SYNTHESIS AND CHARACTERIZATION OF POLY ( VINYL ALCOHOL )-HYALURONIC ACID BLENDED HYDROGEL MEMBRANES

Poly(vinyl alcohol)PVA is a hydrophilic polymer and water soluble . It is used in many biomedical and pharmaceutical applications, due to its advantages such as: non-toxic, non-carcinogenic, and biodegradable characteristics with the ease of processing. Physically cross-linked hydrogel membranes composed of different amounts of hyaluronic acid (HA) blend with (PVA) were prepared by freeze–thawing method. This freezing–thawing cycle was repeated for three consecutive cycles. Properties of (PVA–HA) hydrogel membrane such as gel fraction, swelling, mechanical properties(tensile strength, elongation to break),degradation and protein adsorption were investigated. With the increasing of HA content, the gel fraction, the maximum tensile strength and elongation at break(%) of (PVAHA) hydrogel membranes were decreased. Furthermore, with the increase of HA content, the swelling, the protein adsorption and the hydrolytic degradation of PVA-HA hydrogel membrane were increased.After soaking of hydrogel membrane for three days in phosphate buffer saline (PBS), the maximum weight loss of PVA–HA hydrogel membranes ranged between 18% and 70% according to HA content, this indicates that they are biodegradable.

"ultrapure" network without the using any toxic cross-linking agents with tunable mechanical properties (Yokoyama et al., 1985).Mechanical properties of the gel increase with the temperature, number of freezing-thawing cycles, polymer solution concentration and its molecular weight hence; different mechanical properties and erosion time up to six monthes were obtained (Hassan & Peppas, 2000).
PVA as a hydrophilic polymer is water soluble (Briscoe et al., 2000).It is used in many biomedical and pharmaceutical applications, due to its advantages such as: non-toxic, non-carcinogenic, bio-adhesive and biodegradable characteristics with the ease of processing (Wensheng et al., 2002).
Hydrogles being used as basic materials for manufacturing of wound dressings were invented in 1989 by Rosiak et al.However, some of the hydrogel dressings, due to their low strength and elasticity, did not satisfy the ideal dressing requirements, viz, they might stick to the wound surface or crushed under high stresses (Yoshii et al., 1999) .
The present work is designed to prepare blended hydrogel membranes (PVA-HA) by freezing-thawing method as membranes with good strength and elasticity to be compatible in biomedical application.This is the first time to prepare these membranes by this method for wound dressing.

2.2.Preparation of hydrogel membranes
PVA-HA hydrogel membranes were prepared by freezing-thawing (F-T) cycle according to the reported procedure of Peppas and Stauffer (1991).Briefly, aqueous solution containing 5% (w/v) PVA, 1% (w/v) HA and 0.3% (w/v) of ascorbic acid (AA) were carefully dissolved in distilled water.Different proportions of PVA and HA contents (0%, 10%, 20%, 30%, 40%, and 50%) solutions were mixed, sonicating, and vortexing for one hour.Proper amounts of this mixture were poured in Petri dishes, followed by freezing at -20 °C for 18 hours and thawing for 6 hours at 25 °C for one, two, and three continuous cycles.

2.3.1-Gel fraction
The pieces of PVA-HA hydrogel membrane samples 2 × 2(cm) were dried for 6 hrs at 50°C in an oven and weighted (Wo).They were soaked in distilled water into Petri dishes for 24 hrs up to a constant weight and taken out from Petri dishes in order to remove the soluble parts.The gels were dried again at 50°C in an oven and weighted again (We).The gel fraction percentage was calculated by the following equation (Yoshii et al., 1999 andAjji et al., 2005): Where (W 0 ) and (We) are the weights of hydrogel samples dried for 6 hrs at 50 °C before and after soaking, respectively.

2.3.2-Water uptake
In order to measure the water uptake of PVA-HA hydrogel membranes, the membrane samples were cut into 2x2 (cm) pieces and dried at 50 °C in an oven for 6 hrs, the weight of dried sample were determined (We).The dried samples were soaked indistilled water, maintained and incubated at 37 °C, then weighted (Ws) at specific interval times.The water uptake of PVA-HA hydrogel membranes was determined using the following Eq.( 2 Water uptake (%) = (Ws-We/ We) X 100 (2) Where (Ws) is the weight of swelled sample and (We) is the weight of dry sample.

