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Synthesis of Bioactive Scaffolds Based on Polyurethane and Surface Modified Hydroxyapatite (HA) Nanocomposites for Bone Repairing and Regeneration.


Tissue engineering plays a pivotal role in regeneration of damaged or accidentally injured tissues. Numerous techniques available for the preparation of scaffolds (templates) for this purpose are autografts, allografts, xenografts [1] and a large amount of research efforts are dedicated towards advancement of these techniques. However, inspite of all the efforts, there are major drawbacks like presence of donor sites, tissue volume to harvest, feeble immune response, rejection of grafts and others adverse biofouling response. Latest efforts further favours synthesis of bio-degradable scaffolds with highly porous structure, high biocompatibility and bioactivity [1, 2].

In the recent researches it has been found that the bioactivity of scaffolds can be increased to a greater extent by introducing some special types of ceramics that themselves possess bioactive responses. These ceramics like hydroxyapatite (HA), bioglass, apatite, calcium phosphate, carbon fibers, glass fibers, polymer fibres and silica particles are mostly used for metallic prostheses to enhance biological properties. Among these ceramics HA has exact stoichiometric Ca/P ratio of 1.67 and its chemical structure has closely resemblances to bone tissues. However, it use is restricted due to some limitations such as difficulty in implants owing to its hardness, fragile and inflexible nature. To overcome these limitations, HA is fabricated as biodegradable composite materials with natural polymers like alginate, proteins, collagens, gelatin, fibrins, albumin and synthetic polymers like polyvinyl alcohol, poly(glycolic acid), poly(lactic acid), poly(p-dioxane), poly(caprolactone) polyurethanes and polyorthoesters. Further, biomedical significance of these nano-composite scaffolds can be efficiently enchanceed by morphological modifications or doping of osteoconductive components on to HA [3, 4].

Surface Modification of HA enhances the biocompatibility of these materials which in turn significantly improves and accelerate invasion of bone tissues into the scaffolds. To obtain desired properties for surface of HA particles, use of appropriate coupling agents is required. For example, attachment of organo-functional silanes on HA surface sites results in improved interfacial adhesion between filler and polymer matrix. In recent studies, HA surface has been modified with different coupling agents, such as dodecyl alcohol [5], photocrosslinkable chitosan [6], etidronic acid [7], hexyl, octyl, and decyl phosphate in mixed acetone/ water solvents or using laser [3] to improve the interfacial adhesion between filler and polymer matrix. Treatment of HA with bisphosphonates is also an interesting strategy that can be used in treatment of different bone diseases such as metastatic and Paget's disease, osteoporosis and hypercalcaemia induced by tumor [8]. Bisphosphonates act by inhibiting the osteoclastic resorption of bone tissues. They bind strongly to HA crystals and are retained for a long time in bone, eventually excreted unmetabolized in urine. The chemical structure of the bisphosphonates is analogues to pyrophosphates, which are natural modulators for bone metabolism [9]. The difference in bisphosphonates lies with the presence of oxygen atom that binds pyrophosphates through its two phosphate groups (P-O-P) which are further substituted by a carbon atom (P-C-P). This increases the tendency of these molecules to be less resistant to hydrolysis as compared to pyrophosphates employed sodium alendronate a member of BP family for controlling the drug doses in fractured bone treatment. Recently, used zoledronate acid has been reported in bone tissue engineering and results show an inhibition of osteoclastic resorption, whereas osteoblasts remain unaffected [10].

This research is focused on preparation and characterization of nano-composite synthesised from castor oil based polyurethane (PU) and etidronic surface modified nano hydroxyapatite (mnHA). The bioactivity and biocompatibility of the resultant nanocomposite material has been studied and its possible viability in the bone tissue engineering has been explored. The castor oil based PU is composed of hard and soft segments and this strategy of synthesis of PU has gained more attention due to their excellent physical and biocompatible properties [11]. Porous, biodegradable, biocompatible, non-toxic behavior of polyurethane (PU) further supports its use in cellular infiltration, tissue generation, in subcutaneous, cardiovascular diseases and bone tissue engineering.

Materials and Methods

Regents and treatment

Methylene diphenyl 1, 4- diisocyanate (MDI), castor oil, 1,4 Butane diol (BDO), etidronic acid C[H.sub.3]C(OH)[(P[O.sub.3][H.sub.2]).sub.2] (60% aqueous solution) were purchased from Sigma Aldrich (AR grade). Castor oil was vacuum dried at 70[degrees]C for 2 h prior to its use. nHA particles was synthesized and modified by etidronic acid. Modified nHA particles were used as filler in nano composites. Simulated body fluid was prepared as described in literature and pH of the solution was maintained to 7.4. [12].

