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Polycaprolactone-laponite composite scaffold releasing strontium ranelate for bone tissue engineering applications
Colloids and Surfaces B: Biointerfaces 143 (2016) 423–430
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Polycaprolactone-laponite composite scaffold releasing strontium ranelate for bone tissue engineering applications Bindu P. Nair a,b,∗ , Megha Sindhu a , Prabha D. Nair a,∗ a Division of Tissue Engineering and Regeneration Technologies, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695012, Kerala, India b Department of Chemistry, Mahatma Gandhi College, Thiruvananthapuram 695004, Kerala, India
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Article history: Received 24 September 2015 Received in revised form 16 January 2016 Accepted 10 March 2016 Available online 15 March 2016 Keywords: Clay Laponite Bone tissue engineering Strontium ranelate Osteoporosis
a b s t r a c t We report polycaprolactone-laponite composite scaffold for the controlled release of strontium ranelate (SRA), a drug for osteoporosis. Laponite-SRA complex with electrostatic interaction between the drug and laponite was obtained through an aqueous phase reaction. Structural evaluation verified complexation of the bulky SRA molecules with the negatively charged laponite tactoid surfaces, leading to extended ordering of the tactoids, leaving behind the interlayer spacing of the laponite unchanged. The laponiteSRA complex was solution blended with polycaprolactone to obtain composite scaffolds. The strategy was found improving the dispersibility of laponite in PCL due to partial organomodification imparted through interaction with the SRA. The composite scaffolds with varying laponite-SRA complex content of 3–12 wt% were evaluated in vitro using human osteosarcoma cells. It was confirmed that an optimum composition of the scaffold with 3 wt% laponite-SRA complex loading would be ideal for obtaining enhanced ALP activity, by maintaining cell viability. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis is a very common disease condition among postmenopausal women and is caused when bone cells osteoclasts and osteoblasts fail to create a balance between bone resorption and formation, respectively. This imbalance leads to a disruption of bone microstructure leaving it porous and prone to fracture. The positive effect of strontium, an alkaline earth metal on bone homeostasis has recently been explored for improving bone regeneration [1,2]. Strontium is highly similar to its group antecedent in the periodic table and the most abundant metal of bone- calcium, which enables it to be easily incorporated into the mineral phase of bone. Strontium has been proven to be useful for a balanced remodelling of bone through its effect on bone resorbing osteoclasts and the bone forming osteoblasts [3,4]. Several approaches have been attempted to explore the osseointegrative and bone regenerative properties of strontium on both implants and also in scaffolds for tissue engineering applications [5–8]. By utilizing the mechanism of action of strontium on bone regeneration, strontium ranelate (SRA), consisting of two atoms
∗ Corresponding authors. E-mail addresses: [email protected] (B.P. Nair), [email protected], [email protected] (P.D. Nair). http://dx.doi.org/10.1016/j.colsurfb.2016.03.033 0927-7765/© 2016 Elsevier B.V. All rights reserved.
of strontium stabilized by the organic component ranelic acid has been used as an oral drug for the treatment and prevention of osteoporosis in post-menopausal women, in several European and Asian countries [9–12]. However, disadvantages of SRA as a drug for the oral and systemic treatment of osteoporosis are the occurrence of adverse effects such as the increased risk of venous thrombosis, diarrhoea, nausea, headache and cutaneous hypersensitivity [13]. A scaffold or a drug-delivery system for the localized release of SRA is currently not available in literature. Laponite is a synthetic hectorite clay, consisting of relatively uniform disc-shaped particles of 25 nm diameter and 1 nm thickness and has an empirical formula of Na+ 0.7 [(Si8 Mg5.5 Li0.3 )O20 (OH)4 ]− 0.7 [14–17]. Interactions of biomolecules with the clay minerals have been reported extensively in literature [17–19]. Proteins and peptides were reportedly interacting with the clay through electrostatic and van der Waals’ interactions and cation exchange mechanism, whereas nucleic acids were binding with the clay edges, leaving the interlayer spacing unchanged [19,20]. More recently, clays have been used for therapeutic delivery of many drugs through intercalation and also for localization of biomolecules [21–23]. Studies have also proven that laponite can induce bone formation even in the absence of any osteoinductive factors [24,25]. In this context, in the present study, a complex of strontium ranelate with laponite is attempted for the first time for bone tissue engineering applications, with a view to release the
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drug in a localized and controlled manner, and benefit from the dual effect of laponite and SRA for bone tissue regeneration. The use of the complex for bone tissue engineering application is evaluated by making a composite scaffold of it with polycaprolactone (PCL). It is expected that the strategy can suppress the cytotoxicity of laponite and SRA at higher concentrations by actively protecting it within the slow degrading polymer PCL. In vitro evaluation of the scaffold consisting of dual osteoinductive factors laponite and strontium ranelate, using human osteosarcoma cells is also presented in the article.
The PLS scaffolds were also characterized using scanning electron microscopy, for its morphology. SEM images were taken in JEOL JSM-5600 LV scanning electron microscope using samples provided with a thin gold coating with JEOL JFC-1200 fine coater. Release of SRA from the PLS scaffolds was studied in water using a CaryWin UV–vis spectrophotometer. Sterilized and washed scaffolds of known weight were kept in 1 mL water and the release was studied by withdrawing water at regular intervals, and measuring the intensity of the peak at a wavelength of 318 nm, in comparison with a standard plot of SRA.
2. Experimental
2.2.4. Swelling and degradation studies The swelling capacities of the PLS scaffolds were determined in PBS buffer solution of pH 7.4, at room temperature. Swelling was calculated by weighing the scaffold before and after soaking in PBS. The percentage swelling was calculated as [(Wh − W0 )/W0 ] × 100, where Wh is the weight of the swollen scaffold at regular intervals and W0 is the weight of the dry scaffold. Degradation of the PLS scaffolds were evaluated by immersing the scaffolds in PBS and the weights of the scaffolds were recorded at regular intervals after washing five times with water.
2.1. Materials Strontium ranelate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and PCL (Mw = 70,000–90,000 g/mol) were purchased from Sigma, India. The clays used were a synthetic hectorite, Laponite RD and a smectite aluminosilicate Cloisite-Na+ from Bayville Chemical Supply Company Inc., USA. 1,4 Dioxane was purchased from Merck Specialties Pvt. Ltd., India. Dulbecco’s Modified Eagles Medium, High Glucose (DMEM-HG), phosphate buffered saline (PBS), penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco (USA). Human Osteosarcoma (HOS) cell line was sub-cultured from a stock culture obtained from National Centre for Cell Sciences, India. 2.2. Methods 2.2.1. Preparation of laponite-SRA complex SRA was complexed with laponite (LAP) by treating aqueous laponite suspension (2.2 wt%) with an aqueous solution of SRA such that the final percentage of SRA would be within its solubility limit in water, i.e. 1 mg mL−1 or 0.1 wt% and the weight percentage of laponite would be 1.1%. Concentration of SRA used was arbitrarily fixed as either equivalent to Cation Exchange Capacity (CEC) of laponite i.e., 75 meq/100 g (denoted as LS1) or two times that of CEC (LS2) and the solution was stirred at ambient conditions for 2 h. The turbid and the viscous solution obtained was slow-dried at ambient conditions at 37 ◦ C in an air oven and the thin film-like material obtained was powdered using a mortar and pestle and used for further characterization. For comparative purposes, Cloisite-Na+ (CLO) was also treated with SRA at concentrations equivalent to 1 or 2CEC. The powders obtained were denoted respectively, as CS1 and CS2. 2.2.2. Fabrication of PCL-LS 3D scaffolds PCL-LS composite scaffolds were prepared through solution blending method. PCL was dissolved in 1,4 dioxane to obtain a 7 wt% solution, to which the required amount of LS1 complex was added (3, 6 and 12 wt% of PCL) and the mixture was blended manually. The PCL-LS blend obtained was frozen at −80 ◦ C and lyophilized for about 8 h to obtain a three dimensional porous scaffold. The resulting composite scaffolds were denoted respectively, as PLS3, PLS6 and PLS12. 2.2.3. Structural evaluation of the LS complex and the PLS scaffolds The LS complexes were characterized using X-ray diffraction (XRD), Fourier-Transform Infrared spectroscopy (FT-IR), UV–vis spectroscopy and transmission electron microscopy (TEM) for its structure and morphology. XRD data for 2 between 2.5◦ and 30◦ were collected on a Bruker D 8 advance X-ray diffractometer using Cu K␣ radiation. FT-IR measurements were made on a Nicolet 5700 FTIR Spectrometer in the range of 4000- 400 cm−1 using KBr pellets containing 2 wt% samples. TEM analysis was performed in FEI, TECNAI 30G2 S-TWIN microscope at an accelerating voltage of 100 kV.
