Mina Mekhail , Jamal Daoud , Guillermina Almazan , and Maryam Tabrizian *
Rapid, Guanosine 5’-Diphosphate-Induced, Gelation of Chitosan Sponges as Novel Injectable Scaffolds for Soft Tissue Engineering and Drug Delivery Applications
Chitosan has been emerging as a promising biomaterial for a multitude of applications in tissue regeneration and drug delivery. [ 1 , 2 ] Many fabrication methods have been established to form chitosan-based microparticles, nanoparticles, fi lms, sponges, microfi bers, nanofi bers, and hydrogels; making chitosan one of the most versatile biomaterials ever investi-gated. [ 3–8 ] More specifi cally, chitosan has been widely explored in the growing fi eld of injectable hydrogels. Stimuli such as temperature, pH and UV-irradiation, have been tuned to trigger chitosan gelation in situ following injection. [ 9–12 ] Thermosen-sitive Chitosan/ β -Glycerophosphate hydrogels, extensively studied in the literature, were shown to gel in 4–9 minutes at 37 ºC; [ 13 ] thermosensitive PEG-grafted chitosan was shown to undergo a sol-gel transition in 10 ± 4 minutes; [ 14 ] and pH-sensitive chitosan/PVA hydrogels underwent gelation after 35 minutes. [ 15 ] These examples illustrate that there still remains a need for rapidly-gelling hydrogels. A fast rate of gelation ensures the localization of the hydrogel at the injection site and prevents undesirable fl ow to surrounding tissues, a sus-ceptibility characteristic of slow-gelling hydrogels. Therefore, in comparison to current injectable systems, the proposed inject-able guanosine 5′-diphosphate (GDP)-crosslinked chitosan sponges are superior since they undergo gelation in less than 1.6 seconds without the need for external stimuli. Furthermore, GDP, a cellular component, has not been previously explored as an anionic crosslinker. It was investigated in this study for its excellent biocompatibility and the presence of guanosine, a nucleoside with known therapeutic effects in the central nervous system. [ 16 ]
Chitosan gelation occurs rapidly upon mixing the chitosan and GDP solutions. This mixing-induced gelation is a result of electrostatic attractions between the anionic phosphate groups of GDP and the cationic amine groups of chitosan. For in situ applications, the chitosan and GDP solutions have to be injected into the target site simultaneously using a double-barrel syringe with two independent outlets. This ensures that mixing occurs in situ and not inside the needle.
M. Mekhail, Dr. J. Daoud, Prof. M. TabrizianBiomedical Engineering Duff Medical Building Room 313, McGill, Montreal, H3A 2B4, Canada E-mail: [email protected] Prof. G. AlmazanPharmacology and Therapeutics McIntyre Building, Room 1321, McGill, Montreal, H3G 1Y6 Canada
Tripolyphosphate (TPP), another widely investigated ani-onic crosslinker that induces chitosan gelation, has been pre-viously used to fabricate chitosan nanoparticles, microparticles and fi bers. [ 17–21 ] However, upon examining the cytotoxicity of GDP as compared to tripolyphosphate (TPP) at concentrations ranging from 0.05 mM to 50 mM using 3T3 fi broblasts, GDP was found to be signifi cantly less cytotoxic at a concentration of 5 mM after 24 hours of culture (Figure S1). The pronounced cytotoxicity of TPP at concentrations higher than 5 mM has also been demonstrated in a recent study. [ 22 ] This renders GDP a more cell-compatible alternative.
In order to demonstrate the potential use of this sponge for tissue regeneration and drug delivery applications, the physico-chemical and mechanical properties, as well as cellular com-patibility, were investigated. Four GDP-crosslinked chitosan sponges were fabricated ( Figure 1 A, B, C, and D). Two concen-trations of chitosan (3 mg/ml and 6 mg/ml) were prepared in a 0.01M HCl solution. The pH of the chitosan solutions was then adjusted to 5 or 6 using a 1M sodium bicarbonate solu-tion. [ 23 ] The four chitosan solutions were designated acronyms in the form of C(X)PH(Y), where ‘X’ and ‘Y’ represent the chi-tosan concentration and solution pH respectively, giving the following formulations: C3PH5, C3PH6, C6PH5 and C6PH6. A GDP solution with a concentration of 100 mg/ml was pre-pared in distilled water. Each chitosan formulation (1.7 ml) was supplemented with 0.3 ml of the GDP solution (fi nal GDP con-centration of 34 mM) through rapid injection, instantaneously producing a GDP-crosslinked chitosan sponge. It is important to ensure that the pH of the distilled water used to make the GDP solution is within the range of 6.8-7. Moreover, it was crit-ical that the pH of the chitosan solutions be maintained within a maximum range of ± 0.05 to avoid inconsistencies in sponge properties. This pH range was chosen since electrostatic attrac-tions between the phosphate and amine groups were found to occur rapidly and form an intact chitosan sponge. In addi-tion, the concentrations of 3 and 6 mg/ml were selected since lower concentrations did not promote the formation of intact sponges, while higher concentrations yielded viscous chitosan solutions, making it diffi cult to produce the sponge through mixing.