Synthesis of Surface Modified nHA polyurethane (m-nHA/PU) nano-composite

The surface of n-HA was modified as reported in literature [10]. In brief the nHA particle was added to aqueous solution of 0.1M etidronic acid where pH was adjusted to 10 with addition of N[H.sub.3]. Afterwards, hydroxyapatite was suspended in etidronate solution and stirred at room temperature for 24 h. Then, the mixture was washed 5-6 times by deionized water to remove non-grafted acid. Finally, the obtained material was filtered and dried at 60[degrees]C for 24 h.

m-nHA/PU nano-composite scaffold were prepared in situ using castor oil and MDI in 1:1 ratio and 30% (by wt.) m-nHA using foaming method. Initially, modified nHA was dispersed in castor oil using a high shear homogenizer under an inert nitrogen atmosphere with stannous octoate (SO) as catalyst at 80[degrees]C for 4 hrs. MDI was then added drop wise at 60[degrees]C for 2-3hr till the solution started becoming viscid as the foaming started, the mixture was stirred again for half an hour and. poured in a mould as the foaming started. It was then kept in oven for 5 hrs at 80-100[degrees]C till the reaction was complete. The moulds were kept at room temperature for remaining reaction to occur. The prepared scaffolds were washed 3-4 times with deionized water for removal of residual reactant and then dried overnight at 80-100[degrees]C

Characterization of m-nHA/PU nano-composite

The chemical structures of m-nHA/PU nano-composites scaffold were characterized before and after immersion in simulated body fluid (SBF) by Fourier transform infrared spectroscopy (FT-IR) in Perkin Elmer RZX spectrometer using KBr technology. FTIR spectra were recorded in a spectral range of 4000-450 [cm.sup."1] with a resolution of 2 [cm.sup."1] with two scans for each sample. Phase analysis and crystallinity of nanocomposite was performed using a Philips X'Pert Pro X-ray diffractometer system. The radiation was Cu Ka (e = 0.15406 nm) with 40 kV voltage and 40 mA intensity. JSM JEOL-6100 Scanning electron microscope was used to study surface morphology and pore size of nano-composites. The nanocomposites were gold coated in order to make the surface conductive.

The compressive strength was evaluated using Instron test machine (4466 Instron Instruments, UK) at room temperature. The cylindrical specimens were compressed at a rate of 1 mm/ min (ASTM F451-95) with load cell 10kN. The results were evaluated with Blue Hill 2 software. The given value is means of 7 measurements. The porosity of the nano composite, a, was calculated from the measured the skeletal density ([D.sub.S]) and apparent density ([D.sub.a]) by a formula[13]:

[epsilon] = (l - [D.sub.a]/[D.sub.S]) X 100% (1)

Cell Culture

U-2 OS, a human derived osteogenic sarcoma cell line was used to study the effect of m-nHA/PU nano-composite on cell proliferation and survival. Base medium for U-2 OS cells was McCoy's 5A medium with 2mM L-glutamine. For complete medium preparation, 10% FBS was added along with 100 U/ mL penicillin and 100 [micro]g/mL streptomycin. Growing cells were cultured in tissue culture grade T-25 flask at 37[degrees]C and 5% C[O.sub.2] in a humidified incubator. Culture medium was changed every alternate day. Cells with at least 95% viability confirmed by Trypan Blue exclusion, were used for proliferation and viability assay.

Proliferation and viability assay: MTT based assay, where reduction of yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is allowed to interact with cells, was used to asses proliferation and viability of cells grown on nano-composites. Viable cells convert yellow colored soluble MTT to purple colored water insoluble formazan crystals, which can be measured at 570 nm using plate spectrophotometer. Intensity of purple color thus formed could be projected to measure number of metabolically active viable cells. Method reported by Dong et al was followed with certain modifications[14]. Briefly, nHA/PU, m-nHA/PU composite were prepared in a size of 10 x 10 x 1 [mm.sup.3] and sterilized by exposure of UV light for 2 h in a biosafety cabinet. Sterilized nano-composite were placed in tissue culture grade 24 well plate and cells were seeded at a density of 20 x [10.sup.3] cells per well. Cells grown on plastic surface, without any composite were used as an experimental control. Media of the cells was changed on alternate day followed by microscopic imaging. MTT solution (5mg/mL) was added at a final concentration of 10 %v/ v in well containing each scaffold on 2nd, 4th, 6th, and 8th day. After 4 h, media containing MTT was removed and formazan crystals were dissolved in 200 [micro]L DMSO. Dissolved formazan was aspirated and transferred into 96 well plate and optical density was measured at 570 nm. Optical density of each condition was compared with respective day's no-composite control. Data from 4 replicates was analyzed and plotted as bar-graph. Difference between measured optical density of different groups was compared using Kruskal-Wallis nonparametric statistical test at [??] = 0.05 using Graph Pad Prism (version 6.01).

