS only particles. Height image is shown in figure 2A and the 3-D representation of this image is shown in figure 2E. Dark areas of this image shows deeper areas on the surface while light colored areas show peaks compared to the initial contact point of the probe tip at the center of the image. The darker region along the left boundary, however, shows the curvature of the particle rather than actual pores on the surface. As the probe tip moves away from the center of the image, unlike contact mode, image tends to turn darker due to the curvature of the particle. The intensity of deformation, shown in deformation mapping (Fig 2B) along the surface of particle, appears to be uniform regardless of the location of the topography image. Adhesion map (Fig 2C) shows the marked contrast in adhesive force between the porous and non-porous region. The adhesion force was higher along the deeper region compared to superficial regions. The average adhesion force between the tip and CS only particle surface was 1.28±0.09 nN with the average maximum force of 14.2±0.94 nN. The dissipation map shown in figure 2D looks similar to the adhesion map with more energy dissipation taking place along the deeper regions. The average energy dissipation was 128.75±10.07 eV for these particles.

Figure 2.

Figure 2

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AFM images of CS particle captured using PeakForce QNM: (A) the topography of the particle, (B) the deformation of the particle during the scanning, (C) the adhesion force on the probe tip during the scanning of the image, (D) the energy dissipated or used to move the probe tip over the particle. (E) 3D topography image of height image A.

Figure 3 shows the images obtained from different channels for CS/nHA particles. Figure 3A shows the height image in the range of 280 nm and its 3D representation is shown in figure 3E. The surface of these particles, compared to that of CS only particles, had irregular ridges. There was slight contrast in the deformation (Fig 3B) of the particle along the surface with the peaks having slightly bright regions. The adhesion map showed a contrast in adhesion force between the peak regions and deeper regions. As in CS only particles, the adhesion force was higher on the deeper regions. The adhesion force, however, was higher (p<0.05) for CS/nHA particles compared to CS only particles with average adhesion force of 2.37±0.73 nN and the average maximum force of 28.2±6.49 nN. The dissipation map (Fig 3D) looked similar to the adhesion map with regions having higher adhesion causing probe to dissipate more energy. The average energy dissipation was 387 eV±90.5 which was higher than that for CS only particles.

Figure 3.

Figure 3

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AFM images of 2% nHA/CS particle captured using PeakForce QNM: (A) the topography of the particle, (B) the deformation of the particle during the scanning, (C) the adhesion force on the probe tip during the scanning of the image, (D) the energy dissipated or used to move the probe tip over the particle. (E) 3D topography image of height image A.

Degradation of Particles

Degradation of particles was simulated by immersing particles in PBS and keeping the vials on a plate shaker in an incubator. Experimental results show 2% nHA/CS particle samples (20 mg) have lost 15.82% of initial weight of the particles after 120 days. Particles degradation rate is slower at first 30 days such that only 1% of initial weight has been lost. Particles have accelerated the degradation after 30 days and after 60 days average weight of particle sample is 18.73 mg. The degradation trend shows that particles continue to degrade at same rate till 120 days (Figure 5).

Figure 5.

Figure 5

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Degradation of 2% nHA/CS particles over 120 days (A) weight remaining of particles (B) Weight loss of particles.

Growth Factor Release

The cumulative release data in Figure 6A shows that about 87% of total BMP-2 encapsulated within the particles was released by third week of experiment period. For the first week of experiment, the release was higher with the release of more than 50% encapsulated BMP-2. The released amount of BMP-2 ranged from 0.8-1.2 ng for this time period with no any abrupt change in the amount released (Fig. 6B). The release, however, slowed down during the second and third week with cumulative release % staying lower than 30% of total encapsulated BMP-2. The released amount of BMP-2 was lower for this time period with amount closer to 0.6 ng and similar to first week no abrupt change in the released amount was observed for this time period as well.

Figure 6.

Figure 6

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(A) Cumulative release of BMP-2 as a percentage of total encapsulated amount on 10 mg of particles over three weeks period and (B) Amount of BMP-2 released at different time points.

DISCUSSION

In this study, the coacervation method was used to ionically crosslink nHA/CS droplets that dripped into the crosslinking media by needle and syringe. The positively charged amino groups of CS present in nHA/CS droplet and negatively charged ions in crosslinking medium instantly crosslink to produce a bead [13]. The longer crosslinking time produces particles with higher structural integrity. We showed in our previous study that the freeze drying technique used in this study allowed to develop a spherical scaffolds with distributed pores which is important for cell ingrowth and diffusion of nutrients into the scaffold. We also showed that the incorporation of nHA into the microparticles improves the compressive strength of the microparticles and that with 2% nHA had the highest strength [24]. In this study, we compared the nano-mechanical surface properties of these 2% nHA/CS microparticles with CS only microparticles. One of the major advantages of using microparticles based scaffold systems in tissue regeneration applications is the ability to effectively encapsulate the growth factors and bioactive agents into them and release them at target site [4]. In this study, we showed that 2% nHA/CS was able to release the BMP-2 in a controlled manner in in vitro conditions.

