Physically and chemically-crosslinked L-arginine-loaded polyvinyl alcohol- hyaluronic acid- cellulose nanocrystals hydrogel membranes for wound healing: influence of crosslinking methods on biological performance of membranes in-Vitro

Two types of polyvinyl alcohol-blended hyaluronic acid and cellulose nanocrystals (PVA/HA/CNCs) hydrogels membranes were developed as delivery agents for L-arginine using chemical/and/physical crosslinking methods for exploring biological performance of the obtained membranes. Chemically and physically/crosslinked membranes were fabricated by solution-casting and freeze-thawing techniques, respectively. The characterization of the crosslinked PVA/HA/CNCs/L-arginine membranes were conducted by SEM, FTIR, TGA, swelling ratio, and in—vitro L-arginine release. Furthermore, applying two types of crosslinked membranes in vitro were investigated in terms of cytotoxicity, hemocompatibility, and cell adhesion affinity of HFB-4 cell line. Chemical and physical-crosslinked membranes were verified through the esterification and hydrogen bonds interactions, respectively as shown the FTIR spectra. The chemically-crosslinked membranes showed more thermal and mechanical stability than the physically-crosslinked ones. Besides, the chemically-crosslinked hydrogels exhibited biphasic release behavior of L-arginine reached 50% in the first hour and 92% after 48 h. Notably, chemically-crosslinked membranes a somewhat affect the cell viability %, while both crosslinked membranes exhibited excellent cell adhesion affinity and low hemolysis. However, the physically- crosslinked members showed high cell viability ~ 172%, compared to the chemically-crosslinked membranes at 98.5%; and low hemolysis ca. 0.84 versus 1.34 for chemically-crosslinked membranes. Accordingly, both types of crosslinked membranes have a potential for applying in biomedical applications, particularly for designing promising wound dressing biomaterials.