2.2.5. Water contact angle measurements The water contact angles on the surface of PLS thin films were measured using sessile drop technique (Data Physics, OCA10). A water droplet (3 L) was placed onto the surface of the PLS films on a glass slide, and the contact angle between the water and the surface was measured immediately by taking pictures of the droplet using an optical microscope and averaging the right and left angles using surface contact angle software (SCA20, Data Physics). The values reported are mean of at least five measurements. 2.2.6. Cytotoxicity evaluation PCL and PLS scaffolds were sterilized by immersing in ethanol and washed with sterile water thrice for removing traces of ethanol. Each washing was given 2 h duration using an incubator shaker. The cytotoxicity of the control PCL and the PLS scaffolds were evaluated in vitro by MTT assay using HOS cells and the method is reported elsewhere [26]. The scaffold pieces were incubated at 37 ◦ C in DMEM (10 mg/mL) for 48 and 72 h and the extract of the scaffolds in DMEM were added to a monolayer of the HOS cells in a 96 well plate, and incubated for 24 h. The formazan crystals formed by incubating the cells with MTT were dissolved in DMSO and absorbance at 570 nm was measured using a plate reader (Finstruments microplate reader). Cells treated with medium, was used as the negative control. 2.2.7. In vitro evaluation of the scaffolds The sterilized and washed scaffolds of volume 52 mm3 were then placed in 24-well culture plates, and pre-wetted using 50 L of DMEM-HG prior to cell-seeding and incubated for 1 h. HOS cells were then seeded onto scaffolds as a concentrated droplet at a density of 1000 cells/mm3 of the scaffolds. The cells were allowed to adhere on the scaffolds by incubating for about 30 min and supplemented with 1 mL DMEM-HG containing 10% FBS and 1% penicillin-streptomycin. The medium was changed every 2 days, and the cells on the scaffolds were cultured for up to 8 days. 2.2.8. SEM analysis of the cell-seeded scaffolds The cell-seeded scaffolds were fixed in paraformaldehyde solution (4% w/v) for 4 h, and the morphology of the cells was examined by FEI Quanta 200 E-SEM FEG scanning electron microscope. 2.2.9. Cell viability The cell viability on the scaffolds was assessed using the Live/Dead staining kit (Molecular Probes, Eugene) and the pro-
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cedure is reported elsewhere [27]. The scaffolds were rinsed in PBS and incubated in 4 mM calcein-AM and 2 mM ethidium homodimer-1 for 45 min at room temperature. The samples were again rinsed in PBS, and images were obtained on a Nikon A1R confocal microscope. Blue auto-fluorescence from the scaffolds was also measured and the overlapped image was used for distinguishing the cells and the scaffold. 2.2.10. Alkaline phosphatase activity ALP activity was determined by colorimetric endpoint assay which measures the enzymatic conversion of p-nitrophenyl phosphate (pNPP) to the yellowish product p-nitrophenol (pNP) in the presence of ALP and the procedure is reported elsewhere [27]. The cells on the scaffolds were lysed using 0.2% Triton X-100 and were centrifuged at 10000 rpm for 10 min. To 50 L of the supernatant, a 200 L portion of pNPP solution (4 mg/mL) was added and incubated for 15 min. The reaction was stopped by adding 1 N NaOH, and the absorbance was measured at 405 nm on a plate reader. All samples were run in triplicate and compared to p-nitrophenol standards. The ALP activity was normalized by the amount of protein obtained from the Pierce BCA Protein Assay Kit (Thermoscientific) and was expressed as micromoles of pNP/g of protein/min. For measuring the cellularity on the scaffolds, the cells were first lysed by incubating in cell-lysis buffer containing 0.05% Triton X-100 (Sigma Aldrich) at 37 ◦ C in a shaker. The DNA amount in the lysate was quantified using the Qubit dsDNA BR Assay Kit and a Qubit 2.0 Fluorometer. ALP activity was also normalized by the amount of total DNA content and expressed as micromoles of pNP/g of DNA/min. 2.2.11. Statistical analysis All measurements were performed in triplicate and expressed as mean ± standard deviation. For all experiments, statistical significance of differences between groups was determined using one-way ANOVA with the Tukey post hoc test in Vassar Stats: Website for Statistical Computation. In all the cases, when the difference was significant, symbols are used to indicate the difference. 3. Results and discussion An aqueous solution of laponite was treated with SRA at two concentrations, one and two times equivalent to the cation exchange capacity of laponite, with the aim of exploring the intercalation of clay with strontium ranelate. A 0.1% aqueous solution of SRA and 1.1% suspension of laponite appeared transparent, whereas upon mixing the laponite and the SRA solutions, turbidity and increase in viscosity of the solution become evident immediately, indicating the interaction of SRA with the laponite (Fig. S1, Supplementary data). The laponite-SRA reaction products were retrieved by slow drying, powdered and analyzed using XRD. XRD patterns of the pure laponite and the SRA treated laponites (LS1 and LS2) in comparison with the SRA are given as Fig. 1. LAP showed a relatively amorphous peak at 7◦ 2, corresponding to an interlayer spacing of 12.5 Å (d001 ). Interestingly, the interlayer spacing of the LAP remained unchanged for both LS1 and LS2, whereas the peak intensity corresponding to structural ordering of the laponite increased 3.6 fold for LS1 and 2.6 fold for LS2. It was also confirmed that increase in the structural ordering of the laponite was occurring at the expense of the loss of crystallinity of the SRA. Pure SRA showed characteristic sharp crystalline peaks, whereas none of these peaks were evident for LS1 and LS2. The property of cation exchange capacity of clay minerals has been conventionally used for removing metal ions and transporting radionuclides and also organomodify the clays for improving its dispersion in polymers [28–32]. It has been reported that the cation exchange of sodium ions available in the interlayer space of
Fig. 1. X-ray diffractograms of laponite (LAP) and the SRA-treated laponites (LS1 and LS2).