2.3.3-Mechanical properties
The maximum tensile strength and the elongation to break of PVA-HA blend hydrogel membranes have been conducted using a tensile test machine (model: AG-I / 50 N -10 KN,Japan).PVA-HA membranes were cut into specific adog-bone shape (5 cm long, 1.5 cm wide at the ends and 1 cm in the middle).The analysis was performed at stretching rate 10 mm / min.The thickness of membrane samples were measured with an electronic digital micrometer before examination (Alencar et al., 2003).

2.3.4-Protein adsorption study
The amount of adsorbed bovine serum albumin (BSA) was detected by UV-visible spectrophotometer .In order to establish the relationship between the visible absorbance of BSA at 630 nm and the concentration of BSA, a calibration curve was drawn for standard solution of BSA ranging from 3.1-60 mg/ml.All standard solutions were prepared with distilled water.From the calibration curve a study was made restricting the curve to the linear part that followed linear part that followed Beer's law

A= acL
Where A the absorbance, c is the concentration, a is a proportionality constant and L is the path -length which is constant (Queiroz et al., 2001).
Pieces of PVA-HA hydrogel membranes cut into 1 x1 (cm) were immersed in 10 ml phosphate buffer saline (PH 7.4), and incubated at 37 °C for 24 hrs until reach to equilibrium swelling weight.
The swollen hydrogel pieces were transferred to buffer solution containing BSA (30 mg/ml) and shacked for 4hrs at 37 °C.After protein adsorption, the hydrogel pieces were gently removed.The protein adsorption for each sample were calculated by the difference between protein concentrations before and after immersing hydrogel pieces in protein/ phosphate buffer solution using albumin reagent kit ( absorbance range at 630 nm), this procedure has been adapted and modified from the procedure of Lin et al. (2006).

2.3.5-Hydrolytic degradation
The degradation activities were determined by the hydrolytic degradation method (Xiao and Zhou, 2003).This method is based on gravimetric determination study of the weight loss % of the gel.Procedure: dry 2 x 2 (cm) membrane samples were weighed and immersed in 10 ml phosphate-buffer saline (PBS) (0.1 M, pH 7.4) at 37°C.The samples were removed at timed intervals, blotted with soft paper to remove surface water, dried at ambient temperature and weighed.  1) Shows the gel fraction of the PVA hydrogels membrane with different HA content.The results clearly indicated that with the increase of HA content, the gel fraction of the hydrogel membrane decreased.During F-T cycles, the cross-linking strength of HA was weaker than that of PVA, even though HA formed a cross-linking bond with PVA in the gel.Generally, as the gel fraction decreased, the strength of the gel was weakened (Ajji et al., 2005 andKim et al., 2008).There fore, HA could be used to control the strength of hydrogel because it reduced the cross-linking reaction and, consequently, the gelation process.This is in agreement with

3.2-Water uptake
As shown in Fig .(1) the water uptake of PVA hydrogel membrane increases with increasing HA content in hydrogel membranes.This due to, the high hydrophilicity of HA in PVA film which increases the water uptake character of the studied hydrogel.This coincide with Hwang et al. (2010) and Abou El-Enin (2013).They found that the swelling of PVA-dextran and PVA-alginate blended hydrogel membranes increased with increasing dextran and alginate contentes respectevily.

3.3-mechanical properties
To investigate the influence of HA on the mechanical properties of the hydrogels, their tensile strength and elongation to break were evaluated (Fig. 2 ).The maximum tensile strength and elongation at break of hydrogel membranes decreases with increasing HA content.As HA was blended with PVA, the cross-linking density of the gel was decreased.These results are coincide with that of

3.4-Protein adsorption
The protein adsorption onto PVA-HA blend hydrogel membranes has been conducted via in vitro experiments.

3.5-Hydrolytic degradation
The hydrolytic degradation of PVA hydrogel membrane at different hyaluronic acid content in Phosphate buffer saline(PBS) is showed in fig.(4).The results indicated that the hydrolytic degradation of PVA hydrogel membrane increases with increasing HA content in hydrogel membranes.This phenomenon can be ascribed to the degradation of PVA-HA hydrogel membranes that are predominantly the cleavage of cross-linking segments of PVA and is consistent with the fact that the degradation of PVA is quite slow (Takasu et al., 2002), whereas the degradation of PVA-HA is quite high.In addition, as PVA and HA are nontoxic, the PVA-HA hydrogel and its degradation by-product might be expected to be nontoxic too.