Introduction
Several research approaches have been applied for innovating and designing more than one ideal bio-scaffolds for critical applications e.g.tissue engineering and organs regeneration.Whereas, bio-scaffolds have provided normal healing and regeneration process mimicking of tissue microenvironment [1].Among numerous tissue engineering scaffolds, hydrogel membranes that consist of three-dimensional (3D) network structures from chemically or physically-crosslinked natural or synthetic polymers.Hydrogels are considered an important class of tissue engineering materials due to their unique characteristics mimicking the native extracellular matrix (ECM) microenvironment, excellent swelling ability and tunable degradation rates [2].Furthermore, hydrogels provide a convenient alternative to traditional gels to immobilize cells, antibacterial agents, variable growth factors, amino acids and other bio-macromolecules.Hence, hydrogels are applied in skin tissue engineering due to their ability to establish optimal microenvironment for skin repair.Besides, their ability to sustain release of bioactive materials allowing integration between cells in a proper manner [3].
L-arginine is bioactive molecule due to its antioxidant and antimicrobial characteristics, thus, it plays the prime role in wound healing processes [4,5].In addition, L-arginine acts as the main substrate for production of nitric oxide molecules (NO), that induces many cellular processes such as; angiogenesis and epithelialization [1].Moreover, L-arginine participates in collagen synthesis through its transformation by the arginase pathway into proline that is the main precursor in collagen synthesis processes [6].Furthermore, L-arginine has stimulatory effect on the release of growth hormones and growth factors that are essential for healing process as insulinlike growth factor-one (IGF-1) [7].Also, L-arginine has a recognized antibacterial activity due to its positive charge that can electrostatic interact with the negative charge of bacterial cell-wall, leading to membrane damage and disruption through a lytic mechanism [8].Despite of L-arginine contributes in several regeneration process such as the skin wound healing process, the body can't provide a sufficient amount of L-arginine during the new tissue formation and regeneration [7].Therefore, the external administration of L-arginine has been required to compensate its deficiency leading to improving and accelerating the healing process.However, the exogenous administration may have a negative effect on the body by disturbing the body homeostasis, resulting in toxic side effects.Also, L-arginine administrated by local transdermal route is more exposed to rapid metabolism [4].Therefore, there is need to develop an L-arginine delivery system to provide its sustained release, increasing its bioavailability and avoiding its rapid metabolism and toxicity.
Nowadays, hydrogels serve as a promising drug deliver scaffold owing to their crosslinked network structure, which allows sustained and controlled release of a drug to tissues [9].Recently, hydrogels have been introduced as effective wound dressings offering valuable advantages like adjusting wound moisture, absorbing a large amount of wound exudates, and permitting sufficient oxygen supply.Moreover, hydrogels based-wound dressings can provide structural integrity supporting tissue by their ECM mimicking properties [4,7].
To date, several types of natural and synthetic polymers have been incorporated in fabrication of hydrogel basedwound dressings.Among the synthetic polymers, polyvinyl alcohol (PVA) has been involved in various biomedical applications, especially tissue engineering [10].PVA is a linear hydrophilic homopolymer, consisting of polyvinyl acetate.In fact, PVA-based hydrogels would act as suitable candidates for tissue engineering applications such as; transdermal patches, wound dressings and cosmetic owing to its biocompatibility, non-toxicity, biodegradability and hydrophilicity [11].Interestingly, PVA hydrogels can be obtained by various methods such as; chemical crosslinking and physical crosslinking by solution-casting and freeze-thawing approaches, respectively.In biomedical applications, the freeze-thawing technique is preferred over chemical crosslinking owing to its easy synthetic method with fewer number of intermediate chemical reagents and solvents that might have acute toxic effect on cells [12].
Another promising biopolymer that has been utilized in the development of polymer-based scaffolds for regeneration and skin tissue engineering, is hyaluronic acid (HA), it is the main component of ECM.HA is involved in wound management through its interaction with cell surface receptors, which in turns stimulates cell migration and re-epithelization, enhances angiogenesis, modulates the inflammatory response, and induces collagen synthesis at the wound site [13,14].In the last decade, many PVA/ HA composite hydrogels have been synthesized for drug delivery application.Recently, PVA/HA hydrogel blendedpullulan has been developed with a chitosan nanoparticles (NPs) delivery scaffold for acceleration wound healing [15].In general, crosslinking between synthetic polymer as PVA and polysaccharides as HA doesn't give the desired physicochemical properties for wound dressings.For instance, HA based hydrogels have poor mechanical properties that can limit their capacity to initiate a biomechanical signal, which is required for cellular proliferation.In order to overcome this problem bio-filler or reinforcing 1 3 agents are being necessary to incorporate into HA-based hydrogel membranes [12].
Cellulose nanocrystals (CNCs) or nano-whiskers are renewable, a natural bio-reinforcing agent due to their strong excellent mechanical properties and high crystallization [16].Recently, CNCs have been involved in fabrication of several biomaterials for tissue engineering purposes.In addition, CNCs can provide a high surface area mechanical support that enhance cell attachment, migration and proliferation [14].In this context, Yina et al. reported a significant enhancement in mechanical performance of gelatin-HA composite hydrogel after blending with CNCs that in turn had a great impression on wound healing [17].Further, Dehkordi et al. demonstrated the positive impact of using CNCs as bio-fillers on the mechanical and biological properties of HA-chitosan nanoparticles based hydrogel loaded with Granulocyte macrophage colony stimulating factor [14].
The aim of the research described here as synthesis of L-arginine delivery membranes based on physically or chemically-crosslinked PVA/HA hydrogels, mechanically reinforced by incorporation of CNCs as bio-nanofillers.The physiochemical properties of the crosslinked membranes were determined by swelling index, hydrolytic degradation rate and in vitro L-arginine release profile, while the cytotoxicity, cell adhesion, antimicrobial activity, and hemocompatibility were also assessed in vitro.

Chemical crosslinking method
The solution casting approach was used to prepare chemically crosslinked PVA/HA/CNCs/L-arginine hydrogel membrane, as described previously [18].Typically, (10%, w/v) PVA solution and (2%, w/v) HA solution were prepared individually at 25 °C.Then, the PVA/HA polymer blend membrane was fabricated through mixing PVA and HA solutions together at volume ratios of (80:20) for 3 h at 25 °C in a closed vial to eliminate bubbles formation and for assuring full homogeneity.CNCs as nanofiller (0.5%, w/v) were then incorporated into the PVA/HA solution, the PVA/HA/CNCs mixture was kept under sonication for 15 min for ensuring good dispersion of CNCs.Glycerol (1.0 ml) and CA (10%, w/v) were added to the PVA/ HA blend mixture as a plasticizer and crosslinker, respectively with gentle-stirring for one hour at room temperature.Finally, L-arginine (0.25% w/v) was added to the PVA/ HA/CNCs with further stirring for a half hour.This mixture was ultra-sonicated for 5 min to get rid of air bubbles, and then the resultant homogeneous solution was poured in a glass Petri dish, followed by thermal treatment in an oven at 60 °C for 18 h for launching crosslinking reaction, followed by 80 °C for two hours.

