Introduction
The neural disorder is an essential health challenge which can be caused by accident or disease. Due to the complication of neural regeneration and predomination of neural disease, the evolution of the novel treatment approaches has been performed by researchers [1]. Tissue engineering or regenerative medicine is one of the multidisciplinary fields that could recompense the pain from organ loss. It comprises the synthesis of tissue from biological materials and is substituted with the malfunctioning tissue. Indeed, tissue engineering improves tissue function by combining a suitable supportive matrix, biological materials and cells [2, 3]. Polymer scaffolds have significant roles as supportive matrices with an aim to develop tissue engineering. Hydrogels as an excellent class of biomaterials demonstrate the advantages in many ways for their adjustable biodegradation rate, biocompatibility with host tissue, proper porosity for the carrying of nutrients and wastes, and tunable mechanical properties. In addition, they are networks that can absorb water, and as a result, cells can adhere, proliferate and differentiate into the hydrogels. Also, their structure is similar to the native extracellular matrix so they can be a good carrier for drug/gene delivery systems. Hydrogels in biomedical applications have two categories: (1) natural polymers like chitosan, gelatin and agarose (2) synthetic polymers such as polycaprolactone (PCL), polylacticacide (PLA), polyurethane (PU) [4].
Proliferation and differentiation of cells depend on the surface properties of scaffolds. Recent studies have shown that conductive polymers, as a new generation of materials, have demonstrated a beneficial use by sending cellular signaling. Because of the intrinsic properties of neural tissue, the conductivity is more striking. Signals have transferred in neural tissue through action potential phenomenon, and these conductive polymers can mimic this action. The soft nature of conductive polymers causes a good connection with cells and better biocompatibility in comparison with metals and inorganic materials. Moreover, they can promote the adhesion, migration, proliferation, differentiation, and shape of cells, with or without electrical stimulation. Conductive polymers including polyaniline (PANI), carbon nanotubes (CNTs), polythiophene and polypyrrol (PPY), are appropriate materials in biomedical applications such as tissue engineering, drug delivery, neural prostheses, biosensors and artificial organ; because of the feasible processability and biocompatibility. Although poor solubility, poor degradation, and chronic inflammation are their drawbacks. In order to overcome these disadvantages, aniline oligomers have been utilized which have both biodegradability and solubility with proper electroactivity. Oligo anilines can be consumed by macrophage and filtered with kidney [5]. Because of the light toxicity and brittleness of oligo anilines, they have been grafted with natural biodegradable macromolecules such as chitosan, gelatin, agarose, and alginate; and create bio conductive polymers [6]. Chitosan is a linear bio-based cationic polysaccharide which is driven from deacetylation of its parent polymer chitin; and has received a great attention because of its biocompatibility, biodegradability, low toxicity and the ability to promote cell adhesion. All these properties of aniline oligomers and chitosan make them a great bio conductive scaffold for demanding tissue engineering and drug delivery [7].
Recently, injectable thermosensitive hydrogels that are soluble at room temperature, and become gel after injection into the body, have received a great attention in tissue engineering and biomedical applications. Injectable hydrogels have advantages such as they can be implanted into the body without surgery, which is a noninvasive approach; they can encapsulate cells or drugs in situ; they can take the shape of damaged tissue; and the ease of administration. Thermosensitive gelation is generally used to produce injectable conductive hydrogels, through chemically grafting conductive segments onto the end of thermosensitive materials. Pluronic® F127 is a triblock copolymer poly (ethylene oxide)-b-poly (propylene oxide)-b-poly (ethylene oxide) (PEO-PPO-PEO), which has sol-gel transition above its LCST point, because of hydrophobic interaction in PPO [8].
