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Mechanically stiff, zinc crosslinked nanocomposite scaffolds with improved osteostimulation and antibacterial properties Rekha Rani Sehgal, Edmund Carvalho, and Rinti Banerjee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02740 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016
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Mechanically stiff, zinc crosslinked nanocomposite scaffolds with improved osteostimulation and antibacterial properties Rekha R Sehgala, Edmund Carvalhoa, Rinti Banerjee*a a
Department of Biosciences and Bioengineering, IIT Bombay, Mumbai, India. Fax: +91-22-
2572-3480; Tel: +91-22-2576-7868; E-mail:
[email protected] *Corresponding author: Prof. Rinti Banerjee, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai-400067, Phone: + (91-22) 2576 7868 (Direct); 2572-2545 (ex-7868), Fax: + (91-22) 2572 3480, Email:
[email protected] Keywords: Nanocomposite scaffolds, bone tissue engineering, zinc, gellan, bioglass nanoparticles, antibacterial property.
Abstract Nanocomposite scaffolds are studied widely due to their resemblance with the natural extracellular matrix of bone; but their use as bone tissue engineered scaffold is clinically hampered due to low mechanical stiffness, inadequate osteoconduction, and graft associated infections. The purpose of the current study was the development of a mechanically stiff nanocomposite scaffold using biodegradable gellan and xanthan polymers reinforced with bioglass nanoparticles (nB) (Size: 20-120nm). These nanocomposite scaffolds were crosslinked with zinc sulfate ions to improve their osteoconduction and anti-bacterial properties for the regeneration of a functional bone. Compressive strength and modulus of optimized nanocomposite scaffold (1% w/v polymer reinforced with 4%w/v nB nanoparticles, crosslinked with 1.5mM zinc sulfate) was 1.91 ± 0.31 MPa and 20.36 ± 1.08 MPa respectively, which was comparable to the trabecular bone and very high compared to nanocomposite scaffolds reported in earlier studies. Further, in vitro simulated body fluid (SBF) study suggested deposition of biomimetic apatite on the surface of zinc crosslinked nanocomposite scaffolds confirming their 1 ACS Paragon Plus Environment
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bioactivity. MG 63 osteoblast-like cells cultured with the nanocomposite scaffolds responded to matrix stiffness with better adhesion, spreading and cellular interconnections compared to the polymeric gellan and xanthan scaffolds. Incorporation of bioglass nanoparticles and zinc crosslinker in nanocomposite scaffolds demonstrated 62% increment in expression of alkaline phosphatase activity (ALP) and 150% increment in calcium deposition of MG 63 osteoblast-like cells compared to just gellan and xanthan polymeric scaffolds. Furthermore, zinc crosslinked nanocomposite scaffolds significantly inhibited the growth of gram positive Bacillus subtilis (70% reduction) and gram negative Escherichia coli (81% reduction) bacteria. This study demonstrated a facile approach to tune the mechanical stiffness, bioactivity, osteoconduction potential and bacteriostatic properties of scaffolds, which marked it as a potential bone tissue engineered scaffold. 1. Introduction Bone tissue engineering has emerged as a potential technique to repair the damaged bone injured by trauma, infection, tumors, and aging, to handle the scarcity of autografts.1 An ideal bone tissue engineered scaffold should mimic the basic properties of the bone like porosity, adequate mechanical stiffness and optimal degradability as well as should have bioactive cues to stimulate the osteogenesis process.1 Clinically available strategies involved the incorporation of bioactive growth factors within the scaffolds to enhance bone formation2, still orthopedic implants failures occurred due to poor bioavailability of the growth factor and implant associated bacterial infections.3,4 Furthermore, infections were associated with the adhesion of different bacteria such as Staphylococcus species, Escherichia coli and Bacilli species to the implant surfaces causing formation of a biofilm layer which subsequently induced resistance to antibiotics necessitating the surgical removal of the implant to prevent chronic infection.3-5 Hence, new tissue engineering strategies must be explored which can stimulate healing of the fracture by promoting osteogenesis as well as can provide controlled release of the antibacterial molecules to prevent bacterial infections. One of the most attractive strategies to provide osteostimulation as well as antibacterial property is the incorporation of metallic ions like zinc, copper or cobalt within the tissue engineered scaffolds, which is cost effective, provide osteostimulation at very low concentration; and are chemically stable.6-8 Zinc is the preferred choice because it is the natural ingredient of the human 2 ACS Paragon Plus Environment