Physical crosslinking method
Physically crosslinked PVA/HA/CNCs/L-arginine hydrogel membranes were prepared by consecutive freezingand-thawing cycles as previously described by Peppas and Stauffer [19].Briefly, an aqueous solution containing 10% (w/v) PVA, 3% (w/v) of HA, 0.5% (w/v) of CNCs, and 0.25% (w/v) of L-arginine was dissolved in 15 ml distilled water and mixed well.Then, one ml of glycerol as a plasticizer was added to the later mixture.The aforementioned solution was kept under stirring for two hours at 25 °C to ensure the mixture homogeneity.The mixture was then ultra-sonicated for 15 min to guarantee removal of any formed air bubbles, and after that the resultant homogeneous mixture was poured to plastic Petri dishes, followed by freezing (F) at -20 ºC for 20 h, and then thawing (T) at 25 °C for two hours.The F-T cycles were repeated for three consecutive cycles.Finally, the obtained PVA/HA/ CNCs/Larginine membrane was dried by an oven at 70 ºC for 24 h.

Instrumental analysis of hydrogel membranes
FTIR: The chemical structure compositions of the prepared membranes was analyzed by FTIR model (Bruker Vertex 70, Germany) using transmittance modes of the spectra between (4000-400 cm −1 ).SEM: The morphology of the membrane's surface was investigated by scanning electron microscope (SEM) model (FESEM, Quattro S, Thermo-Scientific, USA) at an accelerating voltage of 5 kV and original magnification 500, 4,000 and 10,000X.
Mechanical properties: The membrane's tensile strength was measured by a standard universal tensile testing machine model (AG-I/50 N-10 KN, Shimadzu, Japan).
TGA: The thermal stability of obtained membranes was evaluated by thermogravimetric analysis (TGA) devise model (TGA-50, Shimadzu, Japan).The sample was heated under N 2 gas with a constant heating rate 10 °C/min from 25 to 500 °C.

Swelling ratio (%)
For each of the chemically and physically crosslinked hydrogel composite membranes, the swelling ratio was determined by submerging of the composite hydrogel membranes separately in a Petri-dish containing distilled water, pH 7.0 at 37 ºC.After specific time intervals, samples were re-weighted after drying the excess water.Then, the degree of swelling or water uptake of the membranes was calculated as a function of time, using the following formula [14].
Where, (W s ) is the weight of swollen sample and (W e ) is the weight of dried sample after soaking.

L-arginine release profile
The release profiles of L-arginine from the two types of crosslinked PVA/HA/CNCs/L-arginine hydrogel membranes were tested by the membrane diffusion method [20].Typically, 50 mg of PVA/HA/CNCs/L-arginine membranes were added to a dialysis tube (12-14 kDa, MWCO Visking dialysis tubing SERVA, Germany).The dialysis tubewas then soaked in 50 mL of PBS at pH 7.4 as release medium while being kept in a shaker incubator (50 rpm, at 37 °C).At chosen time intervals, the absorbance of one mL of the release medium was measured using a spectrophotometer (Agilent technologies, Cary series UV-Vis-NIR, Korea) set at 280 nm.According to the previously discussed, the concentration of L-arginine at different time intervals was calculated dependent on standard curve calculation of L-arginine as previously discussed [20].

In vitro hydrolytic degradation
Dried composite membranes with measured weight and specified dimension (4 cm × 4 cm) were immersed in PBS solution to determine their hydro-degradability.The weight of the tested membranes was measured initially and again at appointed timespan after removal of solution on their surfaces [13,21].Then, the hydrolytic degradation (1) was estimated in term of weight loss % versus the time, dependent on the following equation.
Where, W 0 is the original weight of the membrane, and W t is the weight of the membrane after removal from PBS at specified incubation intervals.
Cell viability percentages of both types of crosslinked membranes were accessed using colorimetric MTT-assay on the HFB-4 cell line at serial dilutions of (0, 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml) [22].The percentage of relative cell viability was calculated by substition in the following equation: Where, OD S the mean optical density of the sample, OD C the mean optical density of the control.
Graphpad Prism 7 software was used to display the results graphically as a percentage of cell viability.versusthe concentrations of the materials being tested.

