CHAPTER 1 INTRODUCTION Tissue Engineering approaches generally aim towards the development of biological substitutes which in turn can be used in combination with cells and growth factors to facilitate the regeneration of tissue of interest by mimicking the native extra cellular matrix

CHAPTER 1
INTRODUCTION
Tissue Engineering approaches generally aim towards the development of biological substitutes which in turn can be used in combination with cells and growth factors to facilitate the regeneration of tissue of interest by mimicking the native extra cellular matrix (ECM). Recently Hydrogels have been the leading candidates in the field of tissue engineering due to their ability to mimic the extracellular matrix (ECM) that would essentially control the cell fate process (Ferris et al. 2013).

Hydrogels are 3D networks of hydrophilic polymer, crosslinked either through physical intramolecular or intermolecular attractions or through covalent bonds (Coutinho et al. 2010). Since water is the major component in the human body, a hydrogel which has the ability to swell without dissolving by in taking huge amounts of water has a wide range of application. Compared to other types of biomaterials, hydrogels offer many advantages like biocompatibility, tunable biodegradability, porous structure etc. However, due to its low mechanical strength and fragile nature, the feasibility of hydrogel is still limited as a tissue engineered construct. In order to enhance the properties of hydrogels, the polymer chains are modified with stimuli responsive functional groups. Hence, hydrogel properties can be varied by stimuli including pH, temperature, light etc (Fenn et al. 2016).

1.1. Thermoresponsive property
Thermoresponsiveness, a term used to imply a drastic and critical change in the physical properties over a small temperature range. Thermoresponsive polymers exhibit a miscibility gap, a region where the mixture exists as two or more phases (Kushwaha et al. 2012). Upper critical and lower critical solution temperature is determined by the presence of miscibility gap at high or low temperatures respectively. Lower critical solution temperature (LCST) formulation is liquid at room temperature (20-25°C), undergoes gelation in contact with body fluid (35-37°C). Whereas Upper critical solution temperature (UCST) formulation is liquid at higher temperatures (50-60°C), undergoes gelation in contact with body fluid (35-37°C). However these thermoresponsive gels can crosslinked either through physical or covalent interactions.

1.2. Crosslinking
In case of covalently linked gels, the formed three dimensional construct merely swells in solvents. They exhibit a discontinuous change of degree of swelling with temperature (chai et al. 2017). These gels are mostly preferred for drug delivery applications. In swollen state, the drug gets released by means of simple diffusion. Whereas physical gels unlike covalently linked gels can dissolve under appropriate conditions. The gels are held together by weak secondary forces like hydrogen bonding, vanderwaal’s forces etc (Grasdalen et al.1987). These gels are mainly exploited as injectable gels for tissue engineering applications.

1.3. Photocrosslinking
In order to enhance the mechanical stability of hydrogels, photo crosslinking is preferred. Among the crosslinking strategies employed (chemical crosslinking (UV, Visible) and physical crosslinking (ionic)), visible light mediated photocrosslinking offers the advantages of reduced cytotoxicity and rapid crosslinking (free radical polymerization).The efficiency of the crosslinking depends on the monomer, photo-initiator concentration, beam wavelength, and the exposure time.

A dye molecule (initiator) usually absorbs light and undergoes transition from a low level to higher excited states. In the presence of a co-initiator, some of the dye molecules abstract an electron from the co-initiator molecules (example: amine molecules in amine co-initiator) to form radical anion/cation pair. These molecules are held by electrostatic attractive forces and subsequently results in the formation of a pair of radicals upon ion transfer. These free radicals then attach to the monomers through the respective functional groups thus inducing rapid polymerization between the monomers (Shin et al. 2012).

1.4. Gellan gum
Gellan gum is a linear anionic hetero-polysaccharide and has a high molecular weight of 1000 kDa. It consists of glucose- glucuronic acid – glucose- rhamnose as repeating unit (Paceli et al. 2015). This tetra-saccharide polymer exists as random coils when dissolved in water and exhibits a sol-gel transition at ~ 40 °C. Even though Gellan gum is biocompatible and exhibits the potential to form stable gels, it is least explored as a tissue engineered construct due to its poor mechanical properties. Hence, the aim this project aims to enhance the stability of the Gellan gum based hydrogels in addition to incorporate stimuli responsive sol-gel transition.

1.5. Acrylation of Gellan gum
Modification of gellan gum (Methacrylation) was done using Methacrylic anhydride. This functionalization occurs through the free -OH groups present in the monomer (Correia et al. 2011). Methacrylation is done to increase the reactivity of the polymer. Also, it is these functional groups that facilitate the addition of free radicals to the monomers hence paving way for free radical polymerization.