In our previous work, a novel drug loaded colloidal hydrogel by reacting carboxyl capped oligo-aniline and gelatin was synthesized with tunable properties for neural tissue engineering. Colloidal hydrogels have been used for regulating the neural interface modulus, but they did not have enough conductivity, therefore, these hydrogels can be apt for neural tissue engineering [9]. Also, alginate-aniline tetramer-agarose was synthesized and exhibited applicable interaction with PC12 cells. Agarose, an inert and non-immunological material, was used to prevent the toxicity of crosslinking agent. In spite of its good mechanical features, agarose decreased the ionic conductivity because of the large size molecule which acts as a barrier between conductive units. In addition, Aniline tetramer in alginate structure, augments the conductivity. Because of the ion mobility improvement, ionic conductivity is increased and activation energy is reduced with temperature being incremented [10]. In addition, Colloidal hydrogels have been applied for regulating the neural interface modulus, however, their inadequate conductivity hindered their applications to some extent, so our group has reported a bio-conductive colloidal hydrogel based on agarose-aniline pentamer, which improved the cellular activity and amended the nerve regeneration. For the reason that conductivity, on-demand drug release, mechanical features and self-gelling properties; this hydrogel can be a good option for neural electrodes [5].
Recent cases reported by Gue et al. also support the hypothesis that in comparison with traditional scaffolds, the injectable hydrogels with electrical stimuli not only are feasible to synthesize, but also there is no need to use invasive surgical operations. They developed injectable conductive hydrogels for drug delivery with electro and pH responsibility based on chitosan-poly aniline, and oxidized dextran as a cross-linker. The in vitro and in vivo studies demonstrated inherent antibacterial activity and suitable cytocompatibility [11]. Also, Dong et al. developed a series of injectable conductive self-healing hydrogels based on aniline tetramer-graft-chitosan and dibenzaldehyde-terminated poly ethylene glycol, and exhibited their possibility as cell delivery vehicle for myocardial infarction for cardiac cell therapy [12]. Moreover, they have developed an injectable dopamine based hydrogel by NaIO4 as the oxidizing agent for oxidizing a mixture of chitosan, gelatin and dopamine. This system could deliver dopamine and inflammatory drugs, which they could help treatment of Parkinson’s disease [13]. In a similar case, a series of in situ forming electroactive biodegradable hydrogels based on gelatin-graft-poly aniline and genipin, as a cross-linker, were synthesized. They demonstrated a linear release profile of diclofenac sodium as a drug. The in situ forming convenience administration of these materials in a non-invasive way [14].
Material and methods
Materials
Dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), N-hydroxysuccinimide (NHS) and N, N-di cyclohexyl carbodiimide (DCC), ammonium peroxidisulfate (APS), camphor sulfonic acid and succinic anhydride were purchased from Merck. Medium molecular weight Chitosan, N-Phenyl-p-phenylenediamine, p-Phenylenediamine, Pluronic F127 and β-glycerophosphate was received from Sigma-Aldrich.
Experimental methods
NHS-capped Aniline Pentamer Synthesis
NHS-capped Aniline Pentamer was synthesized as the same way in the references. Carboxyl capped aniline pentamer was synthesized by reacting of N-Phenyl-p-phenylenediamine and succinic anhydride to produce carboxyl capped aniline dimer, and then p-phenylenediamine was added to the mixture to make carboxyl capped aniline pentamer. APS was added dropwise to the mixture. Finally, N-hydroxysuccinimide (NHS) and N,N-di cyclohexyl carbodiimide (DCC) was added to achieve NHS-capped Aniline Pentamer [15].
Synthesis of Chitosan -graft-NHS capped aniline pentamer copolymer
The presence of amine groups in the structure of Chitosan (CS) facilitates the reaction with carboxyl group of aniline pentamer (AP) using carboiimides. 0.5 gr of CS were dissolved in 20 ml camphor sulfonic acid. Also, 0.1 gr NHS-capped-AP were dissolved in 5 ml DMSO. Subsequently, the two solutions were mixed and stirred for 24 h at 50 °C temperature under a nitrogen atmosphere [16].
Synthesis of CS-graft- NHS capped AP / Pluronic F127
The product was mixed with 0.5, 0.25, 0.12 gr pluronic with was dissolved in 5 ml DMSO and stirred at room temperature under nitrogen atmosphere.
Preparation of CS-g-NHS capped AP/Pluronic F127/ β-glycerophosphate hydrogel
0.5 gr β-glycerophosphate was dissolved in 5 ml of deionized water and then added to the aforementioned mixture to reach intended hydrogel.