In-vitro cell adhesion test
The cell adhesion and spreading on the surface of the two tested crosslinked membranes were investigated by the Wash-assay [23].The prepared membranes were collected on glass cover slips.The sterilized membranes were placed into 24-well plate prior to seeding of the cells, while plain cover slips without any treatment were served as a control.HFB-4 cells (1.0 × 10 5 ) were seeded into 24-microwell plate containing DMEM media supplemented with (10%, w/v of FBS at 37 °C) and kept for 2 and 4 h.After incubation, ( 2) The relative cell viability (%) = [OD S ∕ OD C ] × 100 the wells were rinsed softly with PBS to eliminate nonadherent cells.The attached cells were calculated by adding 100 µl/well of 0.1 (w/v, %) crystal violet solution and incubated for one hour at ambient conditions.Subsequently, the cells were rinsed twice with 1.0 M PBS and the absorbance was measured using a micro-plate-reader (CLARIOstar, BMG Labtech-Germany) at 570 nm.

Hemolysis test
The hemolysis-assay was used to evaluate the level of hemocompatibility of the chemically and physically crosslinked PVA/HA/CNCs/L-arginine [24].The hemolysis level was accessed by measuring the absorbance at 545 nm for the tested sample versus a negative control, and positive control, respectively [24].

Antimicrobial activity
The antimicrobial potential of the two membranes was screened for five strains of pathogenic bacteria and yeast (Escherichia coli NCTC10418, Pseudomonas aeruginosa ATCC9027, Vibrio cholerae ATCC700, Candida albicans ATCC 700, and Klebsiella pneumoniae ATCC13883) by the disc-diffusion method, as previously described [8].Spreading over the plates' surfaces by each pathogenic microbe using sterile cotton swab and leaving till complete dryness.After that, the tested samples were individually added to the surface of each pathogen-inoculated plate.After incubation for 24 h, the formed clear zones that indicate the antimicrobial activity of the compound were measured and photographed.

Preparation of chemically and physically crosslinked PVA/HA/CNCs/L-arginine hydrogel membranes
The chemically and physically-crosslinked PVA/HA/ CNCs/L-arginine hydrogel membrane showed uniform, homogenous, continuous and smooth surface shapestructures, as seen in SEM images.It was observed that, the chemically crosslinked PVA/HA/CNCs/L-arginine membranes displayed more homogenous and denser structure than the physically-crosslinked membranes.This is due to the effect of chemical crosslinker (i.e.citric acid) on changing the orientation of PVA, HA and CNCs polymers chains, hence, exposing abundant hydroxyl group to from large number of intermolecular hydrogen bonds, that enhanced the miscibility of the polymers with each other [25].In addition, CA could enhance the hydrogen bonding interaction between the polymers chains [26], beside its mediation crosslinking effect through the esterification process between -OH groups in PVA and -COOH groups in CA [27].On the other hand, the physically crosslinked membranes showed a less homogeneous and thinner structure than the chemically crosslinked ones.

SEM
SEM photographs of the tested composite hydrogel membranes are displayed in Fig. 1.Both membranes presented rough surfaces, and this could improves their biocompatibility by enhancing the cellular affinity, the cell attachment, migration and proliferation [28].Furthermore, SEM images of the two tested membranes surfaces indicates a good miscibility between membranes components, e.g., PVA, HA, CNCs, L-arginine and glycerol through displaying a homogenous, organized and crackles surface morphology.Moreover, a few round scattered pores were clearly observed in random locations as a consequence of the heating process [29] or by direct bonding interaction between L-arginine structure and synthetic polymer moieties.Previously Duceac et al. reported that the preparation of porous chitosan/acrylic hydrogel membranes containing variable pores dimensions was dependent on the L-arginine content [30].