the clay with bivalent cations leads to an increase in the interlayer spacing by a few angstroms [30]. However, in the present study, the interlayer spacing remained unchanged despite having visible evidence of interaction between laponite and SRA (Fig. S1, Supplementary data). Therefore, curious over the above observation, SRA was also treated with an aluminosilicate clay, montmorillonite. The XRD results obtained for the SRA-modified Cloisites (CS1 and CS2) were the same as that for the LS1 and LS2 (Fig. 2a). XRD pattern of CLO showed a crystalline peak corresponding to an interlayer spacing of 12.1 Å, whereas CS1 revealed an increase in the intensity of the same peak by 2.9 fold, without changing the interlayer spacing of CLO. Similar to the SRA-modified laponites, the structural ordering of CS2 was getting reduced when compared to CS1, but the crystallinity was still 1.8 fold higher than that of CLO. In order to confirm that the loss of crystallinity of SRA was not due to dilution in laponite, a blend of laponite and SRA at the same composition of LS1 was made by solid state mixing, using a mortar and pestle. XRD analysis of the LS1-blend clearly showed the crystalline peaks of the SRA and also the nearly amorphous peak of laponite (Fig. 2b). This result further confirmed that the molecular level interaction of SRA and the laponite were occurring only during mixing their aqueous solutions. Therefore, to explain the interaction between laponite and SRA, FT-IR spectra of the SRA, laponite and the SRA-treated laponites were analyzed (Fig. 2c, Magnified version of the FT-IR is given as Fig. S2, Supplementary data). FT-IR spectra of laponite showed bands due to Si–O–Si asymmetric stretching of silicate layer (1030 cm−1 ), and structural hydroxyls and adsorbed water (3300–3600 cm−1 and 1650 cm−1 ). The peak at 1579 cm−1 for SRA was due to the characteristic C O stretching vibrations for a carboxylate salt. It was evident that for both LS1 and LS2, this peak showed a shift to a lower value of 1522 cm−1 . The most plausible explanation for this shift is the electrostatic interaction of the positively charged strontium ion of SRA with the negatively charged clay tactoid surfaces. This interaction would lead to a partial satisfaction of the positive charge on strontium, which in turn would decrease the polarization of carbonyl group of SRA, causing a shift in carbonyl stretching frequency to a lower value. From the XRD results it was evident that the interaction of SRA with the clay was occurring without affecting the clay interlayer distance and hence the negatively charged clay tactoid surfaces alone were interacting with the SRA. Interaction of the negatively charged clay edges with biomolecules, without affecting the clay interlayer spacing has been reported in literature [20].
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Fig. 2. (a) X-ray diffractograms of SRA-modified Cloisites, CS1 and CS2, in comparison with Cloisite-Na+ (CLO) and SRA, and (b) laponite-SRA blend (LS1-blend) in comparison with SRA, (c) FT-IR spectra of laponite (LAP), SRA and the SRA-modified laponites (LS1 and LS2) and (d) UV–vis spectra of SRA and LS1.
It was confirmed from the UV–vis spectra that the electrostatic interaction of the SRA with the laponite does not lead to any structural change of the SRA (Fig. 2d). UV visible spectra of SRA exhibited characteristic absorption maximum at 318 nm due to thiophene chromophore in conjugation with a carboxylate and an imine groups [33]. LS1 revealed these characteristic peaks of SRA unaltered. The observed un-altered peak position of SRA for LS1 complex could also be due to the fact that three out of four carboxylic acid groups in the SRA molecule are not in conjugation with the thiophene component. In order to further elucidate the
evidence for the SRA-laponite interaction, both laponite and the LS1 were analyzed using TEM. Fig. 3 shows the TEM images of laponite and LS1. From the TEM images, it was evident that the laponite consists of randomly distributed clay platelets, whereas good structural ordering was evident for LS1. Extended ordering of the laponite platelets as observed in the TEM images further supported the increased crystallinity of laponite in LS complexes. The observed localized ordering as given in the TEM image is due to the distribution of the sample at different heights on the grid, which
Fig. 3. HR-TEM images of (a) laponite and (b) SRA-modified laponite LS1.
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Fig. 4. (a) Chemical structure of SRA (b) schematic representation of laponite and (c) scheme of interaction between laponite and SRA in LS1 and LS2.
made it difficult to focus together. More TEM images of the LS1 are given as Fig. S3, Supplementary data. Based on the above findings, it is proposed that the bulky SRA molecules are unable to intercalate in the clay interlayer through the cation exchange mechanism. Rather, the SRA molecules when added to laponite at a concentration equivalent to the CEC will interact with the negatively charged surface of the clay tactoids, through electrostatic interaction. This interaction of SRA with clay
tactoid surfaces was sufficient to disturb the crystallinity of SRA, whereas extended ordering of laponite was taking place as shown in Fig. 4. However, when the concentration of SRA was increased two equivalents to that of the CEC, a gradual decrease in the structural ordering was evident. This could be due to the excess of SRA molecules over that required to achieve structural ordering, which could disturb the extended ordering of clay tactoids as shown in Fig. 4.
Fig. 5. SEM images of the horizontal cross-sections of the scaffolds (a) PCL (b) PLS3 (c) PLS6 and (d) PLS12.
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Fig. 6. Release profile of the SRA from PLS3, PLS6 and PLS12.
Since LS1 or LS2 as such do not have the characteristic properties to form a scaffold for bone tissue engineering, and also a high dose of laponite or SRA can cause cytotoxic effects, the LS1 complex was made into a composite scaffold form, by blending it with the biodegradable polymer PCL using the solvent 1,4 dioxane and the technique of freeze drying. The strategy was found to enhance the dispersibility of laponite in PCL due to partial organomodification imparted through interaction with the SRA. In order to find the optimum loading of the LS1 complex for bone tissue engineering, scaffolds with 3, 6, and 12 wt% LS1 were prepared. Morphological features of the horizontal cross-sections of the scaffolds PCL, PLS3, PLS6 and PLS12 are given in Fig. 5 and that of the vertical cross-sections are given as Fig. S4, Supplementary data. The scaffolds were made out of solution containing 7 wt% PCL and from the SEM images of the horizontal cross-sections, the pore sizes of the scaffolds were found to vary between 30 and 150 m. Vertical cross-sections revealed characteristic morphology of the pores produced through the lyophilization of dioxane crystals. It was confirmed through the contact angle measurements that the hydrophilicity of the PLS composites were increasing with LS loading, when compared to PCL. The average contact angles obtained for the surfaces PCL, PLS3, PLS6 and PLS12 were respectively, 121.2 ± 4.5, 84.1 ± 4.1, 82.4 ± 2.8 and 75.8 ± 4.8 (Supplementary data, Fig. S5). However, this increase in surface hydrophilicity was not directly reflected in the water retention capacity of the PLS scaffolds. When compared to the hydrophobic PCL, the PLS3 and PLS6 scaffolds didn’t show a significant increase in water retention capacity in 96 h, whereas, PLS12 showed significant increase, almost two fold when compared to the pure PCL scaffold (Fig. S6a, Supplementary data). Contradictory to the report that nanoclay could enhance degradation of PCL, no significant difference in degradation rate was observed for the PLS scaffolds (Fig. S6b, Supplementary data) [34]. The scaffolds also exhibited a slow degradation rate in 28 days, which could be due to the slow degrading nature of the high molecular weight PCL. Release of SRA from the PLS scaffolds was studied in water and it was confirmed that the cumulative release of the drug exhibited inverse relation with the loading of the LS1 complex in the PLS scaffolds (Fig. 6). Percentage cumulative release from the PLS3 scaffolds was found to be the highest, by releasing a maximum of 40% of the total drug loaded in 21 days, whereas that from the PLS6 and the PLS12 scaffolds exhibited significantly reduced release rate and released respectively, a maximum of only 32 and 28% in 21 days. However, it is worth mentioning that the cumulative amount of the drug released from the scaffolds at a time was following the order PLS12 > PLS6 > PLS3 i.e. when the amount was considered instead of the percentage release. The reduced release rate of the drug with higher LS complex content indicates that the drug is being retained
