Synthesis and Growth of HAp Crystals

Synthesis and Growth of HAp Crystals Results and discussions Synthesis of HAp/Chitosan/Dopamine films Two types of HAp-chitosan composite films were prepared: HAp-chitosan films with and without dopamine (Figure 1). The weight ratio between HAp and chitosan in the films was fixed to 50 wt% since the HAp content in human bones is about 50-70wt% in dry weight28-30. In comparison with the 50 wt% HAp-chitosan, 70 wt% HAp-chitosan films containing dopamine were also generated, but this composite was too brittle to be used as hard tissue substitute. HAp-chitosan films were successfully casted and the films were slightly yellow in color. Dopamine containing HAp-chitosan films were also casted and the color was changed to dark brown. The color alteration of the films was due to the oxidation of dopamine that formed phenolic tanning compounds19,31. Based on the previous studies, wet precipitation methods were carried out to synthesize a nano-HAp in the presence of chitosan and its derivatives13,16. Chitosan strongly interacted with HAp and regulated the anisotropic growth of crystalline HA. I n addition, it was revealed that dopamine can also facilitate the growth of the HAp crystals along the c-axis21. Therefore, the synthesis and growth of HAp crystals in the presence of both chitosan and dopamine were examined by FTIR, XRD and SEM before conducting the mechanical analysis and water uptake analysis. FTIR analysis and Wide-angle X-Ray Diffraction FTIR spectra of the composite films are shown in Figure 1. Generally, hydroxyl and phosphate bands of HAp and characteristic peaks of chitosan are present in all of the composites FTIR spectra regardless of dopamine addition. More specifically, FTIR spectra of the composites showed the identity bands of HAp; stretching and bending vibration modes from the phosphate groups () were identified at absorption bands of 898 1095 cm-1 and 477 660 cm-1 respectively. The combination band of hydroxyl (O-H) bending and libration modes was observed at 630 cm-1. Furthermore, observed band at about 3600 cm-1 indicated the O-H stretching. On the other hand, several typical chitosan absorption peaks of 1150, 1375, 1640 cm-1and 2900 cm-1were observed in the chitosan containing composites. Peak at 1150 cm-1 was indicating glucosamine unit. The peak observed at 1640 cm-1 represents amide I (C=O) and anti-symmetric NH3 deformation. The band appearing at 1599 cm-1is attributed to amide II bands. Both am ide I and amide II show a hydrogen bond between –NH2 and –OH of HAp. Peak at 2900 cm-1 represent the –CH2 backbone. In the dopamine containing composite, the polyphenolic content resembled by phenolic COH peak that was discerned at 1260 cm-1. Moreover, both of aromatic C=C and COO bands were also observed at 1600-1650 cm-1 FTIR is an appropriate technique to observe the composite constituent interaction. It measures the frequencies at which chemical functional groups absorb as the result of the sample’s chemical interaction. In this regards, the appearance of glucosamine unit band at 1150 cm-1 that overlap with the stretching vibrations bands of HAp indicated that HAp crystals were formed on the chitosan molecules through certain interaction. In addition, chitosan interaction with ions by means of phosphorylation were also identified by the emergence shoulder at 1220 cm-1 and an increased at 1064 cm-1 absorption peaks of the chitosan containing composites spectra12. Chitosan has great affinity to react with ions without pH dependent13. This interaction makes chitosan tend to undergo phosphorylation in acid, basic and neutral solutions. In the presence of the phosphorilated groups, chitosan can strongly bind with intermediate form of HAp, amorphous calcium phosphate (ACP), and impose constrain ts of ACP subsequently lead to crystalline HAp formation13. Furthermore, chelation of calcium ions by phosphate functionalities may also induce the formation of crystalline HAp. The XRD spectra of the samples with or without dopamine also support the existence of HAp crystalline phase in the composite films. Most peaks in the XRD spectra of the samples could be indexed to the known HAp structure (Ca10(PO4)6) with characteristic peaks at 2ÃŽ ¸ regions of 26Â º, 29Â º, 32-34Â º, 40Â º, 46-54Â º, which are consistent with HAp phase (JCPDF #09-0432), confirming that the phase was formed in all samples13,32. However, the crystallinity of HAp in the chitosan-HAp composite films was lower than 100% HAp powder due to the presence of chitosan. The broad peak around 20Â º is an indicative peak for chitosan in the composite film regardless of dopamine