Scanning Electron Microscopy (SEM) images of the GDP-crosslinked chitosan sponges revealed a 3D struc-ture with heterogeneous pore sizes and excellent pore inter-connectivity. The sponges were formed of densely packed nanometer-sized poly mer aggregates with an average size of 140 ± 19 nm. Moreover, there were no apparent differences in the micro-structure of the four sponge formulations
Figure 1 . Characterization of GDP-crosslinked injectable chitosan sponges. A), B), C) and D) are SEM images of C3PH5, C3PH6, C6PH5 and C6PH6 respectively; E: Water retention measured using Equation 1 ; F) Gelation time (seconds) measured using impedance spec-troscopy; G) FTIR spectra of C6PH5, which was similar to the other sponges (results not shown), as compared to GDP and chitosan powder; H: Crystallinity index (%) measured using Equation 2 ; I: XRD spectra of the four chitosan sponges.
(Figures 1 A, B, C, and D). However, the micro-structure appeared signifi cantly different from that of freeze dried sponges reported in the literature. [ 24–26 ] Overall, the sponges’
porosity and pore-interconnectivity makes them structurally sound for tissue engineering applications.
Chitosan sponges were found to retain water up to ten times their own weight (Figure 1 E; Equation 1 ). The lower chitosan concentra-tion (3 mg/ml) yielded higher water reten-tion, which is attributed to the ability of water to easily infi ltrate the sponges and cause more swelling. C3PH5 possessed the highest water retention (1037% ± 43), which was signifi cantly higher than C6PH5 (679% ± 44) and C6PH6 (784% ± 57) (P < 0.05). However, there were no statistical differences between C3PH5 and C3PH6 (938% ± 46) or between C6PH5 and C6PH6. The capability for high water reten-tion demonstrates the ability of these sponges to release encapsulated drugs effectively into the surrounding environment via diffusion. In addition, it allows for nutrient and oxygen diffu-sion into the sponge when used for tissue engi-neering applications.
Impedance spectroscopy was used to measure the gelation time after mixing the chitosan and GDP solutions (Figure S2). This method is more reliable than the ‘inverted tube test’ that has been widely used in the literature. [ 27 ] All sponges formed in less than 1.6 seconds (Figure 1 F). C6PH5 yielded the shortest gelation time (1.06 ± 0.0384 seconds), which was signifi -cantly more rapid than the other three sponges (P < 0.05). On the other hand, C6PH6 was the slowest forming sponge (1.58 ± 0.00667 sec-onds) and was signifi cantly slower than C3PH6 and C6PH5 (P < 0.05), but was not signifi cantly slower than C3PH5. A correlation was observed between water retention and gelation time. Sponge formulations using 3 mg/ml of chitosan had a slow gelation time and allowed for more water retention. A similar correlation could be observed in sponges formed using 6 mg/ml of chitosan; however, there was no signifi cant dif-ference between water retention capabilities of C6PH5 and C6PH6. This rapid gelation will ensure the localization of sponges at the site of injection, which is a key requirement for drug delivery and tissue regeneration applications.
FTIR analysis was used to confi rm the incor-poration of GDP within the chitosan structure (Figure 1 G). The spectra of GDP-crosslinked chitosan sponges showed peaks specifi c to GDP (777 cm − 1 , 905 cm − 1 , 1178 cm − 1 , 1229 cm − 1 , and 1533 cm − 1 ) within the chitosan peaks, thus confi rming the crosslinking of chitosan using GDP. X-Ray Diffraction (XRD) patterns and the Crystallinity Index (C r I 100 ) revealed a decrease
in crystallinity and an increase in the amorphous phase in all chitosan formulations compared to the dry chitosan powder (Figure 1 H, I; Equation 2 ). The crystallinity of the as-purchased
Figure 2 . A) The force-displacement curves of the four sponge formulations and an image of the miniature loading cell used; B) The moduli of elasticity of the sponges calculated using Equation 3 with signifi cant differences assigned by ∗ and † (P < 0.05); C) D) E) and F) are SEM images of 3T3 fi broblasts cultured on C3PH5, C3PH6, C6PH5 and C6PH6 respectively for 3 days.
chitosan powder was similar to previous reports, [ 28 ] and inter-estingly the crystallinity trend of the four sponges followed those of the water retention and gelation time. The low crystal-linity index of C6PH5 can be attributed to the fast rate of gela-tion that fi xes chitosan chains in non-crystalline conformations thus inhibiting the slower crystalline formations that take place through diffusion.