in the scaffolds by the highly hydrophilic laponite. At lower LS loading, the proportionately lower amount of laponite and higher hydrophobicity of the scaffold were causing the drug to get released at a faster rate. The higher laponite content in the PLS12 scaffold and hydrophilicity and retention capacity of the clay might have led to the lower release rate of SRA. Cytotoxicity of the PCL and PLS scaffolds were evaluated in vitro by measuring the cell-viability of HOS cells after 24 h incubation with the extracts of the scaffolds in cell-culture medium, using MTT-assay. Both the PCL and PLS scaffolds gave a cell-viability close to 100%, upon incubation with the extract obtained at 48 and 72 h, proving that the scaffolds were non-cytotoxic (Fig. S7, Supplementary data). The scaffolds were further evaluated in vitro for its cytocompatibility using live/dead assay. Fig. 7 shows the confocal micrographs of the HOS cells on the scaffolds stained using live/dead assay dyes, on eighth day. In Fig. 7, each panel shows live cells on the scaffold stained using calcein AM, dead cells using ethidium homodimer-1 (EB), blue autofluorescence from the scaffold and the merged image of the three images. Confocal images of lower and higher magnifications are given as Fig. S8–S10, Supplementary data. From the confocal micrographs, the cells appeared mostly live on the control and the PLS scaffolds. The cells on the PCL and the PLS3 scaffolds appeared to have spread morphology; whereas with increase in the LS content, cells appeared with round morphology. The SEM images of the cell-seeded scaffolds showed the fast growing cancer cells appeared embedded in the extracellular matrix (Fig. S11, Supplementary data). A plausible reason for this change in morphology of the cells is the increase in hydrophilicity of the scaffold with increase in LS content, which is being highest for PLS12. With incorporation and increase in LS content, the fluorescence from EB was also increasing. In the autofluorescent images of the scaffolds, the dense fluorescent areas were due to the LS complexes. The observed red fluorescence was also from the same region indicating that this fluorescence is due to the EB intercalated within the clay. EB due to its ammonium salt form can get intercalated in to the laponite through cation exchange reaction. This was further proven by taking confocal micrographs of the PLS6 scaffold not seeded with cells, after staining with the live-dead assay dye (Fig. S12, Supplementary data). The regions of the scaffolds rich in LS complex appear to adsorb both EB and calcein AM, and therefore in the live-dead assay images also, the red fluorescence which was in superimposition with the dense fluorescence in the autofluorescent images was predicted as due to dyes adsorbed by the laponite. In other words, the dead cells were less and comparable for both the PCL and the PLS scaffolds indicating the cytocompatibility of the composite scaffolds. The strategy of solution blending, adopted for dispersing the LS complex in PCL was leading to micron-sized domains of the LS complex in the PCL matrix and the method was adopted with a view not to disturb the structure of the LS complex. This distribution of the LS-complex at the micron-scale was found to have not much effect on the behaviour of the cells on the scaffolds, as evident from the confocal and the SEM micrographs. It must be presumed that apart from the micro-domains of LS complex as seen in the confocal micrographs, nano-domains are also present, distributed in the PCL matrix and this dispersion is sufficient to provide a uniform surface texture to the scaffold. The cells would be exposed to the released components from the scaffolds into the medium, which was found sufficient to exert a uniform effect on its physiological behaviour. Evaluation of the cellularity on the scaffolds revealed that the DNA content showed a gradual increase with increase in LS complex content; however this increase was significant only for PLS12, when compared to the control scaffold (Fig. 8a). This could be due to the favourable environment for cell proliferation on PLS12 scaffold due to a combined effect of LS complex content and hydrophilicity [3,4,24,25]. This increase in cellularity was not reflected in the
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Fig. 7. Confocal micrographs of the cell seeded scaffolds PCL, PLS3, PLS6 and PLS12 on day 8. Live cells were stained using calcein AM, dead cells using ethidiumhomodimer1(EB) and the blue autofluorescence was used for visualizing the scaffolds. Scale bar represents 50 m.
total protein content on the scaffolds; total protein content on the scaffolds showed no significant difference (Fig. 8b). As the scaffolds were cytocompatible, to elucidate which composition would be the best for bone tissue engineering, ALP activity of the cells on the scaffolds was measured. Since osteosarcoma cells were used for the study, measurable ALP activity was expressed by the cells on all the scaffolds. However, when compared to the control PCL scaffold, the total ALP activity on the PLS scaffolds showed significant enhancement (Fig. S13, Supplementary data). Upon normalization with respect to the amount of the total protein, ALP activity on PLS3 and the PLS6 scaffolds showed significant enhancement when compared to the control scaffold and there was no significant difference between PLS3 and PLS6. Normalization of ALP activity using the total DNA content revealed that the significant enhancement was limited to PLS3 alone (Fig. 8c and d). In other words, ALP content in the total protein expressed by a given number cells on the PLS scaffolds showed significant enhancement only for the PLS3 scaffolds. This could be due to an optimum favourable environment for bone formation on the PLS3 scaffold, through a combined effect of laponite, strontium ranelate and the surface hydrophilicity. This study was an attempt to explain the structural aspects of the laponite-strontium ranelate complex and also elucidate an ideal composition of PCL-LS complex for sustained release of strontium ranelate and a scaffold for bone tissue engineering. From the above
observations, it is suggested that a 3 wt% loading of the LS complex in PCL is sufficient to obtain enhanced ALP activity, which makes that composition ideal for bone tissue engineering. 4. Conclusions In conclusion, a complex of strontium ranelate with the osteoinductive mineral laponite was attempted for the first time with a view to overcome the drawbacks of the oral and systemic administration of strontium ranelate, and also for tissue engineering applications. Strontium ranelate was complexed with laponite through an electrostatic interaction between the negatively charged clay tactoid surfaces and the strontium cation. Polycaprolactone composite scaffolds containing varying amount of the laponite-strontium ranelate complexes were obtained through solution blending and lyophilization. Release profile of strontium ranelate from the scaffolds exhibited an inverse relation on the drug-clay complex content, due to enhanced hydrophilicity and retention capacity of the composite scaffolds. In vitro evaluation of the scaffolds using HOS cells verified that an optimum composition of 3 wt% loading would be sufficient to obtain enhanced ALP activity, by maintaining cell viability. The study warrants further exploration of PCL-LS complex scaffolds for bone tissue engineering applications.
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Fig. 8. (a) Total DNA, (b) total protein, and (c) and (d) normalized ALP activity relative to total DNA and total protein, respectively on day 8. Values are expressed as mean ± standard deviation from three different replicates. The symbols ¤ indicate p < 0.05 for PCL vs. PLS12; * indicate p < 0.05 for PCL vs. PLS3; ∧ and # indicate p < 0.05 respectively for PCL vs. PLS3 and PCL vs. PLS6.