Characterization and experiments
Fourier transformed infrared spectroscopy (FT-IR)
FT-IR spectra were obtained to study the chemical interactions between the various functional groups within the components present in the CS, NHS-capped AP, CS/NHS-capped AP/Pluronic and final hydrogel, using a Bruker instrument with the KBr disc, made by Germany, working in the range of 4000-600 cm-1 at a resolution of 4 cm – 1 at ambient temperature.
Electro activity and conductivity
UV-visible (UV-Vis) was used to evaluate the AP concentration and transition states of the copolymer with a spectrophotometer (Shimadzu, Kyoto, Japan). To do so, AP has dissolved in DMSO/deionized water (1:1) solvent mixture and the spectra of the undoped AP using camphor sulfonic acid, as a dopant, were assessed at 340 nm. The AP content (C) in the sample was evaluated by dividing of the slope of samples concentration (P) on pure AP slope (P0)(C% = P/P0 * 100) [17].
Micro auto lab type ш apparatus was used for cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Carbon pasted electrode (CPE) modified with 20% polymer was the working electrode, platinum wire was the counter electrode, Ag/AgCl was the reference electrode and indium tin oxide (ITO) electrode was the working electrode. All of which were put in the 1 M HCl solution and the 50 mVs−1 was applied. Also, EIS was performed at 0.01 Hz. EIS by the neural model which consist of one capacitance and two resistors. With EIS records, Nyquist plot and conductivity properties were obtained [5].
The conductivity of the hydrogel was determined by measuring the resistances using the four-probe method [18]. Samples were prepared in pellet then volt and ampere were applied, the value of conductivity was obtained by the Eq. (1):
σ = 1/R d/S (1)
Where σ is the conductivity, d is the thickness, R is the resistance, and S is the area of the sample, respectively. Ionic conductivity was obtained at the ultimate swollen state in various temperatures using Eq. (2):
σ =σ_0/T exp〖(- E_a/RT)〗 (2)
In which, σ0 is the initial conductivity value before the sample being swelled, T is the absolute temperature at which test is performed, Ea is activation energy and R is the molar gas constant.
Conductivity is related to mobility and hole concentration according to (3):
σ=q× μ_p×p (3)
Where q, μp and P are mathe gnitude of electronic charge (1.6 × 10-19 C), mobility and hole concentration [10, 19].
Morphology
The surface morphology, porous structure and cell adhesion of cells to scaffolds the freeze-dried hydrogel were studied using scanning electron microscopy (SEM). Before the examination, the surface of the samples was coated with a gold layer.
Thermal properties
Thermal characteristics of the samples were conducted with differentiation scanning calorimetry (DSC) under a nitrogen atmosphere with a heating rate of 10 °C/min which is swept from 0 to 250 °C and 250 to 0 °C for heating and cooling, respectively.
Furthermore, Thermogravimetric analysis (TGA) of hydrogels were persuaded using a Perkin–Elmer Pyris-1 TGA apparatus, with a heating rate of 10 °C/min from 35 to 900 °C under air and nitrogen atmosphere [20].
Rheological characterization
The gelation time and storage modulus of these hydrogels were measured with MCR 300 Anton Paar Rheometer with cylinder geometry. Temperature sweep test was done in the range of 15°C to 70°C, a frequency 0.1 Hz and strain of 1%. Time sweep test was conducted with a frequency 1 Hz and a strain of 1% in 30 °C. Also, a dynamic frequency sweep test with frequency ranging from 1 to 100 rad s-1 at a shear amplitude of 0.1% was employed to perform the measurement.
Contact angle
Owing to the fact that the surface characteristics are connected with the hydrophilicity of materials, it is a vital factor for cell attachment that has to be measured. Contact angles of hydrogels in room temperature were determined using a data physics contact angle system OCAH 200 device, functioning in static mode. A drop of distilled water around 0.1–0.2 μl was introduced onto the surface of the dry hydrogel. Images of the water droplet were documented within 5 s and the contact angles were measured on both sides of the hydrogels and averaged. Digital pictures were analyzed by computer for angle determination.
Porosity
The porosity of the hydrogels was determined by liquid displacement method. The hydrogels were immersed in Specified volume (V1) of hexane in a graduated cylinder for 24h. Hexane was selected because it permeates inside scaffolds without causing a high shrinkage and swelling in comparison with other liquids unlike ethanol, etc.