Hence, in this project, gellan gum is modified using Methacrylic anhydride and was photo crosslinked at visible light (520-530nm) and the hydrogel properties were be characterized for its mechanical strength (Uniaxial Tensile Strength), rheological properties (Rheometer) and swellability.
CHAPTER 2
REVIEW OF LITERATURE
One of the main purposes of methacrylation is to facilitate free radical polymerization. Methacrylation results in functionalization of Gellan gum. The vinyl group of the methacrylic anhydride facilitates photo crosslinking on irradiation with visible light. The radical co-initiator molecules are generated through pair separation reaction with the photo excited dye. This then interacts with the double bond of the vinyl group of methacrylic anhydride, results in the abstraction of a proton, thus generating a growing macro free radical. Two such radicals interact to form a stable crosslinked network. Hamcerencu et al have reported the use of acryloyl chloride for acrylation of gellan gum. Dimethyl form amide (DMF) and TEA (Triethyl amine) were used in addition to acryloyl chloride in order to facilitate this process. But one major drawback faced while using this reagent is that HCl is produced as the byproduct, hence repeated rinsing with IPA is required to ensure that the acidic pH does not alter the innate properties of the polymer. In addition to this, the degree of the substitution of the developed construct was also found to less, hence result in insufficient crosslinking. On the other hand, Correia et al have reported the use of Glycidyl Methacrylate (GMA) for acrylation of gellan gum. The reaction is facilitated by a ring opening in the GMA monomer at pH 8.5. Although the process of acrylating gellan gum is feasible, the byproduct produced is reported to be carcinogenic. Dialysis is performed in order to remove the unreacted GMA and byproducts. Even though cytoxicity evaluated through MTS assay proved that the developed construct is less toxic, its efficacy in in vivo studies is still yet to be explored. Hence Methacrylic anhydride was chosen since the byproduct produced are insoluble products which are removed during dialysis. Coutinho et al have reported development of ionic and chemical crosslinked gellan gum hydrogel disc. The stability of ionic crosslinking is less due to weak vanderwaal’s and hydrogen intermolecular forces. Whereas in chemical crosslinking Irgacure 2959 was used as the initiator and exposed to UV light for 10 minutes. Even though crosslinking was achieved at faster rate, prolonged exposure to UV light can damage the DNA. Hence, visible light mediated photo crosslinking (520-530 nm) was chosen to reduce the cytotoxic effects as evident with the usage of UV light (360-400 nm). Here, Eosin y (photo initiator) was chosen since it gets excited only when exposed green light (520-530nm).The excited Eosin y molecule in the presence of a suitable co-initiator (Tri ethanol amine (TEOA)) and catalyst (N vinyl pyrrolidone) results in the formation of chemically crosslinked stable gel.

CHAPTER 3
SCOPE OF THE PROJECT
Even though Gellan gum is thermoreversible, possess excellent biocompatibility and has the capability to form stable gels, it has very brittle in nature. This project aims to improve the Mechanical Stability of Gellan gum, enhance it properties by carrying out the following specific aims:
Specific Aim 1: Modification of gellan gum using methacrylic anhydride.

Specific Aim 2: Fabrication of acrylated gellan gum hydrogel.

center47180500Specific Aim 3: Characterization of gellan gum hydrogel.

center3921760Fig 1. Work plan
00Fig 1. Work plan

CHAPTER 4
EXPERIMENTAL PROCEDURES
4.1. EXPERIMENTAL MATERIALS
Low acyl gellan gum (GG, SIGMA), Methacrylic anhydride (MA, SIGMA), Sodium hydroxide (NaOH, MERCK), Sodium Chloride (NaCl, MERCK), Potassium Chloride (KCl, MERCK), Disodium hydrogen Phosphate (Na2HPO4, MERCK), Potassium dihydrogen Phosphate (KH2PO4, MERCK). Eosin y (SIGMA), Tri ethanol Amine (MERCK), N-vinyl pyrrolidone (SIGMA), Dialysis Membrane (Mw cut off: 12-14 kDa, HI MEDIA)
4.2. EXPERIMENTAL METHODS
4.2.1. Acrylation of Gellan gum
Methacrylated gellan gum (GG-MA) was synthesized by reacting low acyl gellan gum (GG, Gelrite®, Sigma, Mw=1000kDa) with methacrylic anhydride (MA, Sigma) based on a previously described reaction mechanism (Paceli et al. 2015) and according to the schematic representation in Fig 1. Briefly, GG (1% (w/v)) was added to distilled water and heated at 90 °C for 20-30 minutes to obtain a homogeneously dispersed solution. The temperature was cooled down to ~50°C and then appropriate amount of methacrylic anhydride (3ml) was added to it. The reaction was continued for 4 hours .Periodically pH (8.0) was maintained by addition of 5.0 N NaOH solution. The modified gellan gum was purified by dialysis (HI media, membrane with a molecular weight cut off of 12 -14 kDa) for 3 days against distilled water at 4°C to remove residual MA. The purified GG-MA was obtained after lyophilization and stored in a dry place until further use. The ratio of gellan gum: methacrylic anhydride was also varied to observe the effect of methacrylation and polymer concentration on the properties of the fabricated material.