Acknowledgements This research was supported by INSPIRE Faculty fellowship and research grant (IFA-11-CH-19) to BPN from Department of Science and Technology (DST), India. MS is also thankful to DST for Research Fellowship through IFA-11-CH-19. The authors also thank Director, SCTIMST and Head, BMT Wing, SCTIMST for providing the facilities. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.03. 033. References [1] E. Gentleman, Y.C. Fredholm, G. Jell, N. Lotfibakhshaiesh, M.D.O. Donnell, R.G. Hill, M.M. Stevens, Biomaterials 31 (2010) 3949. [2] O.Z. Andersen, V. Offermanns, M. Sillassen, K.P. Almtoft, I.H. Andersen, S. Sørensen, C.S. Jeppesen, D.C.E. Kraft, J. Bøttiger, M. Rasse, F. Kloss, M. Foss, Biomaterials 38 (2013) 5883. [3] F. Yang, D. Yang, J. Tu, Z. Qixin, L. Cai, L. Wang, Stem Cells 29 (2011) 981. [4] S. Peng, X.S. Liu, S. Huang, Z. Li, H. Pan, W. Zhen, K.D.K. Luk, X.E. Guo, W.W. Lu, Bone 49 (2011) 1290. [5] Y. Xin, J. Jiang, K. Huo, T. Hu, P.K. Chu, ACS Nano 3 (2009) 3228. [6] Y. Li, Q. Li, S. Zhu, E. Luo, J. Li, G. Feng, Y. Liao, J. Hu, Biomaterials 31 (2010) 9006. [7] L. Zhao, H. Wang, K. Huo, X. Zhang, W. Wang, Y. Zhang, Z. Wu, P.K. Chu, Biomaterials 34 (2013) 19. [8] J.W. Park, Y.J. Kim, J.H. Jang, Clin. Oral Implants Res. 21 (2010) 398. [9] J. Buehler, P. Chappuis, J.L. Saffar, Y. Tsouderos, A. Vignery, Bone 29 (2001) 176. [10] E. Bonnelye, A. Chabadel, F. Saltel, P. Jurdic, Bone 42 (2008) 129.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Polycaprolactone-laponite composite scaffold releasing strontium ranelate for bone tissue engineering applications Bindu P. Nair a,b,∗ , Megha Sindhu a , Prabha D. Nair a,∗ a Division of Tissue Engineering and Regeneration Technologies, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695012, Kerala, India b Department of Chemistry, Mahatma Gandhi College, Thiruvananthapuram 695004, Kerala, India
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Article history: Received 24 September 2015 Received in revised form 16 January 2016 Accepted 10 March 2016 Available online 15 March 2016 Keywords: Clay Laponite Bone tissue engineering Strontium ranelate Osteoporosis
a b s t r a c t We report polycaprolactone-laponite composite scaffold for the controlled release of strontium ranelate (SRA), a drug for osteoporosis. Laponite-SRA complex with electrostatic interaction between the drug and laponite was obtained through an aqueous phase reaction. Structural evaluation verified complexation of the bulky SRA molecules with the negatively charged laponite tactoid surfaces, leading to extended ordering of the tactoids, leaving behind the interlayer spacing of the laponite unchanged. The laponiteSRA complex was solution blended with polycaprolactone to obtain composite scaffolds. The strategy was found improving the dispersibility of laponite in PCL due to partial organomodification imparted through interaction with the SRA. The composite scaffolds with varying laponite-SRA complex content of 3–12 wt% were evaluated in vitro using human osteosarcoma cells. It was confirmed that an optimum composition of the scaffold with 3 wt% laponite-SRA complex loading would be ideal for obtaining enhanced ALP activity, by maintaining cell viability. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis is a very common disease condition among postmenopausal women and is caused when bone cells osteoclasts and osteoblasts fail to create a balance between bone resorption and formation, respectively. This imbalance leads to a disruption of bone microstructure leaving it porous and prone to fracture. The positive effect of strontium, an alkaline earth metal on bone homeostasis has recently been explored for improving bone regeneration [1,2]. Strontium is highly similar to its group antecedent in the periodic table and the most abundant metal of bone- calcium, which enables it to be easily incorporated into the mineral phase of bone. Strontium has been proven to be useful for a balanced remodelling of bone through its effect on bone resorbing osteoclasts and the bone forming osteoblasts [3,4]. Several approaches have been attempted to explore the osseointegrative and bone regenerative properties of strontium on both implants and also in scaffolds for tissue engineering applications [5–8]. By utilizing the mechanism of action of strontium on bone regeneration, strontium ranelate (SRA), consisting of two atoms
∗ Corresponding authors. E-mail addresses: [email protected] (B.P. Nair), [email protected], [email protected] (P.D. Nair). http://dx.doi.org/10.1016/j.colsurfb.2016.03.033 0927-7765/© 2016 Elsevier B.V. All rights reserved.
of strontium stabilized by the organic component ranelic acid has been used as an oral drug for the treatment and prevention of osteoporosis in post-menopausal women, in several European and Asian countries [9–12]. However, disadvantages of SRA as a drug for the oral and systemic treatment of osteoporosis are the occurrence of adverse effects such as the increased risk of venous thrombosis, diarrhoea, nausea, headache and cutaneous hypersensitivity [13]. A scaffold or a drug-delivery system for the localized release of SRA is currently not available in literature. Laponite is a synthetic hectorite clay, consisting of relatively uniform disc-shaped particles of 25 nm diameter and 1 nm thickness and has an empirical formula of Na+ 0.7 [(Si8 Mg5.5 Li0.3 )O20 (OH)4 ]− 0.7 [14–17]. Interactions of biomolecules with the clay minerals have been reported extensively in literature [17–19]. Proteins and peptides were reportedly interacting with the clay through electrostatic and van der Waals’ interactions and cation exchange mechanism, whereas nucleic acids were binding with the clay edges, leaving the interlayer spacing unchanged [19,20]. More recently, clays have been used for therapeutic delivery of many drugs through intercalation and also for localization of biomolecules [21–23]. Studies have also proven that laponite can induce bone formation even in the absence of any osteoinductive factors [24,25]. In this context, in the present study, a complex of strontium ranelate with laponite is attempted for the first time for bone tissue engineering applications, with a view to release the
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drug in a localized and controlled manner, and benefit from the dual effect of laponite and SRA for bone tissue regeneration. The use of the complex for bone tissue engineering application is evaluated by making a composite scaffold of it with polycaprolactone (PCL). It is expected that the strategy can suppress the cytotoxicity of laponite and SRA at higher concentrations by actively protecting it within the slow degrading polymer PCL. In vitro evaluation of the scaffold consisting of dual osteoinductive factors laponite and strontium ranelate, using human osteosarcoma cells is also presented in the article.
The PLS scaffolds were also characterized using scanning electron microscopy, for its morphology. SEM images were taken in JEOL JSM-5600 LV scanning electron microscope using samples provided with a thin gold coating with JEOL JFC-1200 fine coater. Release of SRA from the PLS scaffolds was studied in water using a CaryWin UV–vis spectrophotometer. Sterilized and washed scaffolds of known weight were kept in 1 mL water and the release was studied by withdrawing water at regular intervals, and measuring the intensity of the peak at a wavelength of 318 nm, in comparison with a standard plot of SRA.
2. Experimental
2.2.4. Swelling and degradation studies The swelling capacities of the PLS scaffolds were determined in PBS buffer solution of pH 7.4, at room temperature. Swelling was calculated by weighing the scaffold before and after soaking in PBS. The percentage swelling was calculated as [(Wh − W0 )/W0 ] × 100, where Wh is the weight of the swollen scaffold at regular intervals and W0 is the weight of the dry scaffold. Degradation of the PLS scaffolds were evaluated by immersing the scaffolds in PBS and the weights of the scaffolds were recorded at regular intervals after washing five times with water.