Results and Discussion

FTIR spectra of m-nHA/PU nano-composite before and after soaking in SBF are shown in figure 1. From the figure 1 (a) it can be seen that the peaks at 3345 and 1729 [cm.sup.-1], respectively belong to the stretching vibration of urethane amino group (NH) and carbonyl group (-C=O). The peaks at 2925 and 2854 cm-1 correspond to asymmetric and symmetric stretching vibrations of -C[H.sub.2], respectively. Small peak at 1639 [cm.sup.-1] corresponds to carbonyl (-C=O) with C=O-NH arrangement of substituted urea which may be present due to the reaction of excess isocyanate with moisture in air [15]. Peaks at 1460 and 1413 [cm.sup.-1] belong to carbonic group (-C[O.sup.3.sub.-2]) of n-HA and bands at 815, 962 and 1160 [cm.sup.-1], respectively are attributed to v(P-C P), (P -O), (P=O) of phosphate group whereas very weak bands around 3000 [cm.sup.-1] are attribute to the C-H and the O-H stretching vibration mode of etidronate grafted on nHA[16]. Disappearance of hydroxyl (-OH) peak at 3571 [cm.sup.-1] in m-nHA/PU nano-composite may be due to the chemical linkage between hydroxyl groups of nHA and isocyanate groups.

From Figure 1(b), it can be seen that after soaking of nanocomposite in SBF solution some new peaks appear and intensities of some peaks change slightly. A new peak of carboxylic acid due to the hydrolysis of ester radical in SBF appears at 1642 [cm.sup.-1] and another peak at 3177 [cm.sup.-1] is due to C-NH groups of hydrolytic biuret. The peaks at 3727 and 1287 [cm.sup.-1] are due to -NH and C-N stretching vibrations of amine. Also the peak at 1725 [cm.sup.-1] becomes implausible due to the urethane hydro lyzation. Peak at 2921 [cm.sup.-1] corresponds to C-H stretching vibration of the methyl group due to the breakage of the long carbon chains of castor oil into short chains molecules. In SBF, urethane degradation take place chemical cleavage of castor oil and hydrolysis of urethane bonds joining the hard and soft segments [17]. From FTIR analysis it can be corroborated that the surface of the porous m-HA/PU nano composite changes greatly in SBF because of hydrolysis and deposition of Ca/P [18].

Figure 2 shows XRD patterns of m-nHA/PU nano composites before and after soaking in SBF. The characteristics peaks of modified HA are shown at 2[??] = 25.9[degrees], 31.9[degrees], 33[degrees], 34[degrees] and 40[degrees]. Figure 2(a) shows that incorporation of nHA into PU nanocomposite does not change the nature of modified HA. A broad peak at 2[??] = 21[degrees] corresponds to hard segment of segmented structure of polyurethane which gets narrow and immense after soaking in SBF as seen in figure 2

(b). The intensity of modified HA peak at 31.9[degrees] increases which may be due to Ca and P ions from SBF getting deposited on the scaffold [10, 14].

Surface morphological analysis of m-HA/PU nano composite is an important part of this study that provides evaluation of the porous nature of the material. Fig. 3 shows SEM micrographsps of the m-nHA/PU nano-composites with surface modified HA nano particles. Published studies have insisted on the fact that about 78% of the porosity is must for any scaffolds, as it provides large surface area and appropriate space for circulation of nutrients and cell waste, growth of the cells in the process of formation of bone tissues and vessels [19]. The obtained SEM images of m-nHA/PU nano-composites clearly depicts a high level of porosity in the material which amounts to 85%. Further, the pores are homogenous, macroporous and interconnected that ensures a incessant flow of nutrients in the scaffold as well good inhibition of cells of various origins. It has been found that the m-nHA/PU porous nano-composites (Fig. 3a) not only consist of macropores of 100-600 im size but also a lot of micropores on the edges of macro-porous structure. The nHA particles are aligned on the surface of the pores giving a high strength to the porous structure. Compressive strength (Fig. 4) of segmented PU nanocomposite was found to be 22.4 MPa that was close to that of Human trabecular bone [20].