Moduli of elasticity (E) of the chitosan sponges were measured using a stainless steel ball indenter (Ø = 1 mm; Figure 2 A, B; Equation 3 ). Sponges prepared using chitosan formulations at pH 5 had the most protonated amine groups, providing more sites for electrostatic crosslinking with GDP, and were therefore the stiffest. One of the reasons C6PH5 (E = 0.867 ± 0.0931 MPa) was not signifi cantly stiffer than C3PH5 (E = 0.789 ± 0.0656 MPa) may be attributed to the satu-ration of the crosslinking reaction, since a similar amount of GDP was used in both sponge formations. Moreover, C3PH6 (E = 0.432 ± 0.0478) was signifi cantly softer than C3PH5 and C6PH5 (P < 0.05) due to less protonation of amine groups at a higher pH. C6PH6 (E = 0.606 ± 0.0880) also had less amine protonation as compared to C6PH5, and was thus softer; how-ever, this difference was not signifi cant (P = 0.09). Mechanical characterization revealed that the sponges are soft biomaterials
that can be used for tissue regeneration of various soft human tissues such as articular cartilage, [ 29 ] spinal cord, [ 30 ] brain [ 31 ] and heart muscle. [ 32 ]
In order to investigate the cell compat-ibility of the sponges and whether pH neutralization by washing in DMEM is essential, 3T3 fi broblasts were cultured on DMEM-washed and unwashed sponges for 3 days. SEM images revealed fi broblast attachment and spreading on the surface of DMEM-washed (Figures 2 C, D, E, and F) and unwashed sponges (Figure S3). To further investigate the cytocompatibility of the sponges, fi broblasts were seeded on the sponges for 1, 5 and 7 days. At each time-point, the cell nuclei were stained with DAPI and Ki-67 (a nuclear protein found in prolif-erating cells). Counting the total number of nuclei and Ki-67-positive nuclei per volume (mm 3 ) provided an indication of the overall proliferation over 7 days and the proliferative state at each time point ( Figure S4 ). Results indicated that C6PH6 showed signifi cant fi broblast proliferation after one week as compared to the other sponges. In addi-tion, there was no signifi cant cell death in any of the sponges after 7 days of culture. These results are a strong indication of the cytocompatibility of all sponges, and dem-onstrate that C6PH6 is the best candidate for tissue regeneration applications utilizing fi broblasts.
In this communication we introduced a methodology to rapidly form chitosan sponges using GDP as a novel anionic
crosslinker. The proposed injectable system possesses excel-lent physicochemical properties that render it an optimal injectable scaffold relative to current alternatives. These prop-erties include: rapid gelation (t < 1.6 sec), a micro-structure that is highly porous with inter-connected pores, the ability to retain water up to 10 times its weight, mechanical properties resembling those of human soft tissue, and fi nally, desirable cytocompatibility.
Experimental Section Materials : High Molecular Weight Chitosan (Degree of Deacetylation
> 90%; 3000 cp viscosity) was purchased from MP Biomedicals, LLC (CAT No. 150597). GDP was purchased from Sigma Aldrich (CAT No. G7127). Hydrochloric Acid (50% v/v) was purchased from LabChem Inc (CAT No. LC15130-3).
Scanning Electron Microscopy (SEM) : Chitosan sponges were dehydrated to ethanol, and then to amyl acetate. Critical point drying (CPD) was performed using Leica EM CPD030. The sponges were then coated with Gold/Palladium and imaged using a Hitachi S-4700 FE-SEM at 2 KeV and a current of 10 μ A.
Fourier Transform Infrared Spectroscopy (FTIR) : GDP-crosslinked chitosan sponges were washed thoroughly in distilled water and dried at 60 ° C overnight prior to FTIR analysis. Infrared measurements
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were performed using a Perkin Elmer FTIR spectrometer with an ATR attachment (Pike Technologies). The spectra were collected in absorption mode, using 64 scans, and a resolution of 4 cm − 1 .