The entire volume of hexane and the hexane saturated hydrogel was recorded as V2. The hexane-saturated hydrogels were then removed from the cylinder and the residual hexane volume was documented as V3 [21]. The porosity of the scaffold ε was obtained by:
ε= (V_1-V_3)/(V_2-V_3 ) ×100 (4)
Swelling/deswelling behavior of hydrogels
Swelling ratio of hydrogels with similar size and shapes was measured over a day at room temperature in distilled water (pH=7.2). First, Hydrogel samples were freeze-dried and then they were immersed in distilled water and over a specified period of time, they were taken out, and the weight was determined after sweeping excess water on the hydrogel’s surface, until a swelling equilibrium was reached. The swelling ratio was calculated using equation (5), in which SR, Ws, Wd are swelling ratio, swollen weight of sample and dry weight of the sample.
SR=(W_s 〖-W〗_d)/W_s ×100 (5)
The equilibrium water content (EWC) and the rate parameter were intended according to equation (6) and (7).
EWC=(W_e 〖-W〗_d)/W_e ×100 (6)
S_t =S_e (1- e^((-t)⁄τ)) (7)
Equation (7) is called Voigt equation, and We, St and Se are the weight of swollen hydrogel at equilibrium state, swelling ratio at time t and equilibrium swelling ratio.
In order to assess deswelling behavior, after hydrogels were reached swelling equilibrium, the samples were weighted immediately in specific time periods. The water retention (WR) was measured from Equation (6). Where Wt is the weight of hydrogel at a certain time of deswelling
WR=(W_t 〖-W〗_d)/(W_e 〖-W〗_d )×100
In vitro degradation of hydrogel
The degradation assay was performed with phosphate buffered saline (PBS) at pH 7.4 and 5.5 in 37 °C. Hydrogel bulks were immersed in PBS, the PBS was updated every week and at the interval time point, the hydrogels were taken out and rinsed with DI water to remove excess salinity, and they were then dried in an oven at 60 °C for 48 h and weighed to determine the degradation rate.
weight remaining ratio (WRR)=W_t/W_0 ×100
Where Wt and W0 were the weight of the degraded hydrogels at different time intervals and the weight of the hydrogels which were at swelling equilibrium state, respectively.
In vitro cell compatibility and cytotoxicity of hydrogels
Cell proliferation and biocompatibility were carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. The samples were sterilized by UV for 20 mins and then ethanol was poured into them, then they immersed in cell culture media for 1 day. After rinsing with PBS, the culture medium was applied to the samples and incubated overnight. the pheochromocytoma (PC12) cells in Dulbecco’s modified eagle medium(DMEM) with 10% fetal bovine serum (FBS) and 105/L penicillin were seeded on the samples, then incubated at 37 °C in 95% moisture and 5% CO2. The MTT test was performed in 1, 3 and 5 days to appraise the biocompatibility of hydrogels.
In order to evaluate the toxicity of hydrogels, the cytotoxicity test was conducted. Samples were incubated at the same condition for 24 hours to reach their secreted liquid. 96-well plate seeded PC12 cells were incubated with various concentrations of medium (1, 5, 10, 20, 50 mg/ml). Subsequently, the cell viability was graphed by using MTT test. For cell attachment assessment, the cells were fixed on the samples which displayed the best compatibility using the glutaraldehyde and alcohol gradients.
Drug release behavior
To evaluate the drug release behavior of the samples in stimulated conditions and passive, 50 mg of dexamethasone was dissolved in 50 ml methanol, and 0.5 g of dry samples was added to methanol solution and stirred for 24h at ambient temperature. Then, the drug loaded hydrogel was washed to remove the weak drug bonds on its surface, and the sample was submerged in PBS (pH 7.4, 37 °C) and at particular interval times the same amount of fresh PBS was replaced. Dexamethasone is a kind of corticosteroid family that demonstrate the immune-suppressant and anti-inflammatory properties. To evaluate the drug release profile, UV-Vis (λ=237 nm) was utilized. In stimulated release, electrical current was used to the hydrogel and after that, the release pattern was determined.