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bone and is present at 0.012% to 0.025% by weight in bone. This amount is comparatively high compared to the zinc content in other adult tissue (0.003% by weight in fat free tissue) and plasma (0.78- 1 mg/L).9 Earlier studies have suggested that slow release of zinc ions from synthetic scaffolds have shown stimulatory effects on osteoblasts differentiation, osteoclastic resorption, and development of blood vessels both in vitro and in vivo10-14, while higher concentrations of released zinc ions have shown cytotoxic effect on cells proliferation.7,11 Moreover, some reports have claimed the antibacterial properties of the zinc substituted scaffolds.7 Different kinds of zinc doped tricalcium phosphate, calcium silicate, borosilicate and nanosilicate scaffolds have been reported in the literature to improve the physicochemical and biological properties of the scaffolds.9-13 However, the brittleness nature of zinc doped scaffolds made them inappropriate for the load bearing conditions. In addition to this, it has been reported in the literature that although doping of zinc in silicate based scaffolds improved the chemical stability of scaffolds; it also reduced their bioactivity at high concentrations because of the decrease in the dissolution rate of calcium and silicon ions from the zinc doped scaffolds compared to the undoped scaffolds.12 In recent years, there has been emerging interest to develop nanocomposite scaffolds by incorporating biomimetic nanoparticles within polymeric chains. These nanoparticles have provided the feasibility of tuning the scaffold physicochemical properties such as porosity and strength by changing the concentration and composition of polymers and nanoparticles15-17; and improved the cellular function due to the high surface area of nanopatterned scaffolds.18,19 Although these nanocomposite scaffolds have shown promise for bone tissue engineering, still their clinical use is restricted due to poor mechanical properties compared to the native bone.16,18,19 In the current study, we have proposed gellan/xanthan/bioglass nanocomposite scaffolds in an attempt to obtain a scaffold with improved mechanical properties, adequate bioactivity and osteoconduction capability. In the present study, zinc has been introduced as a crosslinker of the nanocomposite matrix rather than doping of the nB nanoparticles, which may circumvent the problems associated with the less solubility and poor bioactivity of the zinc doped bioglass, in addition to its effect on osteoblast function and antibacterial performance of the scaffolds. This is 3 ACS Paragon Plus Environment
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the first report dealing with a bioglass-nanocomposite crosslinked with zinc sulfate as a bioactive and osteoconductive scaffold for bone tissue engineering applications. The nB nanoparticles were selected because of its highly resorbable nature and ability to form the biomimetic hydroxyapatite in vivo leading to enhanced osteointegration.1,20 Many reports have shown the positive effects of bioglass on osteoblasts attachment, proliferation and differentiation.20,21 In addition, some reports have claimed the antibacterial properties of bioglass.22 Gellan is a biocompatible and biodegradable polymer which can form a hydrogel by coil (high temperature) to double helix (low temperature) transition.23 Reports have shown that the mechanical properties of the gellan polymer have enriched by blending them with other natural polymers and by the introduction of divalent cations as crosslinker.24-25 In the present study, gellan was blended with xanthan gum, a natural non-gelling polysaccharide, to improve its elasticity. Earlier reports have shown that xanthan improved the elasticity of gellan by enhancement of the viscosity