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Fig 2. Modification of Gellan gum using Methacrylic anhydride
4.2.2. Thermoresponsiveness
GG and GG-MA, (1% (w/v)) solution was prepared by dissolving in Phosphate buffered saline (PBS, pH 7.4) under constant agitation. In order to study the thermoresponsive property of GG and GG-MA, the solutions were varied over a range of temperature (45 to 25°C) and was measured using a thermometer. The solutions were maintained at different temperatures for 15 minutes to identify the sol gel transition temperature. The sol gel transition was confirmed by tube tilting method.

4.2.3. Fabrication of Hydrogel discs
GG discs were fabricated by dissolving gellan gum powder (1% (w/v)) Phosphate buffered saline (PBS, pH 7.4) under constant agitation. The solution was heated to 90 °C for 20-30 minutes to obtain a homogenous solution. The solution was then frozen at two different temperatures, sol-gel transition temperature and at 45oC by immersing in liquid nitrogen. Same protocol was followed for preparing GG-MA discs by keeping the heating to 60-70°C.

Photo crosslinked GG-MA discs were prepared by dissolving (1% (w/v)) lyophilized GG-MA sample in Phosphate buffered saline (PBS, pH 7.4) under constant agitation. The solution was heated to ~ 60-70°C for 20-30 minutes to obtain a homogenous solution. To this, 0.1 mM Eosin y (Photo initiator), 1% Tri ethanol amine (co-initiator), and 1% N-vinyl pyrrolidone (catalyst) were added and then exposed to green light (LED array, 4×3) for 10 minutes to ensure complete crosslinking has occurred.

4.3. CHARACTERIZATION OF ACRYLATED GELLAN GUM
4.3.1. Fourier-Transform Infrared Spectroscopy (FTIR)
The chemical modification that occurs on the addition of methacrylic anhydride was compared to the native/unmodified gellan gum. Prior to analysis, Potassium bromide (KBr) was used to prepare pellets followed by uniaxial pressing. The samples were recorded at room temperature on IR-spectrometer (Perkin Elmer). The spectra’s were obtained at a resolution of 8 cm-1 in the range of 4000 – 400 cm-1 for an average of 48 scans.

4.3.2. 1H-NMR Spectroscopy
The efficiency of methacrylation was also studied using Proton Nuclear magnetic resonance Spectroscopy (1H-NMR). The 1H?NMR spectra of GG and GG–MA samples were recorded with Brucker AC- 300. GG (unmodified), GG-MA, and GG-MA photocrosslinked solutions were prepared by dissolving 10mg of the powder, lyophilized samples respectively in 1ml of D2O. The 1H-NMR was recorded at 70°C. 1H-NMR for Methacrylic anhydride was carried out by dissolving in chloroform.

The degree of Derivatization (Paceli et al. 2015) of the polymers was calculated from the H-NMR spectra using the following equation:
center188595
Where nOH represents the number of hydroxyl groups present in the repeating monomer units and ICH3 (methacrylate), and I CH3 (rhamnose) represent the Integration of methyl peaks of the methacrylic groups rhamnose respectively.

4.3.3. Physico-chemical characterization of Acrylated Gellan gum
After freeze?drying the gellan gum?based hydrogel discs, morphology of each scaffold was examined using a scanning electron microscope (Vega3 TESCAN) at an accelerating voltage of 5 kV. The samples were sputter coated with gold prior to imaging with SEM.

4.3.4. Swellability
Hydrogel discs were prepared as per the above mentioned procedure (section 4.2.3) and lyophilized afterwards. The freeze dried samples were cut (n=4) and the dry weight (Wdi) was measured. The discs were transferred to a 24 well plate and soaked in 500µl of PBS (pH 7.4). At pre-determined time intervals the discs were removed and the wet weight (Ww) was calculated. The water uptake (WU) of the discs were calculated (Correia et al. 2011) by the following equation:
WU (%) = 100 x (Ww -Wdi) / Wdi.