2.1. Materials Strontium ranelate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and PCL (Mw = 70,000–90,000 g/mol) were purchased from Sigma, India. The clays used were a synthetic hectorite, Laponite RD and a smectite aluminosilicate Cloisite-Na+ from Bayville Chemical Supply Company Inc., USA. 1,4 Dioxane was purchased from Merck Specialties Pvt. Ltd., India. Dulbecco’s Modified Eagles Medium, High Glucose (DMEM-HG), phosphate buffered saline (PBS), penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco (USA). Human Osteosarcoma (HOS) cell line was sub-cultured from a stock culture obtained from National Centre for Cell Sciences, India. 2.2. Methods 2.2.1. Preparation of laponite-SRA complex SRA was complexed with laponite (LAP) by treating aqueous laponite suspension (2.2 wt%) with an aqueous solution of SRA such that the final percentage of SRA would be within its solubility limit in water, i.e. 1 mg mL−1 or 0.1 wt% and the weight percentage of laponite would be 1.1%. Concentration of SRA used was arbitrarily fixed as either equivalent to Cation Exchange Capacity (CEC) of laponite i.e., 75 meq/100 g (denoted as LS1) or two times that of CEC (LS2) and the solution was stirred at ambient conditions for 2 h. The turbid and the viscous solution obtained was slow-dried at ambient conditions at 37 ◦ C in an air oven and the thin film-like material obtained was powdered using a mortar and pestle and used for further characterization. For comparative purposes, Cloisite-Na+ (CLO) was also treated with SRA at concentrations equivalent to 1 or 2CEC. The powders obtained were denoted respectively, as CS1 and CS2. 2.2.2. Fabrication of PCL-LS 3D scaffolds PCL-LS composite scaffolds were prepared through solution blending method. PCL was dissolved in 1,4 dioxane to obtain a 7 wt% solution, to which the required amount of LS1 complex was added (3, 6 and 12 wt% of PCL) and the mixture was blended manually. The PCL-LS blend obtained was frozen at −80 ◦ C and lyophilized for about 8 h to obtain a three dimensional porous scaffold. The resulting composite scaffolds were denoted respectively, as PLS3, PLS6 and PLS12. 2.2.3. Structural evaluation of the LS complex and the PLS scaffolds The LS complexes were characterized using X-ray diffraction (XRD), Fourier-Transform Infrared spectroscopy (FT-IR), UV–vis spectroscopy and transmission electron microscopy (TEM) for its structure and morphology. XRD data for 2 between 2.5◦ and 30◦ were collected on a Bruker D 8 advance X-ray diffractometer using Cu K␣ radiation. FT-IR measurements were made on a Nicolet 5700 FTIR Spectrometer in the range of 4000- 400 cm−1 using KBr pellets containing 2 wt% samples. TEM analysis was performed in FEI, TECNAI 30G2 S-TWIN microscope at an accelerating voltage of 100 kV.
2.2.5. Water contact angle measurements The water contact angles on the surface of PLS thin films were measured using sessile drop technique (Data Physics, OCA10). A water droplet (3 L) was placed onto the surface of the PLS films on a glass slide, and the contact angle between the water and the surface was measured immediately by taking pictures of the droplet using an optical microscope and averaging the right and left angles using surface contact angle software (SCA20, Data Physics). The values reported are mean of at least five measurements. 2.2.6. Cytotoxicity evaluation PCL and PLS scaffolds were sterilized by immersing in ethanol and washed with sterile water thrice for removing traces of ethanol. Each washing was given 2 h duration using an incubator shaker. The cytotoxicity of the control PCL and the PLS scaffolds were evaluated in vitro by MTT assay using HOS cells and the method is reported elsewhere [26]. The scaffold pieces were incubated at 37 ◦ C in DMEM (10 mg/mL) for 48 and 72 h and the extract of the scaffolds in DMEM were added to a monolayer of the HOS cells in a 96 well plate, and incubated for 24 h. The formazan crystals formed by incubating the cells with MTT were dissolved in DMSO and absorbance at 570 nm was measured using a plate reader (Finstruments microplate reader). Cells treated with medium, was used as the negative control. 2.2.7. In vitro evaluation of the scaffolds The sterilized and washed scaffolds of volume 52 mm3 were then placed in 24-well culture plates, and pre-wetted using 50 L of DMEM-HG prior to cell-seeding and incubated for 1 h. HOS cells were then seeded onto scaffolds as a concentrated droplet at a density of 1000 cells/mm3 of the scaffolds. The cells were allowed to adhere on the scaffolds by incubating for about 30 min and supplemented with 1 mL DMEM-HG containing 10% FBS and 1% penicillin-streptomycin. The medium was changed every 2 days, and the cells on the scaffolds were cultured for up to 8 days. 2.2.8. SEM analysis of the cell-seeded scaffolds The cell-seeded scaffolds were fixed in paraformaldehyde solution (4% w/v) for 4 h, and the morphology of the cells was examined by FEI Quanta 200 E-SEM FEG scanning electron microscope. 2.2.9. Cell viability The cell viability on the scaffolds was assessed using the Live/Dead staining kit (Molecular Probes, Eugene) and the pro-
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cedure is reported elsewhere [27]. The scaffolds were rinsed in PBS and incubated in 4 mM calcein-AM and 2 mM ethidium homodimer-1 for 45 min at room temperature. The samples were again rinsed in PBS, and images were obtained on a Nikon A1R confocal microscope. Blue auto-fluorescence from the scaffolds was also measured and the overlapped image was used for distinguishing the cells and the scaffold. 2.2.10. Alkaline phosphatase activity ALP activity was determined by colorimetric endpoint assay which measures the enzymatic conversion of p-nitrophenyl phosphate (pNPP) to the yellowish product p-nitrophenol (pNP) in the presence of ALP and the procedure is reported elsewhere [27]. The cells on the scaffolds were lysed using 0.2% Triton X-100 and were centrifuged at 10000 rpm for 10 min. To 50 L of the supernatant, a 200 L portion of pNPP solution (4 mg/mL) was added and incubated for 15 min. The reaction was stopped by adding 1 N NaOH, and the absorbance was measured at 405 nm on a plate reader. All samples were run in triplicate and compared to p-nitrophenol standards. The ALP activity was normalized by the amount of protein obtained from the Pierce BCA Protein Assay Kit (Thermoscientific) and was expressed as micromoles of pNP/g of protein/min. For measuring the cellularity on the scaffolds, the cells were first lysed by incubating in cell-lysis buffer containing 0.05% Triton X-100 (Sigma Aldrich) at 37 ◦ C in a shaker. The DNA amount in the lysate was quantified using the Qubit dsDNA BR Assay Kit and a Qubit 2.0 Fluorometer. ALP activity was also normalized by the amount of total DNA content and expressed as micromoles of pNP/g of DNA/min. 2.2.11. Statistical analysis All measurements were performed in triplicate and expressed as mean ± standard deviation. For all experiments, statistical significance of differences between groups was determined using one-way ANOVA with the Tukey post hoc test in Vassar Stats: Website for Statistical Computation. In all the cases, when the difference was significant, symbols are used to indicate the difference. 3. Results and discussion An aqueous solution of laponite was treated with SRA at two concentrations, one and two times equivalent to the cation exchange capacity of laponite, with the aim of exploring the intercalation of clay with strontium ranelate. A 0.1% aqueous solution of SRA and 1.1% suspension of laponite appeared transparent, whereas upon mixing the laponite and the SRA solutions, turbidity and increase in viscosity of the solution become evident immediately, indicating the interaction of SRA with the laponite (Fig. S1, Supplementary data). The laponite-SRA reaction products were retrieved by slow drying, powdered and analyzed using XRD. XRD patterns of the pure laponite and the SRA treated laponites (LS1 and LS2) in comparison with the SRA are given as Fig. 1. LAP showed a relatively amorphous peak at 7◦ 2, corresponding to an interlayer spacing of 12.5 Å (d001 ). Interestingly, the interlayer spacing of the LAP remained unchanged for both LS1 and LS2, whereas the peak intensity corresponding to structural ordering of the laponite increased 3.6 fold for LS1 and 2.6 fold for LS2. It was also confirmed that increase in the structural ordering of the laponite was occurring at the expense of the loss of crystallinity of the SRA. Pure SRA showed characteristic sharp crystalline peaks, whereas none of these peaks were evident for LS1 and LS2. The property of cation exchange capacity of clay minerals has been conventionally used for removing metal ions and transporting radionuclides and also organomodify the clays for improving its dispersion in polymers [28–32]. It has been reported that the cation exchange of sodium ions available in the interlayer space of
Fig. 1. X-ray diffractograms of laponite (LAP) and the SRA-treated laponites (LS1 and LS2).