of gellan solution.26 These properties of gellan and xanthan have made them excellent candidates for different tissue engineering applications.24-25 This paper was focused on optimization of various scaffold components to obtain scaffolds with maximum mechanical stiffness, good bioactivity, improved osteoconductivity and antibacterial properties. Mechanical properties were tested on Universal Testing Machine (UTM) and bioactivity was tested by in vitro SBF study. Osteoconductivity of scaffolds was checked by performing in vitro proliferation and differentiation assays with MG 63 osteoblast-like cells. The antibacterial properties of scaffolds were demonstrated by using gram positive Bacillus subtilis and gram negative Escherichia coli as model bacteria under physiological conditions. 2. Experimental section 2.1. Synthesis of bioactive nanocomposite scaffolds First the nB nanoparticles based on SiO2–CaO–P2O5 (Si: Ca: P (Mol) ≈ 66:27:7)) were synthesized by the standard protocol.27 Both polymeric (1%w/v) and nanocomposite scaffolds (1% w/v polymer with 4% nB nanoparticles) crosslinked with varying ratio of crosslinkers (overall 3mM crosslinker ) were synthesized by the ionic crosslinking process. Detailed description of nanoparticles and nanocomposites synthesis has been summarized in supporting
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information (Supporting Info. S1.1-S1.4). The composition of various polymeric and nanocomposite scaffolds has been shown in Table 1. Table 1. Composition of polymeric and nanocomposite scaffolds with varying molarity of crosslinkers Sample name
(9:1w/w) Gellan: xanthan
nB nanoparticles
Crosslinker
%w/v
% w/v
ZnSO4 (mM)
CaCl2 (mM)
G(Z0)
1%
0
0
3
G(Z1)
1%
0
1
2
G(Z1.5)
1%
0
1.5
1.5
G(Z2)
1%
0
2
1
G(Z3)
1%
0
3
0
nGB(Z0)
1%
4%
0
3
nGB(Z1)
1%
4%
1
2
nGB(Z1.5)
1%
4%
1.5
1.5
nGB(Z2)
1%
4%
2
1
nGBZ(3)
1%
4%
3
0
2.2. Characterization of nB nanoparticles and nanocomposites The nB nanoparticles were characterized for size, morphology and distribution by high resolution transmission electron microscopy (TEM) and field emission gun-scanning electron microscopy (FEG-SEM). Surface charge of nB nanoparticles was calculated by measuring Zeta potential. Crystallinity of the nB nanoparticles and nanocomposites was analyzed by powder X ray diffraction analysis (XRD). Finally, the composition of nanoparticles was confirmed by energy dispersive X- ray spectroscopy (EDAX) and fourier transform infrared spectroscopy (FTIR) analysis. The macroporous nature of scaffolds was examined by the environmental scanning electron microscopy (ESEM). To analyze the pore size distribution and the pore-wall thickness, images of the different types of scaffolds were processed by Image J software. % porosity of scaffolds was calculated by the liquid displacement method as described elsewhere using hexane as the solvent.24 Mechanical properties of nanocomposite scaffolds (as prepared freeze-dried scaffolds, after rehydration in SBF solution and after 7 days of SBF solution incubation) were evaluated by measuring the compressive strength and compressive modulus
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using the computer controlled UTM machine. The details of all characterization techniques have been described in supporting information (Supporting Info. S1.5). 2.3. In vitro degradation, swelling and bioactivity screening study The bioactivity of different types of scaffolds was examined by the SBF study according to the standard protocol.12 Briefly, the scaffolds were immersed into 40 mL of SBF solution and incubated in a shaking waterbath at 37°C and 50rpm for a period of 7, 14 and 21 days. After a fixed interval of time, scaffolds were removed from the SBF solution, lyophilized and analyzed. Deposition of apatite on scaffolds was analyzed by various techniques such as FEG-SEM equipped with EDAX and elemental mapping system, XRD and inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis. Swelling rate of scaffolds and their degradation were measured by the standard protocols.28, 29 Dried scaffolds were incubated with 5mL of the phosphate buffered saline (PBS) solution at 37°C, 100rpm in a shaking water bath for the swelling analysis28 and incubated with 5mL solution of (100U/L) amylase in PBS (pH 7.4) for the degradtion.29 Details of the experimental procedure for swelling and degradation has been summarized in supporting information (Supporting Info. S1.6). 