Contact mode imaging showed that the average roughness for CS only particles was higher than that for nHA/CS particles. The microparticles developed are porous with pore size ranging from 2 to 10 μm as observed in our previous study [24]. The decrease of roughness (Rq) in nHA/CS microparticles could be due to the deposition of nHA particles into the pores thereby reducing the depth height of the pores as well as decreasing the base to peak height [25].

The PeakForce QNM technology of the Bruker AFM MultiMode 8 was used to further characterize the surfaces of particles. The deformation of the particles surface was varied slightly and was mostly uniform throughout the map for both groups. While the intensity of particle deformation for CS only particles was mostly in the positive picometer (pm) range, that for the CS/nHA particles was mostly in the negative nanometer (nm) range. nHA distributed along the surface of nHA/CS particles increases the stiffness of the surface thereby decreasing the deformation of the particles. The typical force-separation curve exhibits in Figure 4. The maximum deformation of the sample is defined as the distance from the base of the deformation fit region position to the peak interaction force position caused by the probe. Deformation fit region is defined as 85% of the complete deformation region [21]. Therefore, negative deformation of the particle was due the penetration of the probe tip into the surface.

Figure 4.

Figure 4

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Typical force-separation curve obtained for the sample.

Adhesion and dissipation (Fig 2C&D, 3C&D) images show similar characteristic that the highest energy was dissipated from the spots with the highest adhesion force. The regions along the particle where the adhesion force is higher between the tip and the surface, higher energy is required to lift the tip and thus dissipation is higher. The force curve in figure 4 shows the representation of adhesion and dissipation energy. The dissipation actually represents the hysteresis between the loading and unloading curve and thus the dissipation from a material undergoing elastic deformation is low [26]. The energy dissipation from CS particles was lower than that from the nHA/CS only microparticles. This shows the effect of nHA distribution along the surface of the particles in decreasing the elastic deformation of the sample. However, the highest energy dissipation not occurred uniformly along the surface and mostly occurred from the deeper regions. This suggests that highest energy dissipated from spots where nHA was present and that nHA was mostly deposited along the porous regions of the particles. The nHA spots have higher density than CS which leads to higher forces acting on the probe tip and higher energy dissipation to lift the tip from nHA areas.

Degradation study was conducted to understand how the particles surface degradation occurs in the presence of PBS with continues shaking in an incubator at 37°C (Figure 5). Particles have degraded by 15~16% after 120 days which is promising property for a scaffold. However, our cell culture study has shown that osteoblast seeded CS particles degrade faster rate in the presence of osteoblast cells [23]. Thus, this degradation study needs to be improved by incorporating enzymes that supports degradation of CS in human physiologic conditions. According to Figure 5, the degradation of 2% nHA/CS/BMP-2 particles within 21 days is negligible. Therefore, the release of BMP-2 from the 2% nHA/CS/BMP-2 particles occurred mainly due to the diffusion controlled delivery. No burst release of BMP-2 was observed in cumulative release of BMP-2 plot (Figure 6A).

In conclusion, we have prepared porous CS and nHA/CS particles using a simple coacervation technique. These porous particles were studied with contact mode and peakforce quantitative nanomechanical property mapping mode in atomic force microscopy. The deformation of the particles was mostly uniform along the scanned surface. The adhesion and dissipation, however, varied across these regions with higher adhesion and dissipation observed along the deeper regions. Comparative analysis of adhesion force and dissipation energy showed that both of these parameters were higher for nHA/CS particles compared to CS only particles. The cumulative release data showed that about 87% of total BMP-2 encapsulated within the particles was released within three weeks period. BMP-2 release from the 2% nHA/CS particles was governed by the simple diffusion rather than the degradation of particles.

Highlights.

  • Surface of both types of particles was rough with many nano peaks and pores.

  • Adhesion force was higher along the deeper region compared to superficial regions.

  • Deformation of the particle surface was mostly uniform for both particle groups.

  • About 87% of BMP-2 encapsulated in the particles was released by third week.

Acknowledgments

We are grateful to National Institute of Health (NIH) grant number R01DE023356 for support. This work would not have been possible without their financial support.

Footnotes

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