Water Retention : Chitosan sponges were placed in PBS overnight at 37 ºC. Excessive surface water was removed by gently tapping the sponges on a paper towel and W wet was recorded. Sponges were dried at 60 ºC overnight, and the W dry was measured. The following equation was then used to calculate the percent water retention of the sponges:
Wwet − Wdr y
Wdr y× 100
(1)
Time of Sponge Formation : The time of sponge formation was
measured using an Agilent 4294A high precision impedance analyzer. A constant frequency of 300 KHz and a peak-to-peak AC voltage of 300 mV were applied. GPIB computer data logging produced 400 data points, collected over a span of 20 seconds. The dielectric spectrum of a fi xed volume of chitosan (150 μ l) solution was measured for 1 minute until the signal was stabilized. An equal volume of GDP (150 μ l) solution was then introduced and the change of impedance was recorded. The time of sponge formation was calculated by measuring the time between GDP injection and the re-stabilization of the dielectric spectra. In order to investigate whether the change in impedance is due to an increase in volume or GDP crosslinking, the signal produced by the chitosan solution (150 μ l) was stabilized and an additional 150 μ l of the chitosan solution was added. No change in the impedance was observed after the volume increase, thus concluding that the decrease in impedance observed with crosslinking was due to GDP crosslinking and not to volume increase.
X-ray Diffraction : A Bruker D8 Discovery X-Ray Diffractometer was used to study the crystallinity of as-purchased chitosan and the four sponge formulations using: a 2 θ range from 2 to 60º, a rate of 4 º/min, 40 kV and 80 mA. The Crystallinity index (C r I 100 ) was calculated using the following equation: [ 28 , 33 ]
Cr I100 =
I110 − Iam
I110× 100
(2)
I 110 is the lattice diffraction measured at 2 θ = 20 and I am is the amorphous region diffraction measured at 2 θ = 16.
Compressive Mechanical Properties : Samples were washed thoroughly in PBS prior to any measurements. Indentation using a stainless steel ball (Ø = 1 mm) was used to measure the moduli of elasticity of the chitosan sponges. Measurements were done using a Fullam miniature loading stage in conjunction with a FUTEK 20g load cell. A rate of deformation 5 μ m/s was used and the force was measured until a threshold of 0.15 N was reached.
F =
4
3
E
(1 − v)2δ
32 R
12
(3)
A model ( Equation 3 ) assuming an elastic half space and a rigid
spherical indenter was used to calculate the modulus of elasticity (E) from the force-displacement graphs. [ 34 ] F is force, ν is Poisson’s ratio, δ is indentation displacement, and R is the radius of the spherical indenter. Equation 3 was fi tted to the experimental data with a chi-square tolerance of 10 − 9 and assuming a Poisson’s ratio of 0.5 since the sponge was fully swollen and was assumed to act as a rubber-like material. [ 32 , 35–37 ]
SEM of Fibroblasts on Sponges : Chitosan sponges to be seeded with cells were prepared under sterile conditions. Chitosan and GDP solutions were prepared in sterile water and fi ltered using a 0.2 μ m fi lter after pH adjustments. In order to examine the applicability of using the injectable sponges in situ, two groups of sponges were prepared: sponges that were washed thoroughly with DMEM prior to cell seeding and sponges that were not washed. Fibroblasts (1 × 10 5 cells/ml) were then cultured on the four sponge formulations (washed and not washed) for 3 days using DMEM (containing High Glucose, HEPES and L-Glutamine) and supplemented with 10% Calf Bovine Serum (CBS) and 1% PenStrep. For SEM analysis, sponges containing cells were thoroughly washed with
[ 1 ] M. Prabaharan , J. Biomater. Appl. 2008 , 23 , 5 . [ 2 ] I. Y. Kim , S. J. Seo , H. S. Moon , M. K. Yoo , I. Y. Park , B. C. Kim ,
C. S. Cho , Biotechnol. Adv. 2008 , 26 , 1 . [ 3 ] I. Panos , N. Acosta , A. Heras , Curr. Drug Discov. Technol. 2008 , 5 , 333 . [ 4 ] J. J. Wang , Z. W. Zeng , R. Z. Xiao , T. A. Xie , G. L. Zhou , X. R. Zhan ,
S. L. Wang , Int. J. Nanomed. 2011 , 6 , 765 . [ 5 ] F. J. Pavinatto , L. Caseli , O. N. Oliveira , Biomacromolecules 2010 , 11 ,
1897 . [ 6 ] R. Jayakumar , M. Prabaharan , P. T. S. Kumar , S. V. Nair , H. Tamura ,
Biotechnol. Adv. 2011 , 29 , 322 . [ 7 ] R. Jayakumar , M. Prabaharan , S. V. Nair , H. Tamura , Biotechnol. Adv.