addition13. Interestingly, some evidence which support anisotropic growth of Hap in the presence of dopamine were shown in the XRD spectra. The intensities of HAP diffractions relating to (002), (300) and (211) peaks (at 2ÃŽ ¸ of 26 Â º, 32 Â º, 33 Â º respectively) were measured. The ratio of the measured diffra ction intensity of c-axis (002) to another direction was used to determine the orientation degree. The XRD results after Gaussian Fit indicates that the (002) to (300) intensity ratio of 50% HAp samples with and without dopamine was 0.17 and 0.45 respectively. This indicated the preferential orientation of the HAp growth in the c-axis was significantly increased with the presence of dopamine. Additional broad peaks (~10Â º ~15Â º) were observed in the dopamine containing film. It indicates that the addition of dopamine induced structural changes in d-spacing over 0.6-0.9 nm in the film due to dopamine-mediated crosslinking, or dopamine-mediated HAp growth. Overall, XRD spectra suggest that the aspect ratio and anisotropy increased in the dopamine containing HAp/chitosan composite. SEM, TEM analysis and Cell Test Result To examine the effect of dopamine addition on the surface morphology, the dopamine-containing films were observed under SEM and TEM (Figure 3 and 4). The figures show a presence of nanorod particles in the composites with narrow and uniform particle size distribution in all samples. In the absence of dopamine, this structure is likely formed due to phosphorylation of chitosan which bind with phosphate precursor compounds and modulate the crystallization of HAp13,16. In the presence of dopamine, the aspect ratio of HAp was increased up to ~4.7 fold compared with control HAp in the absence of chitosan nor dopamine (figure x). For a comparison, in the 50 wt% composite, the aspect ratios are 2.4 and 4.5, without and with dopamine crosslink respectively. The dopamine effect is probably because of catecholic group from the dopamine bind with Ca2+ in HAp crystals formation 21. The pKa dopamine is ~8.9, dopamine was added while the pH decreased from ~8 to 4.2 The protonated cathecholic group of dopamine are possibly involved in HAp formation and regulate the one-dimensional growth of HAp crystals. This phenomenon is well agreed as the previous experiment result that polydopamine addition on HAp provides mechanism for surface-anchored catecholamine moieties to enrich the interface with calcium ions, facilitating the formation of hydroxyapatite crystals19.The addition of dopamine not only guides the anisotropic directional growth of hydroxyapatite crystals which increased its aspect ratio, but also changed the homogeneity of the grain distribution and shape of the nanostructure (Figure 5). To study the effect of HAp content on the aspect ratio of the nanostructure, the 25% and 70 wt% HAp-chitosan film with/without dopamine was synthesized. As the results, the aspect ratio is increases with increasing of HAp weight %. The 70 wt% with dopamine containing sample showed the highest aspect ratio. (Supporting figure X). The aspect ratio is a significant property of HAp related to the absorbability and fracture toughness of the samples. Higher aspect ratio known to have better adsorbability since it is proportional to the surface area of rods3, which are beneficial for cell attachment. However, aspect ratio alone is inadequate to identify the cellular affinity of sample, as a previous study suggested that surface roughness can also play an important role33. It was also found that aspect ratio and surface roughness of the composite film have a significant effect on the cell attachment and proliferation1. We tested MC-3T3 cells (mouse pre-osteoblast cell line) proliferation on the HAp-chitosan composite surfaces. To measure the dependence of MC-3T3 cell viability and proliferation on surface materials quantitatively, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] based cell counting assay was performed. WST-8 produced yellow-colored product (formazan) when it was reduced by dehydrogenases in living cells34,35. The levels of cell viability and proliferation on the sample that contains dopamine were slightly lower than others (Figure 6) suggesting that the increasing of HAps aspect ratio is not always beneficial because the cell viability was rather decreased. Nevertheless, the number of viable cells in the sample is still increased, yet at a slightly lower rate than the other sample. This is implying that this material would probably have no strong cytotoxicity. However, in vivo testing is remains to be proved the cytotoxicit y of the dopamine-containing composites.