FTIR analysis
The FTIR spectra showed the nature of interactions between the membrane's polymeric components, the occurrence of crosslinking reactions via CA and the successful loading of L-arginine into the hydrogel membranes.
Figure 2 shows the FTIR spectra of PVA, HA and CNCs, as components of the chemically/physically-crosslinked PVA/ HA/CNCs/L-arginine hydrogel membranes.A noteworthy observation is that the distinctive PVA bands belong to the hydroxyl, alkyl and acetyl groups can be seen at ѵ 3289, 2903 and 1713 cm −1 , respectively [11].
In addition, the vibrations of C = O and N-H in amide II were responsible for the unique bands of HA at ѵ 1683 cm −1 and 1559 cm −1 , respectively; while, other bands correlated to stretching vibrations of OH and C-H were at 3340 cm −1 and 2922 cm −1 , respectively [17].On the other side, CNCs have crystallinity and amorphous bands related to a symmetric -CH 2 bending vibration and C-O-C stretching vibrations of its β-(1 → 4)-glycosidic linkages at ѵ 1427 cm −1 and 897 cm −1 , respectively [31].Furthermore, the presence of sulphate ester peaks at 1060, 1110, 1162 and 1205 cm −1 demonstrate the existence of sulfate esters, which are functionalized CNCs, following the sulfuric acid hydrolysis process [32].
After loading of L-arginine into the chemically and physically-crosslinked PVA/HA/CNCs membranes, characteristic absorption bands were detected at 3151 and 1677 cm −1 that belong to -NH and carboxyl-carbonyl group stretching vibrations, respectively.In addition, the characteristic peak assigned to the guanidine group of L-arginine was presented at 1612 cm −1 .Furthermore, both chemically and physically-crosslinked PVA/HA/CNCs/L-arginine membrane display a broadened and blue shift of the -OH peak from 3312 to 3280 cm −1 and 3301 to 3289 cm −1 , due to effect of L-arginine loading which increased the hydrogen bonds interactions.Besides, the absorption peak of -CH 2 at 2929 cm − 1 became much sharper as consequence of the loading of L-arginine due to increasing the ratio of methylene groups.These results are in line with those published by Song et al., during his successful study on the immobilization of L-arginine onto chitosan hydroxypropyl methylcellose films [33].Moreover, chemically-crosslinked PVA/HA/CNCs/L-arginine membranes showed an obvious enhancement in intensity and sharpness of the peak assigned for the crosslinking reaction at approximately ν 1725 cm −1 , due to crosslinking effect of amino group of L-arginine.This observation is similar to the finding made by Udhayakumar et al., who stated the possibility of using L-arginine as a bio-crosslinking in collagen/chitosan scaffolds for tissue regeneration [34].

TGA thermographs
The TGA results of tested hydrogel membranes are presented in Table 1.It is noted that the overall thermal stability of the chemically crosslinked membrane exceeded that of the physically-crosslinked membranes, owing to the enhanced crystallinity of PVA by chemical crosslinking through the esterification reaction between PVA and CA [35].This result was obviously detected through recording enhancement in the second degradation stage by increasing both the start and end-stage of thermal decomposition from 110-264 oC to 132-396 oC for the physically and chemically crosslinked membranes, respectively.Whereas the T onset values were found at 132 and 110 °C for the chemically-and physically-crosslinked PVA/HA/CNCs/Larginine membranes, respectively.In the same context, the high value of T 50% of the chemically-crosslinked membranes was detected at 305 °C, compared to physically-crosslinked membranes at 280 °C.Similar thermal enhancement effects were observed with CA crosslinked chitosan membranes, due to chemical crosslinking reaction occurred [36].

Mechanical properties of the crosslinked membranes
The mechanical characteristics of the physically and chemically-crosslinked PVA/HA/CNCs/L-arginine composite hydrogel membranes in the wet state were evaluated in terms of Young's modulus, elastic modulus, elongation at break and maximum stress, as listed in Table 2.By comparing the mechanical measurements, it is clearly indicated that the mechanical measurements of the chemically-crosslinked PVA/HA/CNCs/L-arginine exceeded significantly those of the physically-crosslinked membranes.It is noticed that Young's modulus of the chemically-crosslinked membranes increased by 40-folds (~ 188 MPa,) compared to the physically-crosslinked membranes (~ 4.6 MPa.).In addition, the elastic modulus of the chemically-crosslinked membranes was 3 times higher than physically-crosslinked membranes.The enhancement in mechanical performance of the chemically-crosslinked membrane, was related to the influence of the amino group of L-arginine on enhancement of non-covalent interactions beside crosslinking esterification bonds leading to formation of a more dense membrane structure [33].Also, it might be associated with enhancing the extent of crystallinity in PVA by effect of CA as a crosslinker [35].On the other hand, the physical crosslinked membranes had higher values of maximum stress than those of the chemically crosslinked membranes; this was due to the influence of the freezing-thawing cycles on the crystal structure formation and hence increasing the physical crosslinking.Similarly, Lotfipour et al. displayed moderated mechanical wound dressing membranes using oxytetracycline loaded-PVA/chitosan hydrogels by three freeze-thawing cycles [37].