CONCLUSION
The PVA-HA blended hydrogels have been prepared by freezing-thawing technique as a physical cross-linking method.Each of swelling, hydrolytic degradation and protein adsorpition of PVA-HA hydrogel membranes increased with increasing HA content in hydrogels but both gel fraction and mechanical properties (tensile strength and elongation at break) decreased with increasing HA content in hydrogels .Then the addition of HA to PVA hydrogels is expected to improve the benefit of hydrogel membrane for biomedical applications.

Fig. (
Fig. (1) Shows the gel fraction of the PVA hydrogels membrane with different HA content.The results clearly indicated that with the increase of HA content, the gel fraction of the hydrogel membrane decreased.During F-T cycles, the cross-linking strength of HA was weaker than that of PVA, even though HA formed a cross-linking bond with PVA in the gel.Generally, as the gel fraction decreased, the strength of the gel was weakened (Ajji et al., 2005 and Kim et al., 2008).There fore, HA could be used to control the strength of hydrogel because it reduced the cross-linking reaction and, consequently, the gelation process.This is in agreement with Maolin et al. (2002) on their study at PVA-starch blended hydrogels, Long et al. (2003) on PVA-carboxymethylated chitosan blended hydrogels .
Fig. (1) Shows the gel fraction of the PVA hydrogels membrane with different HA content.The results clearly indicated that with the increase of HA content, the gel fraction of the hydrogel membrane decreased.During F-T cycles, the cross-linking strength of HA was weaker than that of PVA, even though HA formed a cross-linking bond with PVA in the gel.Generally, as the gel fraction decreased, the strength of the gel was weakened (Ajji et al., 2005 and Kim et al., 2008).There fore, HA could be used to control the strength of hydrogel because it reduced the cross-linking reaction and, consequently, the gelation process.This is in agreement with Maolin et al. (2002) on their study at PVA-starch blended hydrogels, Long et al. (2003) on PVA-carboxymethylated chitosan blended hydrogels .

Fig. 1 -
Fig. 1-Effect of HA content in PVA hydrogel membranes on gel fraction and water uptake(%).
Rosiak et al. (2001).They referred that the maximum tensile strength of PVA hydrogel decreased with increasing blend materials due to decreased cross-linking density.Hwang et al. (2010) demonstrated that the maximum tensile strength of PVA hydrogel has sharply decreased with increasing dextran portions in the hydrogel and Abou El-Enin (2013) who reported that the maximum tensile strength and elongation at break of PVA-HES hydrogel membranes, sharply decreased with increasing hydroxyethylstarch (HES) contents.

Fig. 2 -
Fig. 2-Effect of hyaluronic acid contentes on mechanical properties of PVA hydrogel m membrane.
Fig.(3) shows the Protein adsorption of PVA hydrogel membrane at different Hyaluronic acid content in PBS.The results appeared that the Protein adsorption of PVA hydrogel membrane increases with increasing HA content in hydrogel membranes, the highest values of protein adsorption on PVA-HA surface have been cleared with the highest values of hydrophilic surface interaction due to the addition of HA contents.The blood compatibility of the hydrogel was evaluated by the amount of plasma protein adsorbed onto the hydrogel surface.When foreign material was placed in contact with blood, the adsorption of protein onto the surface occurred, leading to platelet adhesion and activation (Colm et al.,1982 and Burkatovskaya et al., 2006).Because the albumin adsorption on the synthetic surfaces could inhibit platelet activation, it did not promote clot formation.Generally, as the albumin/fibrinogen adsorption ratio was higher, the number of adhering platelets was lower (DionI et al., 1993).Thus, HA gave less adhesion of platelets to artificial surfaces.This agrees the reported results by Kim et al. (2008) and Hwang et al. (2010),they revealed that the adsorption of protein increased with increasing blended alginate and dextran respectively in PVA hydrogels.

Fig. 4 -
Fig. 4-Effect of HA contents on weight loss of the PVA-HA hydrogel membranes after different degrading times in phosphate buffer saline (PBS) (0.1 M, pH 7.4, at 37 °C).