Organically modified clay supported chitosan/hydroxyapatite-zinc oxide nanocomposites with enhanced mechanical and biological properties for the application in bone tissue engineering
Introduction
In orthopedic implant, bone tissue engineering has become a promising technique for bone regeneration [1], [2] and gradually replaces some traditional methods like allografts and autografts as these traditional methods are associated with various complications such as possibility of infection, inappropriate curing from invasive surgeries, unable to seal gap totally due to inadequate bone donation and donor site morbidity [3], [4]. Now a day, development of synthetic polymeric scaffolds plays a vital role in bone tissue engineering that can mimic the characteristic features of natural bone extracellular matrixes for the repairing and regeneration of tissues. The natural bone mainly consists of inorganic hydroxyapatite (Ca10(PO4)6(OH)2) and organic collagen fibrils. The current trend of research is to develop a synthetic nanocomposite, mimicking the characteristics of natural bone [5], [6]. The ideal nanocomposite material should have biocompatibility, suitable mechanical and antimicrobial properties, nontoxicity and surface functionality to favor cell proliferation [7].
Many organic-inorganic composite materials have been developed for the application in bone tissue engineering. Among the various organic-inorganic composites, chitosan (CTS) and hydroxyapatite (HAP) has been utilized extensively due to their excellent biocompatibility with human body [8]. CTS is a natural biodegradable polysaccharide, serves as a good bioactive biomaterial in bone grafting due to its biocompatibility, biodegradability, non-toxicity and non-immunogenicity [9], [10]. CTS is a natural polysaccharide in which glucosamine and N-acetylglucosamine units are connected via β (1–4) linkage. It is a widely used flexible, biocompatible, biodegradable and heat resistant biopolymer [11]. However, the reports on osteoconductivity of CTS are limited. Gkioni et al. have shown that the addition of HAP to CTS enhances the osteoconductivity by generating sites for calcification [12]. Also, due to chemical compositional similarity like natural bone, HAP has attracted much attention as a biomaterial for bone tissue engineering application. HAP is a highly biocompatible, biostable and bio-adoptable natural ceramic, have osteoconductive properties and non-viral effects [13]. HAP also facilitates new bone formation without resorption and interaction with the living system. Moreover, nanostructured HAP has high surface area and enhanced bioactivity. Nevertheless, incorporation of HAP into CTS results in poor mechanical strength as the addition of HAP into the composite increases its brittleness [14].
Montmorillonite (MMT) is a layered aluminosilicate [Na0.7(Al3.3Mg0.7)Si8O20 (OH)4·nH2O]. It has been reported that the addition of lower content of MMT can also improve the mechanical properties and thermal stability of the polymeric composite [15], [16]. Also, MMT has high specific surface area and large aspect ratio [17]. Thus, along with HAP, organically modified MMT (OMMT) has also been chosen to incorporate into CTS matrix to improve the properties required to mimic natural bone. OMMT has been prepared from MMT using alkyl ammonium salts in order to improve its miscibility with polymer matrix. In addition, strong antimicrobial properties of OMMT were observed due to the presence of quaternary ammonium salt [18].