2.4. In vitro cell response 2.4.1. Cell seeding, live/dead assay, proliferation and morphological evaluation For all cell studies, scaffolds of size 1cm x1cm x 0.3cm were prepared and sterilized by gamma radiation at the standard dose of 25kGray60Co at room temperature. Cells were obtained from the National Centre of Cell Science Pune, India. MG 63 osteoblast-like cells were cultured in complete cell culture media (90% Dulbecco’s modified eagle’s medium (DMEM) + 10% fetal bovine serum (FBS) + 1% antibiotic solution) at 37°C, 5% CO2 condition in a humidified incubator. First, all polymeric and nanocomposite scaffolds were screened for in vitro cytotoxicity by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Supporting info. S1.7). Based on the results of cytotoxicity screening of MG 63 cells with scaffolds, only polymeric (G(Z0), G(Z1) and G(Z1.5)) and nanocomposite scaffolds (nGB(Z0), nGB(Z1) and nGB(Z1.5)) which showed >80% viability were used for further experiments to assess the effect of nB nanoparticles and varying crosslinkers concentrations on the osteoblast 6 ACS Paragon Plus Environment
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proliferation and functionality. Briefly, different kinds of scaffolds were pre-incubated with complete DMEM media for 24 hours at 37°C. After that, 2x104 MG 63 cells (with 25µL media) were seeded on scaffolds and allowed to attach for 4 hours at 37°C in 5% CO2 before the addition of an additional 1mL of the complete media. Cells-scaffold constructs were further incubated for 3, 7, 14 and 21 days at 37°C in the 5% CO2 incubator with media change after each 2-3 days. Cell attachment, viability and cellular infiltration within scaffolds was examined after 3 days and 14 days of incubation by tracking the live cells with calcein AM dye (1µM) and dead cells with propidium iodide (2µM) in the confocal microscope and epi-fluorescent microscope. Cellular proliferation on the scaffolds was evaluated after 3, 7 and 14 days of incubation by MTT assay.24 Briefly, cells-scaffold constructs were incubated for 4 hours with 200µL of MTT solution (5mg/mL) for formazan production and then 1mL of 10% sodium dodecyl sulfate (SDS) (pH 3.7-4.1) solution was added to dissolve the formazan crystals. Absorbance of the formazan solution was measured using 560nm filter with 690nm as the reference reading. SEM images of cells were captured after 14 days of incubation with the scaffolds. For SEM analysis, the cellsscaffold constructs were washed with the Dulbecco’s phosphate buffered saline (DPBS) solution, fixed in 2.5% glutaraldehyde solution, and imaged after removal of unbound dye and scaffolds freeze drying. 2.4.2. Maintenance of osteoblastic phenotype The osteoconduction of MG 63 cells on different scaffolds was assessed by quantifying the cellular ALP activity and calcium contents which indicated early and late differentiation phases of cells. For quantifying the ALP activity, SensoLyte®pNPP Alkaline Phosphatase Assay (Anaspec, USA) was performed according to the manufacturer’s protocol. Briefly, after the fixed incubation time, cells-scaffold constructs were washed with assay buffer and lysed with 0.1% triton X solution followed by sonication. Finally the solution was centrifuged and obtained supernatant was incubated for 30 minutes with p-Nitrophenylphenol (pNPP) solution at room temperature. The color absorbance was recorded by the ELISA plate reader at 405nm and results were represented after normalization with the total protein content. Calcium secretion by MG 63 cells was calculated by the semi-quantification assay originally standardized by Gregory et al.30
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Cells-scaffold constructs and scaffolds without cells (control scaffolds) were incubated for 20 minutes in 40mM of alizarin red solution (pH 4.1). After washing, the alizarin red in samples was extracted by treating with 10% acetic acid solution. Samples were neutralized with 10% ammonium hydroxide solution and color absorbance was read at 405nm using a plate reader and, results were normalized with respect to the total protein content. 