Result and discussion
Synthesis and structural characterization
The FT-IR spectra of CS, AP, CS-AP-Pluronic and the hydrogel CS-AP-Pluronic- β-glycerophosphate were demonstrated as curves in fig.2, respectively. The spectrum of CS displayed typical adsorption peaks at 3435.73 cm-1 due to the partially overlapped distinctive peak of amine (–NH2) and hydroxyl group (–OH) stretching vibrations [22]. Peaks of 1602.27 cm-1 and 1423.89 cm-1 in CS were corresponded to the amide bond (–CONH–) and amine bond (–NH2) stretching and bending vibrations. 1081.88 cm-1 -1157.82 cm-1 were assigned to pyranose (–C—O–) stretching vibrations from CS [23]. The spectrum of AP exhibited peaks at 1626.45 cm-1 and 1574.34 cm-1 due to (C==C) absorption of the benzene ring(–N—B—N) and the quinoid ring (–N==Q==N–) of EMAP. The 1312.06 cm-1 band was assigned to C—N stretching in the proximity of quinoid rings [24]. The peak at 1738.34 cm-1 in curve d was attributed to the C== O asymmetric stretching mode of ester in Pluronic [8]. In addition, the peak at 3385.75 cm-1 in curve e respectively indicated an interaction with the –NH2 group of chitosan [25]. The data confirmed the successful crosslinking and grafting reaction of the hydrogel.
Electroactivity and conductivity
Oligoanilines has three oxidation states: leucoemeraldine (LE), emeraldine (EM), and pernigraniline (PN). Fig. shows the oxidation states of aniline oligomer. Polyaniline and the oligoaniline conductivity occur in the oxidation state. The UV-Vis spectra of undopped sample shows two peaks at (…322…?……583……) nm attributed to the benzene ring π-π* transition and the benzenoid-to-quinoid excitonic transition (πB-πQ), respectively. By doping with camphor sulfonic acid, the peak at 583…………. weakened and the blue shift (solvantochromism effect) of benzene ring due to polaron formation resulted in a peak at (……?………328). In addition, two new peaks at …(……419 and …?…………..800) nm have emerged. The peak at (?800 nm?) is owing to the localization of the radical polaron that indorses the formation of emeraldine salt [26].
Cyclic Voltammetry (CV) was used to investigate the electroactivity of hydrogels. As shown in fig, they exhibit three pairs of reversible redox peaks and the mean peak potentials is E1/2= (Epa + Epc)/2. They that refer to the transition from leucoemeraldine to emeraldine state I, emeraldine state I to emeraldine state II and emeraldine state II to pernigraniline state, respectively. The results indicate that the hydrogels showed good electroactivity. Also, molecular resonance is depicted in Fig.
Oxidation-reduction state of aniline oligomer
The four-probe method was utilized for ionic conductivity measurement and the results at various temperatures are represented in fig. The ionic conductivity of all samples arises with the increase in temperature, due to accelerating ionic mobility at a higher temperature. Also, the increment in AP percentage enhances the absolute conductivity because AP provides a higher number of ions for the hydrogel. According to the fig the conductivity of hydrogels at 37 °C are around 3*10-3 which is adequate for cellular response (proliferation or/and differentiation). The conductivity of hydrogels is determined by two factors: the number of ions and their mobility. Fig shows a line where the slope of the plot multiplied by R represents the activation energy for conductivity and the exponential of the intercept displays the pre-exponential factor which represents the number of ions in the hydrogel. According to eq2, the measurements are given in table and it can be understood that lower activation energy and higher pre-exponential factor lead to more conductivity. Pre-exponential factor increase with the increment of the AP content. Also, activation energy decrease with an increment of AP percentage in hydrogel because less conductive hydrogel required more energy for ion transfer.
According to eq 3 Conductivity is a subordinate of mobility and hole concentration (number of ions). With an increment of AP content, the number of ions increase. On the contrary, with an increment of AP percentage, the swelling ratio decrease which restricts the mobility of ions. Thus, the optimum of conductivity is between higher mobility and good swelling behavior and conductivity increase with rising the AP content until an optimum point and after that, it would decreased. To illustrate, in samples with a specific AP concentration, the entire hole concentration is constant, and the increment in conductivity with temperature is because of the increase in mobility. On the other hand, in a particular temperature, the mobility is constant. Therefore, the conductivity depends on the AP percentage and hole concentration. To sum up, the same conductivity could obtain from a large number of holes with small mobility, or a small number of holes with high mobility [10, 27].