Until now, very limited attempt has been made to fabricate CTS/OMMT/HAP composite [19], [20]. Kar et al. fabricated a CTS/OMMT/HAP composite scaffold that was able to show 0.56 MPa of tensile strength compared to pure CTS (0.36 MPa) and CTS-OMMT (0.38 MPa) [19]. Further enhancement in tensile strength is highly required to obtain suitable bone-like mechanical strength [21]. In addition to nano-HAP, incorporation of nanoclay (OMMT) into the nanocomposite could also lead to its brittleness as reported previously [22]. As both nano-HAP and OMMT are chosen to be incorporated into CTS matrix, achieving good tensile strength for our proposed nanocomposite would be a challenge. It has been reported that the tensile strength of human trabecular bone can be up to 50 MPa [21] and therefore, it is necessary to design a nanocomposite having similar tensile strength for its application in bone tissue engineering. Incorporation of trace metal ions has been found to enhance the mechanical and biological performance of composites [23], [24], [25]. Among various metals, zinc is attractive because it is present abundantly as a trace element in bone minerals; it promotes the bone density and prevents bone loss [26]. We have recently showed that addition of ZnO NPs into CTS, other polymers (PEG and PVA) along with HAP enhanced the mechanical properties [23], [24]. We have obtained maximum 15.83 MPa and 20.25 MPa of tensile strengths after addition of PEG and PVA respectively into CTS-HAP-ZnO nanocomposites. A major obstacle for the use of ZnO NPs is their toxicity as observed in a previous report [27]. Thus, the challenge lies in the development of a nontoxic ZnO integrated nanohybrid composite for bone tissue engineering application. We have hypothesized that the incorporation of a minimum amount (0.1 wt%) of ZnO NPs in the nanocomposites should not possess any toxic effect to the body.
To date, there has been no attempt at putting CTS, OMMT, HAP and ZnO together in a single nanocomposite system. In this work, we have developed novel biomimetic nanocomposites from CTS, OMMT, HAP and ZnO, considering the above properties of these materials. Presence of the individual components into these nanocomposites was confirmed by Fourier transform infrared spectroscopy (FT-IR) and powder X-ray diffraction (XRD). Morphology of these nanocomposites was analyzed using scanning electron microscopy (SEM) and transmission electrom microscopy (TEM). Moreover, mechanical properties, antibacterial properties and in vitro biological studies such as water absorption study, pH study, haemolytic assay and human osteoblastic cell proliferation study were carried out to establish their relevance as a biomaterial for bone tissue engineering applications.
Section snippets
Materials
CTS (molecular weight: 230 kDa, degree of deacetylation: 86%) was purchased from Acros Organics. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), diammonium phosphate ((NH4)2HPO4), and ammonium hydroxide (NH4OH, 25%) were purchased from Merck. Zinc acetate (Zn(OAc)2) and sodium hydroxide (NaOH) were purchased from HiMedia Private Ltd. Montmorillonite (K-10) (MMT) and cetyl trimethyl ammonium bromide (CTAB) were purchased from Aldrich Chemical Company, USA. Human osteoblastic MG-63 cells were
FT-IR studies
In the FT-IR spectra of ZnCMH I, ZnCMH II and ZnCMH III (Fig. 1), absorption bands were appeared around 3369–3262 cm−1, 3360–3271 cm−1, 3370–3264 cm−1 respectively due to the OH stretching frequency of CTS. Presence of OMMT was confirmed by the absorption bands observed in the range of 1455–1472 cm−1. The absorption peaks of OH bending for the interlayer water of OMMT in ZnCMH I–III were observed around 1711 cm−1. In addition, broad bands appeared in the range of 1163–912 cm−1, 1168–913 cm−1 and 1172
Discussion
In this paper, biomimetic CTS/OMMT/HAP-ZnO nanocomposites were developed (ZnCMH I-III) for the first time and characterized by FT-IR, XRD SEM and TEM. The mechanical, antibacterial and water absorption properties along with pH study, haemolytic assay and in vitro cell proliferation study of ZnCMH I-III were investigated to obtain an ideal bone-like composite. A control sample (ZnCH) was also prepared in the absence of OMMT, by taking only CTS and HAP-ZnO and the composite was studied thoroughly
Conclusions
In conclusion, biomimetic CTS/OMMT/HAP-ZnO nanocomposites (ZnCMH I-III) were developed to obtain suitable mechanical and biological properties for bone tissue engineering application. Compositions of these nanocomposites were analyzed by FT-IR and powder XRD. SEM and TEM images showed uniform distribution of OMMT and nano-HAP-ZnO into CTS matrix. Water absorption study of ZnCMH I-III revealed that with increasing the content of hydrophobic OMMT (from ZnCMH I to ZnCMH III), the swelling capacity
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