2010 , 28 , 142 . [ 8 ] N. Bhattarai , J. Gunn , M. Q. Zhang , Adv. Drug Delivery Rev. 2010 ,
62 , 83 . [ 9 ] S. S. Vaghani , M. M. Patel , C. S. Satish , Carbohyd. Res. 2012 , 347 , 76 . [ 10 ] L. Zheng , Q. Ao , H. Y. Han , X. F. Zhang , Y. D. Gong , Biomed. Mater.
2010 , 5 . [ 11 ] L. M. Wang , J. P. Stegemann , Biomaterials 2010 , 31 , 3976 . [ 12 ] Y. Tsuda , M. Ishihara , M. Amako , H. Arino , H. Hattori , Y. Kanatani ,
H. Yura , K. Nemoto , Artif. Organs 2009 , 33 , 74 . [ 13 ] K. E. Crompton , R. J. Prankerd , D. M. Paganin , T. F. Scott ,
M. K. Horne , D. I. Finkelstein , K. A. Gross , J. S. Forsythe , Biophys. Chem. 2005 , 117 , 47 .
[ 14 ] N. Bhattarai , H. R. Ramay , J. Gunn , F. A. Matsen , M. Q. Zhang , J. Controlled Release 2005 , 103 , 609 .
[ 15 ] T. Wang , M. Turhan , S. Gunasekaran , Polym. Int. 2004 , 53 , 911 . [ 16 ] M. Mekhail , G. Almazan , M. Tabrizian , Prog. Neurobiol. 2012 , 96 ,
322 . [ 17 ] N. Csaba , M. Koping-Hoggard , M. J. Alonso , Int. J. Pharmaceut.
2009 , 382 , 205 . [ 18 ] A. Hasanovic , M. Zehl , G. Reznicek , C. Valenta , J. Pharm. Phar-
macol. 2009 , 61 , 1609 . [ 19 ] F. Pati , B. Adhikari , S. Dhara , Carbohyd. Res. 2011 , 346 , 2582 . [ 20 ] X. Shu , K. J. Zhu , Int. J. Pharmaceut. 2000 , 201 , 51 . [ 21 ] F. L. Mi , S. S. Shyu , S. T. Lee , T. B. Wong , J. Polym. Sci. Pol. Phys.
1999 , 37 , 1551 .
sterile PBS, fi xed for 30 minutes in 4% paraformaldehyde and dehydrated to ethanol and then to amyl acetate. Sponges were dried using CPD and were coated with Gold/Palladium and imaged using a Hitachi S-4700 FE-SEM at 2 KeV and a current of 10 μ A.
Statistical Analysis : OriginPro 8.6 was used to perform statistical analysis. All experiments were done in triplicates (n = 3). One way ANOVA was performed followed by the Tukey test to examine signifi cant difference amongst groups with 95% confi dence.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements The authors would like to acknowledge Dr. Francois Barthelat and Dr. Reza Rabiei from The Biomimetic Materials Laboratory at McGill University for allowing us access to their Fullam miniature loading stage. The authors would also like to acknowledge the sources of funding: NSERC and MDIEI.
Received: October 18, 2012 Revised: January 31, 2013
[ 22 ] F. Pati , B. Adhikari , S. Dhara , J. Mater. Sci.-Mater. Med. 2012 , 23 , 1085 .
[ 23 ] H. Zhong , X. Lei , L. Qin , J. Wang , T. Hung , Gene Ther. 2011 , 18 , 232 . [ 24 ] Y. J. Park , Y. M. Lee , J. Y. Lee , Y. J. Seol , C. P. Chung , S. J. Lee , J. Con-
trolled Release 2000 , 67 , 385 . [ 25 ] P. Arpornmaeklong , N. Suwatwirote , P. Pripatnanot , K. Oungbho ,
Int. J. Oral Max. Surg. 2007 , 36 , 328 . [ 26 ] D. J. Griffon , M. R. Sedighi , D. V. Schaeffer , J. A. Eurell , A. L. Johnson ,
Acta Biomater. 2006 , 2 , 313 . [ 27 ] D. Gupta , C. H. Tator , M. S. Shoichet , Biomaterials 2006 , 27 , 2370 . [ 28 ] D. Ren , H. Yi , W. Wang , X. Ma , Carbohyd. Res. 2005 , 340 , 2403 . [ 29 ] R. M. Schinagl , D. Gurskis , A. C. Chen , R. L. Sah , J. Orthop. Res.
1997 , 15 , 499 . [ 30 ] L. E. Bilston , L. E. Thibault , Ann. Biomed. Eng. 1996 , 24 , 67 .