For particular conductive polymers such as AP, although the mobility of the hydrogels should be constant, the value of the mobility fluctuate in the specific content, which is due to the instrumental error. In samples, there is an obvious relation with the hole concentration and AP content in hydrogen, and the more AP, the more hole concentration and consequently a better conductivity.
sample slope intercept Ea (KJ/mol) Pre-exponential factor
CAP1 -1.649 5.293 13.709 198.939
CAP2 -1.566 5.134 13.020 169.695
CAP3 -1.826 6.059 15.181 427.947
Electrochemical impedance spectroscopy (EIS) is an outstanding method to study the conductivity using the neural model which consisted of two resistance and one capacitance in fig. f
C R1 R2
CAP1 1.565E-3 9.236E+1 1.208E+3
CAP2 -6.113E-5 1.308E+4 4.819E+4
CAP3 2.451E-3 3.451E+3 1.198E+2
Gelation time and rheological behavior of hydrogels
The gelation time was determined by the inversion method. The samples were incubated at 37 °C for complete gel formation (fig ) and the gelation time, were measured as the time where samples stopped flowing upon tube inversion for 60 s. Gelation time were around 4,5,7 minutes for CAP1, CAP2, and CAP3 samples. These informations are applicable in In Vivo tests. The gelation time increased with decreasing concentration of Pluronic in hydrogels. Owing to the fact that the content of –CHO is increased with increasing concentration of Pluronic and –CHO had more chance to react with –NH2 of chitosan to form Schiff base in the hydrogels.
Rheological properties are shown in fig . The hydrogels with different content of Pluronic were conducted by a time sweep test to depict the process of the gelation. The storage modulus (G’) surpassed the loss modulus (G”) in a few seconds and highest modulus value was reached due to the rapid reaction between –CHO and –NH2 groups to form the dynamic Shiff base.
In addition, as the percentage of AP in CAP1 to CAP3 increase, gelation was happened in a longer time. It is probably because of the nature of PA which is a radical scavenger and may interrupt the nucleophilic substitution reaction between chitosan and β-glycerophosphate [14].
The elastic modulus of hydrogels reduces by increasing the percentage of AP. Since the presence of AP decrease the accessibility of H bond (because of the special effect) and makes difficulty for chitosan’s movement. This accessibility to H bond explained for two reasons. The first is that the more AP in the reaction, the more amine groups on the CS would consume and this could reduce the electrostatic attraction between the amine group and carboxylate groups. Another reason is that the special effect of rigid AP makes difficult the accessibility of H bond.
By plotting modulus against temperature in fig the thermosensitivity of the hydrogels was noted. At low temperature, G’was lower than G”, indicating a viscous sol state. During the sol-gel transition, a significant increase was observed. Also with reducing the percentage of AP in samples, the gelation temperature increase. This should be ascribed to the hydrophobic interaction, hydrogen bonding and π—π stacking between Pluronic and AP [8].
Furthermore, the storage modulus (G’) and loss modulus (G”) versus angular frequency were detected to analyze the stability of the hydrogels. When the angular frequency changed from 0.1 rad·s-1 to 100 rad·s-1, the hydrogels’ storage modulus (G′) and loss modulus (G′′) demonstrated no noteworthy changes, representing the good stability of our hydrogels
Thermal properties characterization
The thermal stability of hydrogels was carried out by TGA, and the representative curves are shown in Fig. Weight loss of hydrogels took place in two steps: the first step of weight loss is attributed to the decomposition of chitosan which occurred at 170 °C, whereas the second thermal decomposition attributed to aniline pentamer and Pluronic at 330 °C. in comparison with TGA of neat chitosan, the hydrogels showed lower initial decomposition and its due to introduction of Pluronic moiety onto chitosan. In addition with introducing AP to CS, because of the intrinsic hydrophobic of AP content, the amine groups of CS was consumed by the amidation reaction with AP segment and as a result the weight loss decrease in comparison with near CS. Also, the rigid AP segments hinder the movement of the polymer chain [28-30].