Physicochemical characterization
of the chemically and physically crosslinked PVA/HA/CNCs/L-arginine hydrogel membranes

Swelling study
Figure 3 displays the swelling capacity of the hydrogel membranes for 14 days.Both membranes' swelling % values increase as the immersion time was extended, until the equilibrium swelling state is reached.Interestingly, swelling of the physically-crosslinked PVA/HA/CNCs/Larginine composite hydrogel membrane reached its equilibrium after 24 h, with a swelling ratio of ~ 517%, whereas, the chemically-crosslinked PVA/HA/CNCs/L-arginine  composite hydrogel membrane reached to the equilibrium of swelling state after 12 days, with a swelling ratio ~ 660%.
It is clearly noted that, the swelling ratio of the physicallycrosslinked PVA/HA/CNCs/L-arginine was > 4.5fold more than that of the chemically-crosslinked membranes after one hour of swelling.Similar swelling behavior was seen in physically-crosslinked CS-PVA hydrogels that quickly absorbed high quantities of water and reached equilibrium after 20 h [38].
The low swelling capacity of the chemically-crosslinked hydrogel membranes might be correlated with the influence of CA as a crosslinker, obstructing the hydrophilic moieties of L-arginine, resulting in high hydrophobicity [39].Beside, using CA results in highly cross-linked polymeric structure by formation of esterification and hydrogen bonds between polymers chains and L-arginine [40,41]; hence obstacles the water diffusion into the hydrogel and decreases the swelling % of membranes [42].Thus, these findings suggest that physically-crosslinked PVA/ HA/CNCs/L-arginine membranes would serve as a promising wound dressing since it has a high swelling capacity reflecting to its ability to absorb wound exudates during healing process.

L-arginine release profile
Figure 4 displays the L-arginine release profile of chemically and physically-crosslinked PVA/HA/CNCs/L-arginine hydrogel membranes in PBS at pH 7. In chemically and physically-crosslinked membranes, the released L-arginine proceeds by a biphasic release profile starting with immediate burst release followed by a second, slow release.Previously it was reported that the freeze-thawed CS-PVA hydrogels exhibited a biphasic drug release and the amount of released drug was directly proportional to increase in freezing time as entanglement corresponding factor [43].Furthermore, it was clearly noticed that, the release was relatively higher in chemically crosslinked membrane, compared to physical crosslinked membranes.Since chemically-crosslinked PVA/HA/CNCs/L-arginine membrane releases about 50% of L-arginine at the first hour and reached a maximum amount of release ~ 92.3%, at about 48 h.Meanwhile, L-arginine initially 8.7% was released from the physically-crosslinked PVA/HA/CNCs/Larginine membrane with in first hour, reaching 13.6% at 48 h.The first fast release phase implies the loaded L-arginine on the surface of the membrane or entrapped near the membrane's surface [44].Besides, the effect of the prolonged drying conditions caused movement and migration of the drug towards membrane's surface, that in turn led to the burst release behavior [45].While, The slow L-arginine release in the second phase might result from the diffusion of entrapped L-arginine in the membrane that is mediated through polymer erosion [45], or by passing L-arginine through mobile PVA, HA and CNCs polymer chains [46].This slow release of L-arginine has been preferred in pharmaceuticals applications to avoid causing any side effects and disturbances in body homeostasis.