the clay with bivalent cations leads to an increase in the interlayer spacing by a few angstroms [30]. However, in the present study, the interlayer spacing remained unchanged despite having visible evidence of interaction between laponite and SRA (Fig. S1, Supplementary data). Therefore, curious over the above observation, SRA was also treated with an aluminosilicate clay, montmorillonite. The XRD results obtained for the SRA-modified Cloisites (CS1 and CS2) were the same as that for the LS1 and LS2 (Fig. 2a). XRD pattern of CLO showed a crystalline peak corresponding to an interlayer spacing of 12.1 Å, whereas CS1 revealed an increase in the intensity of the same peak by 2.9 fold, without changing the interlayer spacing of CLO. Similar to the SRA-modified laponites, the structural ordering of CS2 was getting reduced when compared to CS1, but the crystallinity was still 1.8 fold higher than that of CLO. In order to confirm that the loss of crystallinity of SRA was not due to dilution in laponite, a blend of laponite and SRA at the same composition of LS1 was made by solid state mixing, using a mortar and pestle. XRD analysis of the LS1-blend clearly showed the crystalline peaks of the SRA and also the nearly amorphous peak of laponite (Fig. 2b). This result further confirmed that the molecular level interaction of SRA and the laponite were occurring only during mixing their aqueous solutions. Therefore, to explain the interaction between laponite and SRA, FT-IR spectra of the SRA, laponite and the SRA-treated laponites were analyzed (Fig. 2c, Magnified version of the FT-IR is given as Fig. S2, Supplementary data). FT-IR spectra of laponite showed bands due to Si–O–Si asymmetric stretching of silicate layer (1030 cm−1 ), and structural hydroxyls and adsorbed water (3300–3600 cm−1 and 1650 cm−1 ). The peak at 1579 cm−1 for SRA was due to the characteristic C O stretching vibrations for a carboxylate salt. It was evident that for both LS1 and LS2, this peak showed a shift to a lower value of 1522 cm−1 . The most plausible explanation for this shift is the electrostatic interaction of the positively charged strontium ion of SRA with the negatively charged clay tactoid surfaces. This interaction would lead to a partial satisfaction of the positive charge on strontium, which in turn would decrease the polarization of carbonyl group of SRA, causing a shift in carbonyl stretching frequency to a lower value. From the XRD results it was evident that the interaction of SRA with the clay was occurring without affecting the clay interlayer distance and hence the negatively charged clay tactoid surfaces alone were interacting with the SRA. Interaction of the negatively charged clay edges with biomolecules, without affecting the clay interlayer spacing has been reported in literature [20].
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Fig. 2. (a) X-ray diffractograms of SRA-modified Cloisites, CS1 and CS2, in comparison with Cloisite-Na+ (CLO) and SRA, and (b) laponite-SRA blend (LS1-blend) in comparison with SRA, (c) FT-IR spectra of laponite (LAP), SRA and the SRA-modified laponites (LS1 and LS2) and (d) UV–vis spectra of SRA and LS1.
It was confirmed from the UV–vis spectra that the electrostatic interaction of the SRA with the laponite does not lead to any structural change of the SRA (Fig. 2d). UV visible spectra of SRA exhibited characteristic absorption maximum at 318 nm due to thiophene chromophore in conjugation with a carboxylate and an imine groups [33]. LS1 revealed these characteristic peaks of SRA unaltered. The observed un-altered peak position of SRA for LS1 complex could also be due to the fact that three out of four carboxylic acid groups in the SRA molecule are not in conjugation with the thiophene component. In order to further elucidate the
evidence for the SRA-laponite interaction, both laponite and the LS1 were analyzed using TEM. Fig. 3 shows the TEM images of laponite and LS1. From the TEM images, it was evident that the laponite consists of randomly distributed clay platelets, whereas good structural ordering was evident for LS1. Extended ordering of the laponite platelets as observed in the TEM images further supported the increased crystallinity of laponite in LS complexes. The observed localized ordering as given in the TEM image is due to the distribution of the sample at different heights on the grid, which
Fig. 3. HR-TEM images of (a) laponite and (b) SRA-modified laponite LS1.
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Fig. 4. (a) Chemical structure of SRA (b) schematic representation of laponite and (c) scheme of interaction between laponite and SRA in LS1 and LS2.
made it difficult to focus together. More TEM images of the LS1 are given as Fig. S3, Supplementary data. Based on the above findings, it is proposed that the bulky SRA molecules are unable to intercalate in the clay interlayer through the cation exchange mechanism. Rather, the SRA molecules when added to laponite at a concentration equivalent to the CEC will interact with the negatively charged surface of the clay tactoids, through electrostatic interaction. This interaction of SRA with clay
tactoid surfaces was sufficient to disturb the crystallinity of SRA, whereas extended ordering of laponite was taking place as shown in Fig. 4. However, when the concentration of SRA was increased two equivalents to that of the CEC, a gradual decrease in the structural ordering was evident. This could be due to the excess of SRA molecules over that required to achieve structural ordering, which could disturb the extended ordering of clay tactoids as shown in Fig. 4.
Fig. 5. SEM images of the horizontal cross-sections of the scaffolds (a) PCL (b) PLS3 (c) PLS6 and (d) PLS12.
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Fig. 6. Release profile of the SRA from PLS3, PLS6 and PLS12.
