Synthesis and Growth of HAp Crystals

Synthesis and Growth of pass off CrystalsResults and discussionsSynthesis of HAp/Chitosan/Dopamine filmsTwo types of HAp-chitosan multiform films were lively HAp-chitosan films with and without dopamine (Figure 1). The angle ratio between HAp and chitosan in the films was fixed to 50 wt% since the HAp mental object 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 in addition generated, but this composite was too brittle to be used as fractious tissue substitute. HAp-chitosan films were successfully casted and the films were slightly yellow in people of colour. 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 foregoing studies, wet precipitation methods were carried out to synthesize a nano-HAp in the s traw man of chitosan and its derivatives13,16. Chitosan strongly interacted with HAp and adjust the anisotropic growth of crystalline HA. In addition, it was revealed that dopamine canful 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 ahead conducting the mechanical analysis and water uptake analysis.FTIR analysis and Wide-angle X-Ray DiffractionFTIR spectra of the composite films be 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 irrespective 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 hydr oxyl (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 de shaping. The band appearing at 1599 cm-1is attributed to amide II bands. Both amide 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. More everyplace, both of aromatic C=C and COO bands were also observed at 1600-1650 cm-1FTIR is an appropriate technique to observe the composite constituent interaction. It measures the frequencies at which chemical functional groups absorb as the nu mber of the samples chemical interaction. In this regards, the appearing 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 by dint of certain interaction. In addition, chitosan interaction with ions by means of phosphorylation were also identified by the emergence bring up at 1220 cm-1 and an increase 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 be given to undergo phosphorylation in acid, basic and neutral solutions. In the presence of the phosphorilated groups, chitosan can strongly bind with liaise form of HAp, amorphous atomic number 20 phosphate (ACP), and impose constraints of ACP subsequently lead to crystalline HAp formation13. Furthermore, chelation of calcium ions by phosphate functionalities may also induce the formatio n 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 grammatical construction (Ca10(PO4)6) with characteristic peaks at 2 regions of 26, 29, 32-34, 40, 46-54, 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 write down than 100% HAp powder due to the presence of chitosan. The broad peak close to 20 is an indicative peak for chitosan in the composite film regardless of dopamine addition13. Interestingly, some tell apart 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 2 of 26 , 32 , 33 respectively) were measured. The ratio of the measured d iffraction intensity of c-axis (002) to another direction was used to take 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 advantageous orientation of the HAp growth in the c-axis was significantly increased with the presence of dopamine. Additional broad peaks (10 15) 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 ResultTo 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 pres ence 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 plausibly 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 resul t 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 homogeneousness 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 plaza of HAp related to the absorbability and fracture toughness of the samples. Higher aspect ratio known to have best(p) adsorbability since it is proportional to the surface area of rods 3, which are beneficial for carrel attachment. However, aspect ratio alone is pitiful 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 computation 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 incre asing 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 body to be proved the cytotoxicity of the dopamine-containing composites.