Hydrolytic degradation of the crosslinked hydrogel membranes
The hydrodegradability degree of membranes was evaluated by direct immersing of the nanocomposite membranes into distilled water for 21 days, as presented in Fig. 5.The chemically-crosslinked PVA/HA/CNCs/L-arginine membranes showed a slow hydrolytic degradation rate; they lose about 50% of their weight by day 20.This slow hydrolytic degradation is due to using a crosslinker that forms strong, crosslinking bonds between the polymer chains which improves the water resistance of hydrogel membrane against hydrolytic degradation [36].Whereas the hydrolytic degradation occurred via a break up of crosslinker parts that bond the polymers chains, followed by disintegration of the CA crosslinked polymeric chains [8].Das, et al. demonstrated CA-crosslinked PVA/starch composite films with the same hydrolytic degradation trend [47].Conversely, physically-crosslinked PVA/HA/ CNCs/L-arginine membranes seem to be a more hydrolytically than chemically-crosslinked membranes; they started undergoing hydrolytic degradation after 24 h of immersion.This is assigned to the PVA hydrogel dissolution, followed by releasing of PVA chains that were not incorporated into the physical structure of entangled hydrogel and don't participate in the formed crystallite zones.[48] 3.4 In-vitro bio evaluation tests

In-vitro cell viability of the hydrogel membranes
The cytocompatibility of the chemically and physicallycrosslinked PVA/HA/CNCs/L-arginine hydrogel membranes were tested by MTT-assay using the HFB-4 cell line after 48 h of cell treatment, as presented in Fig. 6.All crosslinked membranes displayed higher cell viability % of the HFB-4 cell lines with increasing concentration of tested membrane samples till reaching 1.0 mg/ml and 1.5 mg/ml for the chemically and physically-crosslinked membranes, respectively, membranes showed HFB-4 cell viability % of 98.5% and 170%, respectively.The higher cell viability could be arisen from the combined physiological effect of membrane composition of HA, CNCs, and L-arginine on cell growth and proliferation.As described previously, HA participates in the stimulation of cell proliferation, and enhancing the angiogenesis [13].CNCs induce biomechanical signals that stimulate collagen synthesis and neo-epithelialization [49].L-arginine stimulates angiogenesis, collagen synthesis and the cell proliferation process mediated by NO production [20].Interestingly, there was obvious higher cell viability % in the physically crosslinked PVA/HA/CNCs/L-arginine membranes than in the chemically crosslinked membranes for the HFB-4 cell line.This result could be due to using citric acid as the crosslinker that has short-term cytotoxic effects on various cell lines by forming acidic-pH [50].
In both membranes, exceeding 1.5 mg/ml there was a noticeable regression in cell viability %.As seen, on exposing of HFB-4 cells to 2 mg/ml of chemically and physicallycrosslinked PVA/HA/CNCs/L-arginine membranes the cell viability % values reduced to 72% and 83.5%, respectively.The decrease in cell viability % might be owing to the transformation of membrane into gel suspension that obstacles the passage of gases [51].While, the other possible mechanism might be associated with exposure of cells to a high dose of L-arginine and CNCs, that involves in generation of RNS and ROS mediating inflammatory cytokines release, DNA damage and alteration of mitochondrial membrane potential [52,53].

In-vitro cell adhesion tests onto the crosslinked hydrogel membranes
HFB-4 cells adhesion onto the tested hydrogel membranes surfaces are displayed in Fig. 7 at different time intervals (2 and 4 h) with varied concentrations of tested samples (0.5, 1 and 1.5 mg/ml), compared to the control.Results revealed an excellent cell adhesion affinity toward the two tested hydrogel membranes with varied values dependent on contact time between membrane and cells.Since cell adhesion affinity values of chemically and physically-crosslinked PVA/HA/CNCs/L-arginine membranes at a concentration of 1 mg/ml were 1.9 and 2.1, respectively after 2 h.This is owing to, a high cell adhesion affinity toward components of the hydrogel membranes.Whereas, HA possesses cell adhesion properties by interacting with CD44 membrane cell surface receptors [54].While, CNCs can attract a large number of adhesion proteins by their reactive -OH groups, that in turn enhances cell adhesion.Also, CNCs can support cells adhesion and growth by generating a natural strong mechanical environment which is favored for cell adhesion [55].In addition, L-arginine induces overexpression of cell surface adhesion molecules [56]; as