The morphology of porous m-nHA/PU nano-composite soaked in SBF has been shown in Fig.3(c, d). The deposition of a layer of white particles made of Ca and P ions of SBFs can be seen on the surface of porous nano-composite as depicted by the arrows in the SEM images. The obtained layer is a result of 4 weeks of soaking of m-nHA/PU nano-composite in SBF liquid. The study clearly indicates spontaneous degradation of the m-nHA/PU nanocomposites, which is always preferred during resuscitation of the damaged bones [21, 22].

Cell proliferation measurement by MTT (3-{4,5-dimethylthiazol-2yl}-2,5-diphenyl-2H-tetrazoliumbromide) assay is based on reduction of MTT into formazan by NAD(P)H-dependent oxidoreductase enzymes present in cytosol of cell and this reduction depends on metabolic activity of cells. Proliferating cells maintain significantly observable metabolic activity due to NADH or NADPH flux. Here, MTT reduction to assess the cytocompatibility of nano-composite's surface in terms of cells proliferation and viability has been utilized. Cells were allowed to grow on both plain nHA/PU and m-nHA/PU scaffold and were compared with cell culture grade plastic surface. Cells in all the photomicrographs A-I in Fig. 5 suggest well defined irregular spindle shaped morphology. None of these images show any sign of toxicity, except cells grown on bare plastic surface (Image G), where number of cells reduced. It is imperative that, with time, conventional plastic surface will be exhausted and no more surface will be available for cell growth. This scarcity of free growth surface will also induce cell death and as shown in Image G in Fig. 5, wherein numbers of cells were reduced. Further, MTT assay also corroborated these finding and optical density data was plotted against different days of incubation (Fig. 6). Findings of MTT assay suggest that bare plastic surface shows higher rate of cell proliferation initially up to 4 days of incubation as compared to both nano-composites. However, as time progressed, cell number in composite placed wells outnumbered bare plastic surface because of higher proliferation and cell survival offered by HA nano-composite. Growth of cells grown over nano-composite was slow initially, improved with time, which could be attributed to initial normalization of growth conditions to these new environment.


An advance m-nHA/PU nano-composite scaffold containing etidronic acid modified HA was successfully synthesized using foaming method. In vitro tests demonstrated that the highly interconnected porous network and porosity of the nanocomposite provide excellent microenvironment for cell culture, enhanced proliferation and viability for soft tissues. The biodegradable behavior of nano-composite was evidenced by the spontaneous deposition of Ca/P ions on the surface of composite from SBF, confirmed by FTIR. More importantly, the cells were able to populate the inner surface of nanocomposite. The overall study suggests the compressive strength and biological properties of the m-nHA/PU nano-composite are potentially suitable for applications in bone tissue-engineering.


We gratefully acknowledge University Grant Commission and for providing financial assistance through Rajiv Gandhi National Fellowship Program to one of the author and Council of Scientific and Industrial Research (CSIR) , India and Technical Education Quality Improvement Program-II, India for giving financial support for carrying out this research work.


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Lokesh Kumar (1), Rajender Kumar (2), Anupama Kaushik (1) (*)

(1) Dr. S.S. Bhatnagar University Institute of Chemical Engineering, Punjab University, Chandigarh 160014, Indi

(2) UGC Centre of Excellence in Applications of Nanomaterials, Nanoparticles & Nanocomposites, Punjab University, Chandigarh, India 160014

Received 28 March 2017; Accepted 11 April 2017; Published online 31 December 2017

* Coresponding author: Dr. Anupama Kaushik; E-mail: in

Caption: Figure 1: FTIR Spectra of m-nHA/PU nano-composite (a) and soaking in SBF solution for four weeks (b)

Caption: Figure 2: XRD profiles of m-nHA/PU nano-composite (a) and soaking in SBF solution for four weeks (b)

Caption: Figure 3: SEM Micrographs of m-nHA/PU nano-composite (a, b) and Soaking for four weeks in SBF solution (c, d)

Caption: Figure 4: Compressive strength result of m-nHA/PU nano-composite

Caption: Figure 5: Effect of culture surface on cell proliferation and viability. Images A-I, shows growth of cells in No Composite (A, D & G), nHA/PU Composite (B, E & H) and m-nHA/PU Composite (C, F & I)

Caption: Figure 6: Comparisons of cell growth on 3 culture surfaces. Colored lines show linear projection of growth of these cells under respective conditions
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Title Annotation:Original Article
Author:Kumar, Lokesh; Kumar, Rajender; Kaushik, Anupama
Publication:Trends in Biomaterials and Artificial Organs
Date:Apr 1, 2017
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