Swelling/deswelling and hydrophilicity behavior of the hydrogels
The mechanical properties, diffusivity and drug release of hydrogels are related to their swelling and deswelling behavior. Figure represents the swelling ratio of hydrogels versus time at room temperature revealing that all the hydrogels absorbed water fast at the first minutes of incubation and reached to an equilibrium state to permeate into the gel. During the swelling process, water must prevail the osmotic pressure inside the gel in order to permeate to the gel. Moreover, the osmotic pressure is depended on the elasticity, and Hydrogels have low elasticity at the early minutes of swelling, as a result, the swelling rate is very fast at the beginning. The amount of absorbed water in the hydrogel increases leading to higher elasticity and consequently higher osmotic pressure in the hydrogel. Therefore, water needs to overcome higher pressure to permeate inside the gel. Thus, the swelling rate decreases gradually with time. In addition, owing to the inverse correlation between the pluronic content and the AP content in the hydrogels, the swelling ratio of hydrogels decreased with increasing the AP percentage in hydrogels. This may have caused by two reasons: the first one is that with growing AP percentage, less free space exists among polymeric chains which makes diffusion of water molecules inside the hydrogel more challenging. And the second one is because of the hydrophobic nature of AT that hinders the water absorption. Moreover, the rate parameter (τ) can be obtained by rearranging Voigt equation. With plotting ln (1-St-Se) versus time the slop which is -1/ τ could have calculated. In fig the rate parameter calculated. The swelling data was divided into two section because the rate at last minutes becomes slower subsequently. According to R-squared, the data in the first section obeys Voigt equation properly, however, the data in the second section does not. In the initial minutes, as the content of AP increments in the hydrogels, the slope of the trend lines decreases and the τ value increases.
The deswelling trend of hydrogels is demonstrated in fig disclosing the fact that the amount of absorbed water by hydrogels decreases fast at the first minutes during deswelling process, and then become slower until reaching the equilibrium state.
Contact angle
As far as the surface properties are concerned, the hydrophilicity of biomaterials has been exposed to be an important factor for cell attachment. In order to evaluate the hydrophilic character of hydrogels contact angles were determined. The contact angle of samples CAP1, CAP2 and CAP3 were 25.9°, 30.6°, and 33.3°. According to the literature, hydrophobic materials
Have the contact angle between 90 and 150 and they are hydrophilic when the contact angle is between 10° and 90°. Cell adhesion to materials occurs in optimal conditions when the material surfaces present a reasonable wettability, with contact angles ranging 40° to70° [31].
In vitro degradation of hydrogels
Time and ingredient of degradation of hydrogels are two vital factors in tissue engineering because of the chronic inflammation of undegradable materials caused by their long stay in the body. Therefore, the degradation rate of the colloidal hydrogels should synchronize tissue regeneration [32]. The in vitro degradation of the hydrogels was performed in a phosphate buffer solution (PBS) (pH 7.4) at 37 °C with a shaking speed of 100 rpm and the result are exposed in fig. owing to the intrinsic hydrophobic nature of AP, it prevents diffusion of PBS and water molecule by repulsing them; consequently, introducing AP to the network should result in a drop in reduction in hydrogel hydrophilicity and tendency towards water. Hence, as the percentage of the AP in hydrogel increase, water swelling and degradation will decrease.
Morphology and porosity determination
The hydrogels were freeze-dried at -80 °C for SEM assessment. The results demonstrate that hydrogels form a large porous network and pores were widely distributed in a matrix with a diameter distribution around 40 μm that provides a large space for cells to migrate into the hydrogels. Thus, the hydrogels would support exchange of nutrients and metabolite and cells could distribute within the network and as a result, the hydrogels are a prominent candidate for forming a uniform tissue. (fig).
Porosity is an important factor in tissue engineering, owing to the higher porosity provides much surface area that promotes cell proliferation. All the samples showed porosity ranging between 75% and 80% which is suitable for better cell proliferation.