Antimicrobial activity of the crosslinked hydrogel membranes
The antimicrobial activity of the crosslinked hydrogel membranes was tested by the disc-diffusion method against Vibrio cholerae ATCC700, Candida albicans ATCC 700, Pseudomonas aeruginosa ATCC9027, Escherichia coli NCTC10418, and Klebsiella pneumoniae ATCC13883).
Results of the microbial inhibition zones are shown in Table 3. Upon comparison between the two types of crosslinked membranes; it is clear that the most effective membrane in terms of its microbial resistance was the chemically-crosslinked hydrogel, which affectd all tested pathogens indicating by clear zones of diameter ranging from 12 to 20 mm.Meanwhile, physically-crosslinked hydrogel membranes failed to suppress the growth width of all tested pathogenic microorganisms, indicating physically-crosslinked membrane had impaired antimicrobial activity as result of repeated freeze and thaw cycles that cause denaturation and aggregation of L-arginine amino acid by Wang et al [58].
In fact, the most affected pathogen by chemicallycrosslinked PVA/HA/CNCs/L-arginine membranes were the Klebsiella pneumonia that showed 20 mm as a clear or inhibition zone.While, the least affected pathogen was Vibrio cholera that displayed of a10 mm clear zone.This antibacterial activity is suggested to be associated with the composition effect of membranes as HA, CNCs and L-arginine on bacterial growth inhibition.Moreover, the bactericidal effect of CA was mediated through decreasing the intracellular pH and ATP concentration, as well as reducing the DNA content [59].

Hemolysis assay of the crosslinked hydrogel membranes
The hemolysis assay was performed to assess the biocompatibility of the tested hydrogel membranes through representing the extent of dissolution of RBCs up on contact with the tested hydrogel.The study of optical density in response to tested hydrogel membrane, compared to control indicated that the level of blood hemolysis varied dependent on hydrogel type, as listed in Table 3. Chemically physically-crosslinked PVA/HA/CNCs/L-arginine membranes have optical densities of 1.34 and 0.84, respectively, compared to the optical density value positive control of 1.4 (Fig. 8).These findingsrevealed that the physically-crosslinked PVA/HA/CNCs/L-arginine membranes are much safer for wound healing purposes than the chemically-crosslinked hydrogel membrane.Also, these results are consistent with those of Shitole et al., who reported that the freeze-thawing method is a type of physicalcrosslinking method for preparation of hemo-compatible hydrogels without any recorded hemolysis values [60].Also, they provided PVA based hydrogels functionalized with three bioactive materials like pullulan, poly-L-lysine and gelatin with low hemolysis degree [60].

Conclusions
Developing two types of the physically and chemicallycrosslinked PVA/HA/CNC/L-arginine hydrogel membranes presented wide features to be used for wound healing purposes.The Chemical-crosslinked hydrogel membranes were created by solution-casting approach employing 10% (w/v) CA as crosslinker; while physically-crosslinked membranes were prepared by freeze-thawing method through three consecutive cycles.Two types of crosslinked hydrogel membranes showed good morphological, thermal and mechanical properties.Indeed, chemicallycrosslinked hydrogel membranes were more thermal and mechanical stable than physically-crosslinked membranes.Furthermore, two types of the crosslinked membranes exhibited desired physicochemical properties representing in appropriate swelling capacity and sustained hydrolytic degradation, in particular chemically crosslinked membranes.In addition, L-arginine release profile of two crosslinked membranes was proceeded by biphasicrelease profile, starting with burst release followed by immediate release rate.Accordingly, the current study has explored that both crosslinked membranes might have a potential as promising biomaterials for biomedical applications; especially as wound dressings.This finding has been issued from observations of high cell viability %, excellent cell adhesion capacity and low hemolysis degree of each crosslinked membrane.

Fig. 6
Fig.6 In-vitro cell viability of chemical and physical PVA/HA/ CNCs/L-arginine hydrogel membranes on HFB-4 cell line, in the concentration rang (0-2 mg/ml) for 48 h incubation period

Fig. 7 Table 3 Fig. 8
Fig. 7 Cell adhesion behavior of the chemical and physical PVA/ HA/CNCs/L-arginine hydrogel membranes on HFB-4 cells in the concentrations (0.5, 1 and 1.5 mg/ml) after 2 and 4 h incubation periods

Table 2
Mechanical properties of chemical and physical crosslinked PVA/HA/CNCs/L-arginine hydrogel membranes