CHAPTER 5
RESULTS AND DISCUSSION
5.1. MODIFICATION OF GELLAN GUM
Even though gellan gum is biocompatible, temperature and pH dependent it has very poor mechanical stability. Hence, in order to improve the stability of gellan gum, it was modified with
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Fig 3: Modification protocol
methacrylic anhydride (Fig. 3). In this study, gellan gum, acrylated gellan gum, and Photo crosslinked gellan gum discs were developed. Chemical modification of GG by methacrylation of the free carboxylic groups enables the enhancement of mechanical properties by allowing a stronger crosslinking of hydrogel network. The methacrylation efficiency was studied by Fourier?transform infrared (FTIR), 1H?NMR spectroscopic methods.

5.1.1. FTIR Spectroscopy
The methacrylation of GG was also confirmed by the appearance of shoulder peak around 1640 cm-1 corresponds to the C=C bond of the MA, known to be present in methacrylate groups but not
.

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Fig. 4: FTIR of gellan gum, GG-MA (2:3), (1:3), (1:2)

in GG chains (Fig. 4). Characteristic C=O peak of the ester bond appeared around 1770-1680
cm-1 and increased in intensity with increasing degree of methacrylation.
Table 5.1 FTIR spectroscopy peak values of methacrylated gellan gum
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5.1.2. 1H-NMR Spectroscopy
The spectra of GG obtained at 70 °C (Fig. 5A) showed the presence of four characteristic peaks that correspond to -CH of rhamnose (? 5.31 ppm), -CH of glucuronic acid (? 5.01 ppm), -CH of glucose (? 4.88 ppm) and -CH3 of rhamnose (? 1.21ppm).The spectra of methacrylic anhydride (Fig.5B) showed distinctive peaks in the double bond region (? 5.50-7.00 ppm) and a sharp peak that corresponds to the -CH3 of the methacrylate groups (? 2.01 ppm) 1H-NMR spectroscopy confirmed the methacrylation of GG by the appearance of distinctive peaks in the double bond region (? 5.79 and 6.21 ppm) and a sharp peak that corresponds to the -CH3 of the methacrylate groups (? 1.81 ppm) on the modified GG spectra. Fig. 5C, Fig. 5D, and Fig. 5E show the 1H-NMR spectra for different ratios of gellan gum and methacrylic anhydride. The Degree of Methacrylation was calculated as ratio of average intensity of the methyl proton peaks of the methacrylate groups to that of the average intensity of the methyl groups of the rhamnose. The degree of substitution (%) for the different ratios were found to be:
Table 5.2 Degree of substitution for different ratios of GG and MA
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Fig. 5. Unmodified Gellan gum (A), Methacrylic anhydride (B)
Fig. 5. (contd.)
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Fig. 5. Methacrylated gellan gum; 1:2 (C), 1:3 (D), 2:3 (E)
5.2. THERMORESPONSIVE PROPERTY
Gellan gum is a UCST polymer (Upper critical solution temperature), which means that it has the ability to remain in solution state at higher temperatures but gels at lower temperature. It was found that there was no significant variation (36°C) in the sol-gel transition temperature when the concentration of Methacrylic anhydride alone was altered. But gelation occurred at a higher temperature (37°C) when the concentration of gellan gum alone was increased. This proves that it is the innate ability of gellan gum that influences the sol-gel transition temperature. Since gellan gum exhibits the property of thermos responsiveness, it can be exploited for injectable applications.
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Fig. 6. Thermoresponsive sol – gel transition temperature of GG and GG-MA
5.2.1. FTIR Spectroscopy
Methacrylation was confirmed by the appearance of a shoulder peak around 1640 cm-1, corresponds to the C=C bond of the MA and a peak at 1725 cm-1corresponds to the carbonyl stretch of a methacrylic ester. A slight sharpening of peaks were observed in the gels frozen at their sol gel transition temperature than at 45oC .This might be due to the rapid physical crosslinking initiated by decrease in temperature ,thus results in formation of stable gels
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Fig.7. FTIR of Thermoresponsive gels
Table 5.3 FTIR peak assignment for thermoresponsive gel
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5.3. PHOTOCROSSLINKED ACRYLATED GELLAN GUM DISC
center145351500Photocrosslinking of GG-MA (2:3) was carried out as per the procedure mentioned in section 4.2.3. Eosin y on exposure to visible light (green light, 520-530nm) induces free radical ion formation which in turn is responsible for the chemical covalent crosslinking. The stability of the gel was checked by reheating it to 50 0C.