In vitro drug release properties of the hydrogel
Conductive hydrogels are fruitful materials for drug delivery system with redox-responsive properties, and are safer than other methods due to the only voltage stimulation is used and other chemical redox materials are removed. AP and chitosan can captivate drugs and interact with them by the carboxyl and amine groups from ionic interaction with drugs. In addition, conductive hydrogels interact with drugs using hydrophobic, acid-base, π-π interaction, ion exchange, H-bond and polar function. By applying electrical voltage to AP generates oxidation/reduction process and can release the drug which is entrapped in hydrogel [33, 34]. Dexamethasone is a hydrophobic drug which in aqueous solutions encounters repulsion forces and have a tendency to be released soon with a large initial burst. Fig depicts the stimulated and passive release behavior by applying an electrical potential to the hydrogel and electrical stimulation is responsible for rupture in vesicle/chain or polarity differentiation, and as a result by this power release profile can be detected. Conductive hydrogels were sensitive to voltage, voltage applying cause increasing in release amount due to variation in AP’s oxidation/reduction state. This variation cause bond cleavage in hydrogels and results in improvement in drug release amount [35]. The first burst release of the drug loaded CAP2 hydrogel is attributed to the drug molecules located near the surface or those with loosely bond within the hydrogel. Because the AP tends to form vesicles and entrap dexamethasone and form the non-covalent interaction, hydrophobic nature of AP limits fluid permeation and therefore the initial burst release was reduced. In stands to reason that in the passive drug release there is a direct relationship between drug release pattern and swelling/deswelling pattern, where more swelling ratio runs to more drug release ratio. Thus, it is judicious that CAP1, which has lower swelling ratio has less cumulative release [36].
In electro-stimulated drug release, the behavior of dexamethasone by applying electrical potential to the hydrogel was observed. In each step, some of the drug released and the final cumulative release at the end of 75h is 90%, respectively. Moreover, the released amount in the first step is higher than subsequent steps because the concentration of drug is much higher at the first step. The slow release of the drugs without any electrical stimulation was because of the free diffusion driven by a different concentration and a 3-D barrier network of the hydrogel [11]. However, the electro-stimulated drug release of conductive hydrogels is attributed to the electric migration of the charged molecules and a change in the total net charge in hydrogel by oxidation/reduction. Positively charged drugs are released by oxidation and drugs with negative charge are released by reduction.
In order to further study of release mechanism, the well-known exponential Korsmeyer-Peppas equation (eq) which is used to define the drug release behavior from polymeric systems, was used to fit the release profile [37]. This equation is suitable for evaluating CAP hydrogels drug release with high enough correlation (r2 > 0.97). It has been reported that when the diffusion exponent, n, is 0.5, indicates that the release mechanism could be described by Fickian diffusion model, and the dexamethasone was released by Fickian diffusion [13]. The fitted data in fig designated that CAP2 hydrogel obeys Fickian diffusion systems and dexamethasone release by diffusion. In order to describe the drug release rate, the mean dissolution time (MDT) was used, which was determined by Mockel and Lippold (eq) [38].
M_t⁄M_f =k.t^n (eq)
MDT=(n⁄(n+1).k^((-1)⁄n) ) (eq)
In vitro biocompatibility
A hydrogel which is proper for tissue engineering should be biocompatible to support basic cellular activities such as proliferation and adhesion. Biocompatibility and cytotoxicity were evaluated by MTT assessment with neural-like PC12 cells after 1, 3, and 5 days. According to fig, cell viability increment with time and the highest cell viability of the cell was observed after 5 days of culturing. These properties could be attributed to the biocompatibility of a natural polymer chitosan and AP conductivity which enhance cell growth. On the other hand, AP has toxicity and it might have led to cell necrosis, therefore there is an optimum percent of AP usage. In terms of various percentage of AP in samples, CAP2 demonstrate the highest cell viability during the culture days which is ascribed by its highest swelling ratio and electrical ionic conductivity making it suitable for cell proliferation. SEM images of PC12 cells ceded on CAP2 after 5 days of culture is exhibited in fig. it reveals a good adhesion of cells to scaffold, neurite extension, and a deformation from spherical to spindle shape
Therefore, the SEM image and MTT assessment confirmed the biocompatibility of hydrogels.
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