Since LS1 or LS2 as such do not have the characteristic properties to form a scaffold for bone tissue engineering, and also a high dose of laponite or SRA can cause cytotoxic effects, the LS1 complex was made into a composite scaffold form, by blending it with the biodegradable polymer PCL using the solvent 1,4 dioxane and the technique of freeze drying. The strategy was found to enhance the dispersibility of laponite in PCL due to partial organomodification imparted through interaction with the SRA. In order to find the optimum loading of the LS1 complex for bone tissue engineering, scaffolds with 3, 6, and 12 wt% LS1 were prepared. Morphological features of the horizontal cross-sections of the scaffolds PCL, PLS3, PLS6 and PLS12 are given in Fig. 5 and that of the vertical cross-sections are given as Fig. S4, Supplementary data. The scaffolds were made out of solution containing 7 wt% PCL and from the SEM images of the horizontal cross-sections, the pore sizes of the scaffolds were found to vary between 30 and 150 m. Vertical cross-sections revealed characteristic morphology of the pores produced through the lyophilization of dioxane crystals. It was confirmed through the contact angle measurements that the hydrophilicity of the PLS composites were increasing with LS loading, when compared to PCL. The average contact angles obtained for the surfaces PCL, PLS3, PLS6 and PLS12 were respectively, 121.2 ± 4.5, 84.1 ± 4.1, 82.4 ± 2.8 and 75.8 ± 4.8 (Supplementary data, Fig. S5). However, this increase in surface hydrophilicity was not directly reflected in the water retention capacity of the PLS scaffolds. When compared to the hydrophobic PCL, the PLS3 and PLS6 scaffolds didn’t show a significant increase in water retention capacity in 96 h, whereas, PLS12 showed significant increase, almost two fold when compared to the pure PCL scaffold (Fig. S6a, Supplementary data). Contradictory to the report that nanoclay could enhance degradation of PCL, no significant difference in degradation rate was observed for the PLS scaffolds (Fig. S6b, Supplementary data) [34]. The scaffolds also exhibited a slow degradation rate in 28 days, which could be due to the slow degrading nature of the high molecular weight PCL. Release of SRA from the PLS scaffolds was studied in water and it was confirmed that the cumulative release of the drug exhibited inverse relation with the loading of the LS1 complex in the PLS scaffolds (Fig. 6). Percentage cumulative release from the PLS3 scaffolds was found to be the highest, by releasing a maximum of 40% of the total drug loaded in 21 days, whereas that from the PLS6 and the PLS12 scaffolds exhibited significantly reduced release rate and released respectively, a maximum of only 32 and 28% in 21 days. However, it is worth mentioning that the cumulative amount of the drug released from the scaffolds at a time was following the order PLS12 > PLS6 > PLS3 i.e. when the amount was considered instead of the percentage release. The reduced release rate of the drug with higher LS complex content indicates that the drug is being retained
in the scaffolds by the highly hydrophilic laponite. At lower LS loading, the proportionately lower amount of laponite and higher hydrophobicity of the scaffold were causing the drug to get released at a faster rate. The higher laponite content in the PLS12 scaffold and hydrophilicity and retention capacity of the clay might have led to the lower release rate of SRA. Cytotoxicity of the PCL and PLS scaffolds were evaluated in vitro by measuring the cell-viability of HOS cells after 24 h incubation with the extracts of the scaffolds in cell-culture medium, using MTT-assay. Both the PCL and PLS scaffolds gave a cell-viability close to 100%, upon incubation with the extract obtained at 48 and 72 h, proving that the scaffolds were non-cytotoxic (Fig. S7, Supplementary data). The scaffolds were further evaluated in vitro for its cytocompatibility using live/dead assay. Fig. 7 shows the confocal micrographs of the HOS cells on the scaffolds stained using live/dead assay dyes, on eighth day. In Fig. 7, each panel shows live cells on the scaffold stained using calcein AM, dead cells using ethidium homodimer-1 (EB), blue autofluorescence from the scaffold and the merged image of the three images. Confocal images of lower and higher magnifications are given as Fig. S8–S10, Supplementary data. From the confocal micrographs, the cells appeared mostly live on the control and the PLS scaffolds. The cells on the PCL and the PLS3 scaffolds appeared to have spread morphology; whereas with increase in the LS content, cells appeared with round morphology. The SEM images of the cell-seeded scaffolds showed the fast growing cancer cells appeared embedded in the extracellular matrix (Fig. S11, Supplementary data). A plausible reason for this change in morphology of the cells is the increase in hydrophilicity of the scaffold with increase in LS content, which is being highest for PLS12. With incorporation and increase in LS content, the fluorescence from EB was also increasing. In the autofluorescent images of the scaffolds, the dense fluorescent areas were due to the LS complexes. The observed red fluorescence was also from the same region indicating that this fluorescence is due to the EB intercalated within the clay. EB due to its ammonium salt form can get intercalated in to the laponite through cation exchange reaction. This was further proven by taking confocal micrographs of the PLS6 scaffold not seeded with cells, after staining with the live-dead assay dye (Fig. S12, Supplementary data). The regions of the scaffolds rich in LS complex appear to adsorb both EB and calcein AM, and therefore in the live-dead assay images also, the red fluorescence which was in superimposition with the dense fluorescence in the autofluorescent images was predicted as due to dyes adsorbed by the laponite. In other words, the dead cells were less and comparable for both the PCL and the PLS scaffolds indicating the cytocompatibility of the composite scaffolds. The strategy of solution blending, adopted for dispersing the LS complex in PCL was leading to micron-sized domains of the LS complex in the PCL matrix and the method was adopted with a view not to disturb the structure of the LS complex. This distribution of the LS-complex at the micron-scale was found to have not much effect on the behaviour of the cells on the scaffolds, as evident from the confocal and the SEM micrographs. It must be presumed that apart from the micro-domains of LS complex as seen in the confocal micrographs, nano-domains are also present, distributed in the PCL matrix and this dispersion is sufficient to provide a uniform surface texture to the scaffold. The cells would be exposed to the released components from the scaffolds into the medium, which was found sufficient to exert a uniform effect on its physiological behaviour. Evaluation of the cellularity on the scaffolds revealed that the DNA content showed a gradual increase with increase in LS complex content; however this increase was significant only for PLS12, when compared to the control scaffold (Fig. 8a). This could be due to the favourable environment for cell proliferation on PLS12 scaffold due to a combined effect of LS complex content and hydrophilicity [3,4,24,25]. This increase in cellularity was not reflected in the
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Fig. 7. Confocal micrographs of the cell seeded scaffolds PCL, PLS3, PLS6 and PLS12 on day 8. Live cells were stained using calcein AM, dead cells using ethidiumhomodimer1(EB) and the blue autofluorescence was used for visualizing the scaffolds. Scale bar represents 50 m.
total protein content on the scaffolds; total protein content on the scaffolds showed no significant difference (Fig. 8b). As the scaffolds were cytocompatible, to elucidate which composition would be the best for bone tissue engineering, ALP activity of the cells on the scaffolds was measured. Since osteosarcoma cells were used for the study, measurable ALP activity was expressed by the cells on all the scaffolds. However, when compared to the control PCL scaffold, the total ALP activity on the PLS scaffolds showed significant enhancement (Fig. S13, Supplementary data). Upon normalization with respect to the amount of the total protein, ALP activity on PLS3 and the PLS6 scaffolds showed significant enhancement when compared to the control scaffold and there was no significant difference between PLS3 and PLS6. Normalization of ALP activity using the total DNA content revealed that the significant enhancement was limited to PLS3 alone (Fig. 8c and d). In other words, ALP content in the total protein expressed by a given number cells on the PLS scaffolds showed significant enhancement only for the PLS3 scaffolds. This could be due to an optimum favourable environment for bone formation on the PLS3 scaffold, through a combined effect of laponite, strontium ranelate and the surface hydrophilicity. This study was an attempt to explain the structural aspects of the laponite-strontium ranelate complex and also elucidate an ideal composition of PCL-LS complex for sustained release of strontium ranelate and a scaffold for bone tissue engineering. From the above
observations, it is suggested that a 3 wt% loading of the LS complex in PCL is sufficient to obtain enhanced ALP activity, which makes that composition ideal for bone tissue engineering. 4. Conclusions In conclusion, a complex of strontium ranelate with the osteoinductive mineral laponite was attempted for the first time with a view to overcome the drawbacks of the oral and systemic administration of strontium ranelate, and also for tissue engineering applications. Strontium ranelate was complexed with laponite through an electrostatic interaction between the negatively charged clay tactoid surfaces and the strontium cation. Polycaprolactone composite scaffolds containing varying amount of the laponite-strontium ranelate complexes were obtained through solution blending and lyophilization. Release profile of strontium ranelate from the scaffolds exhibited an inverse relation on the drug-clay complex content, due to enhanced hydrophilicity and retention capacity of the composite scaffolds. In vitro evaluation of the scaffolds using HOS cells verified that an optimum composition of 3 wt% loading would be sufficient to obtain enhanced ALP activity, by maintaining cell viability. The study warrants further exploration of PCL-LS complex scaffolds for bone tissue engineering applications.
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Fig. 8. (a) Total DNA, (b) total protein, and (c) and (d) normalized ALP activity relative to total DNA and total protein, respectively on day 8. Values are expressed as mean ± standard deviation from three different replicates. The symbols ¤ indicate p < 0.05 for PCL vs. PLS12; * indicate p < 0.05 for PCL vs. PLS3; ∧ and # indicate p < 0.05 respectively for PCL vs. PLS3 and PCL vs. PLS6.
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