Fig 8. Photocrosslinking of methacrylated gellan gum
5.3.1. Crosslinking Chemistry
Eosin y gets excited when exposed to visible light (520-530nm). This excited dye interacts with a suitable co-initiator (Tri ethanol amine), undergoes electron transfer and results in the formation of a radical ion pair. This entity then undergoes subsequent proton transfer which facilitates the pair separation, results in generation of free radicals. The dye/amine co-initiator radical then interacts with the methacrylate group in the gellan gum monomer. Interaction of two such monomers results in rapid crosslinking, results in stable gels.

5.3.2. FTIR Spectroscopy
center30607000
Fig.9. FTIR of GG-MA and photocrosslinked GG-MA
Eosin y has characteristic peaks at 1422 and 1465 cm-1 which corresponds to the carbonyl and phenyl group respectively (Alwin et al. 2015). Here, the presence of a characteristic peak at 1437 cm-1 in GG-MA photocrosslinked sample validates the interaction of eosin y and acrylated gellan gum.

5.3.3. 1H-NMR of Photocrosslinked gel.

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Fig 10. 1H NMR of GG-MA (2:3), Photocrosslinked GG-MA (2:3)
Eosin y has characteristic peaks at 7.49 and 7.30 cm-1 (Okuom et al. 2013). Here, there is a significant downfield shift observed in the spectra, corresponds to the reduction in electron density caused due to proton transfer. Also, a significant reduction in the intensity of the methacrylic peaks validate that photocrosslinking has taken place.

5.4. PHYSICO-CHEMICAL CHARACTERIZATION
SEM (Scanning electron Microscope) was used to analyze the morphology of Gellan gum and Methacrylated Gellan gum (GG-MA) at their thermoresponsive sol-gel transition temperature. (Fig 11.) Shows typical spongy three?dimensional (3D) morphology, with open macro pores and anisotropic porosity. No differences were found in the morphology of the interior of all gellan gum?based hydrogels analyzed. Whereas when the gels (Fig 12) were frozen at a higher temperature (450C), irregular and distorted pore formation was observed. The sudden freezing of the gels at a higher temperature in liquid nitrogen results in lack of time for the gel to settle, hence results in a distorted morphology. (Fig 13) Shows the three?dimensional (3D) morphology of photocrosslinked gels with reduced pore size.

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Fig 11. Cross section images of frozen samples at sol gel temperature; Gellan gum (A); (B), (C), (D) represent GG-MA (1:2), (1:3), (2:3) respectively taken at 2000 X.

Fig 12.Cross section images of frozen samples at 450C; Gellan gum (A); (B), (C), (D) represent GG-MA (1:2), (1:3), (2:3) respectively taken at 2000 X.

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Fig 13. Cross section images of Photocrosslinked GG-MA (2:3); 500X (A), 750X (B), 2000 X (C).

5.5. SWELLABILITY
(Fig 14) shows the weight uptake capability of the developed hydrogel discs soaked at in PBS at 37°C. The results obtained showed a significant change in the weight of the hydrogel discs tested (GG, GG-MA (2:3), GG-MA ((2:3), Photocrosslinked) over a period of 48 hrs. Reduced water uptake capability of the photocrosslinked disc was observed when compared to GG and GG-MA. (Fig.14), this property attributes to the higher degree of crosslinking that has resulted in a tighter matrix, prevents the entry of the solvent into the pores.

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Fig 14. Water uptake capability of prepared hydrogel.

CHAPTER 6
CONCLUSION
Chemical modification of gellan gum was achieved by reacting it with different concentrations of Methacrylic anhydride. Furthermore, it was identified that there was a slight increase in the Degree of substitution was the concentration of the polymer was increased. Thermoresponsive sol gel transition temperature for the various concentrations were identified and the ratio GG-MA (2:3) was chosen for crosslinking since it forms gel at physiological temperature unlike the rest. Photocrosslinking was carried out for the optimized concentration on exposure to visible light (520-530 nm).This induces free radical polymerization, results in the formation of stable gels. After 48 hrs of study, it was concluded that the water uptake ability of the photocrosslinked gel is less than GG and GG-MA. This is due to the reduced pore size induced due to photocrosslinking. Therefore, the proposed photocrosslinked gellan gum hydrogels can be exploited for development of injectable hydrogels.

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Silva?Correia, J., Oliveira, J. M., Caridade, S. G., Oliveira, J. T., Sousa, R. A., Mano, J. F., ; Reis, R. L. (2011). Gellan gum?based hydrogels for intervertebral disc tissue?engineering applications. Journal of tissue engineering and regenerative medicine, 5(6), e97-e107.

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Hamcerencu, M., Desbrieres, J., Khoukh, A., Popa, M., ; Riess, G. (2008). Synthesis and characterization of new unsaturated esters of Gellan Gum. Carbohydrate Polymers, 71(1), 92-100.