2.5. Antibacterial activity This study was performed to evaluate the bacteriostatic effect of zinc crosslinker and nB nanoparticles in the scaffolds, using Bacillus subtilis and Escherichia coli, respectively. Bacteria were incubated in the nutrient broth overnight at 37°C with orbital shaking. After this, optical density (O.D.) of the nutrient broth was adjusted to 0.5 at 625nm. The bacterial suspension was then diluted with the fresh nutrient broth to obtain 0.05 (O.D.) value. The polymeric and nanocomposite scaffolds with varying concentrations of calcium and zinc crosslinkers were incubated overnight at 37°C in an orbital shaker with 1mL of the bacterial suspension. The O.D. of the bacterial solution without any scaffold was used as the control reading. Bacteriostatic activity of scaffolds was represented as the O.D. of the bacterial solution with scaffolds to the O.D. of the control bacterial solution without any scaffold. 2.6. Statistical analysis All results were represented as means ± standard deviation. One way ANOVA analysis was used for all statistical calculation and p50%) in the size range 60-80nm. The nanoparticles showed a zeta potential of -22 ± 5mV at physiological pH that reached to -44 ± 2.16mV when nanoparticles were suspended in gellan: xanthan polymeric solution (Figure S1B). This behavior indicated improved stability of nanoparticles within polymeric solution. Nanoparticles were composed of 8 ACS Paragon Plus Environment
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Ca, Si, P and O with Si: Ca: P molar ratio of 66: 24: 6 as calculated from EDAX analysis (Figure S1C). XRD analysis of nB nanoparticles showed a single broad curve, ranging from 15-35º with a very low intense peak centered at 30º (Figure 1C), which are characteristics of amorphous bioglass nanoparticles as discussed elsewhere in the literature.27 Amorphous nature of nB nanoparticles can improve dissolution rate, degradation and bioactivity of scaffolds.31 FTIR spectrum of nB nanoparticles showed broad and strong peak at 1100cm-1 that was due to O-Si-O and P-O stretching vibrations (Figure 1D). A sharp peak at 810 cm-1 was because of symmetric stretch vibration of Si-O (absorption band). Small peak at 470cm-1 and 580cm-1 were bending vibrations of Si-O- and amorphous P-O bond, respectively. 3.2. Effect of the nanoparticles and crosslinkers concentrations on the scaffold macrostructure Cross sectional ESEM images of scaffolds showed macroporous structure of scaffolds (Figure 2A). Calculated average pore size and pore wall thickness of nanocomposite scaffolds was higher compared to the G(Z3) polymeric scaffold (p80% viability, while
scaffolds crosslinked with high concentration of zinc sulfate (G(Z2), G(Z3), nGB(Z2), nGB(Z3)) showed poor viability (Figure 4A & 4B). ICP-AES analysis of scaffolds extracts in DMEM media (Table 3) showed that concentration of calcium and silicon ions released in the extract media of all polymeric and nanocomposite scaffolds was far below the toxicity level.7 Concentration of zinc ions releasing in the extract media was increasing with increase in zinc 11 ACS Paragon Plus Environment
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crosslinker concentration in scaffolds. Scaffolds with zinc concentration varying from 0-3.4ppm showed biocompatibility, while scaffolds with higher release of zinc ions (>5.5ppm) created cytotoxic effects which was in agreement of previous literature.7,44 Based on the results, polymeric and nanocomposite scaffolds (G(Z0), G(Z1), G(Z1.5), nGB(Z0), nGB(Z1), nGB(Z1.5) with viability >80% were considered for further characterization. Table 3. ICP-AES analysis of complete DMEM solution after 24 hours incubation with scaffolds Concentration
Ca
Ca
Zn 2
Zn
Si 2
Si 2
P
P
of ion
(ppm)
(ppm/cm )
(ppm)
(ppm/cm )
(ppm)
(ppm/cm )
(ppm)
(ppm/cm2)
G(Z0)
21.7
6.8
0
0
0
0
30.1
9.4
G(Z1)
19.3
6.0
2.4
0.8
0
0
30.5
9.5
G(Z1.5)
17.6
5.5
3.4
1.2
0
0
35.3
11.0
G(Z2)
16
5.0
8.2
2.6
0
0
38.9
12.2
G(Z3)
14.4
4.5
11.8
3.7
0
0
40.2
12.6
nGB(Z0)
99.3
31.0
0
0
104.6
32.7
9.3
2.9
nGB(Z1)
94.4
29.5
1.3
0.4
106.8
33.4
10.5
3.3
nGB(Z1.5)
93.5
29.2
2.5
0.8
100.2
31.3
11.6
3.6
nGB(Z2)
85.7
26.8
5.5
1.7
99.7
31.2
15.8
4.9
nGB(Z3)
83.5
26.1
7
2.2
99.1
31.0
17.8
5.6
3.5. Swelling rate and degradation study Swelling of scaffolds in the body fluid is an important criteria for the bone tissue engineered scaffold as it allows transport of nutrients and different metabolites by scaffolds.37-38 All scaffolds showed significant water uptake in PBS media (Figure 4C). The swelling rate of all nanocomposite scaffolds (nGB(Z0), nGB(Z1), nGB(Z1.5) was significantly less (p
