INTRODUCTION:

Many approaches have been advocated to enhance the surgeon’s ability to achieve a rapid and effective control of wound closures, which is one of the dominating variables in any surgical procedure. A rapid and satisfactory wound closure can minimize the time spent in the operation room; the time patients are under anesthesia, the need for transfusion and complications that occasionally accompany wound closure. To date the use of sutures has been the most widely used method for the wound closure because of the high reliability of closure required for the satisfactory wound healing. However alternative wound closure technique have long been sought since suturing procedure often require highly skill and experience surgeons, a relatively longer time for wound closure, and the need for the post operating removal of non-absorbable suture materials on the skin to reduce any risk that could lead to adverse reaction.

Possible alternative of sutures for wound closure includes clip, staples and bioadhesives^1-2. But clips and staples cause inflammation and wound infection. So a better alternative is the use of bioadhesive. In this study, hemocompatible polyurethane films are synthesized and the possible results to use it as a bioacompatible polymer in many fields are discussed.

MATERIALS AND METHODS:

Materials:

Polyethylene glycol (PEG) and the Potassium salt of hydroquinone sulphonic acid, Isophorone-diisocyanate (IPDI), Tolylene diisocyanate (TDI), 1,6-diisocyanatohexane (HMDI), 4,4’-methylbis(cyclohexyl isocyanate) (H₁₂MDI), 4,4’-methylenebis(phenyl isocyanate) MDI were purchased from Aldrich, USA. Dibutyltin-dilaurate was procured from Fluka Chemical Co and was used as received. N, N-dimethyl formamide (extra pure) and was purchased from Sisco Research Laboratories, Mumbai. Ethyl methyl ketone was purchased from Ranbaxy laboratory limited.

Preparation of polyurethane isomers:

10g of polyethylene glycol (PEG) is taken in a three necked flask and dissolve in MEK. A required quantity of diisocyanates was dissolved in ethyl methyl ketone and added by means of a dropper to the reaction vessel. Dibutyltin dilaurate was used as a catalyst. The reaction was continuing for five hours at 95°C. Bring down the temperature of the reaction to 50°C. Calculated amount of hydroquinone sulfonic acid (chain extender) was added and reaction was continuing for 30 min at 95°C. The final polymer solution was cast in a mould. The film was demoulded and condition as per the standard test procedure methods. All the samples are named as GSA.

Characterization of polyurethane:

The various characterization studies carried out for our PU samples are as follows:

· FT-IR spectrophotometer

· FT-NMR spectrophotometer

· Gel permeation chromatography

· Particle size analysis

FT-IR Spectrophotometer:

Nicolet impact 400 FT-IR spectrophotometer was used to substantiate the formation of the PU ionomer. Polymer was dissolved in suitable solvent and run the spectrum. All the spectra were recorded at a resolution of 4cm^-1with a maximum of 100 scans. A background spectrum was run before running the spectra of the PU samples.

FT-NMR Spectrophotometer:

High-resolution ¹H and ¹³C-NMR spectra were recorded using a Brucker MSL 300 P, 300 MHz FT-NMR spectrophotometer. Deuterated dimethyl sulfoxide (DMSO-d₆) was used as solvent for recording NMR spectra. The proton spectra were recorded using broadband inverse probe where the inner coil is for the protons and outer coil for X nuclei. Solvent suppression was applied in some cases where the solvent signal is very strong compared to the sample signals. ¹³C were recorded in dual (¹³C/¹H) probe where the inner coil is for ¹³C and the outer coil is for protons. The decoupling of protons was done using Waltz-16 sequence. The spatial parameters like number of scans, time domain data points, etc was adjusted depending on the nature of the sample and the relaxation parameters like T₁ and T₂.

Gel permeation chromatography:

The molecular weight and molecular distribution of the polymers was determined by Gel Permeation Chromatography using a Water unit interfaced with NEC (IBM AT Compatible) computer. Waters 510 HPLC pump columns (Ultrastyragel columns 10³Å, 10⁴ Å, 10⁵Å in series) coupled with a Waters 410 differential refractometer. In GPC a porous material is used as the stationary phase and a solvent as a mobile phase. The stationary phase used is a swollen gel of polystyrene and the mobile phase used is HPLC grade DMF. The flow rate of the solvent is 1 ml/min. The polymer to be analyzed is introduced at the top of the column and then is eluted with a solvent. The polymer molecules diffuse through the gel at rates depending on their molecular size. As they emerge from the bottom of the column they are detected by a differential refractometer.

Particle size analysis:

The particle sizes of the polyurethane ionomer were analyzed by the Microtrac S3500 SDC with Tri-laser Technology. The sample is introduced in the reservoir, which consists of wetting agent (water). The sample is dispersed uniformly in the fluid and is pumped to the sample cell. Three laser light sources are incident on the sample cell and it is refracted towards the detectors. After detection the sample is sent back to the reservoir by a recirculating system and a drain valve drains the fluid out. The data is collected by the software provided in the Pentium computer.

Haemocompatibility:

The haemocompatibility was evaluated in vitro and the following categories of blood interactions were studied:

1. Haemolysis

2. Thrombogenicity

3. Platelet Adhesion Test

Haemolysis:

The haemolysis tests were performed as described in ASTM F756-00 standard. The haemoglobin released by haemolysis was measured by the optical density (OD) of the supernatant at 540 nm using a spectrophotometer UV-Vis. The percentage of haemolysis was calculated as described in the following equation.

OD_test- OD_negative_control

% Haemolysis = __________________________

OD_positive_control – OD_negative_control

Thrombigenecity and platelet adhesion test:

The thrombogenicity and platelet adhesion test was performed as described in Ting-Yu Liu et al. and Poussarda et al.

RESULTS AND DISCUSSION:

FT-IR Spectrophotometer:

The infrared spectra of thermoplastic polyurethane ionomer have shown in figure 1. The maximum degree of neutralization was 10%-15% for a copolymer containing 4.1-mol percent acid. Evidence for hydrogen bonding is shown by the shoulder around 2800-2900 cm^-1 [hydrogen bonded for hydroxyl]. There is ionized carboxyl at 1650-1667 cm^-1and asymmetric stretching of the carboxylic ions at 1500-1600 cm^-1. Later this increases with the increasing neutralization. The N-H stretching vibration was observed at around 3400 cm^-1. The bond due to C-N stretching and N-H deformation was absorbed at 1530 cm^-1, while the C-O-C bond stretching of PEG bond was absorbed around 1100 cm^-1.

FT-NMR Spectrophotometer:

NMR spectrum of polyurethane was recorded using DMSO-d₆ as the solvent given in the figure 6-9. Methylene protons of PEG resonate at 1.47ppm and methylene protons of -O-CH₂- of PEG is seen in 3.32ppm. The peak at 3.92-4.2 ppm is due to Methylene protons of -O-CH₂- attached to urethane linkage. Methylene protons of TDI absorbed at 2.08-2.3ppm. Aromatic protons of chain extender and TDI resonate from 6 – 7.9ppm. The NH of urethane linkage and carboxylic acid portion of chain extender was fixed using ionic diol. The NH proton of urethane linkage absorbs at 8-10ppm and carboxylic acid portion of chain extender part resonate as weak signal at 9.8ppm (figure 2).

For ¹³C NMR spectrum, the carbon of methylene protons of PEG resonates at 29 ppm (figure 3). The carbon of -O-CH₂- groups absorbed at 70.2ppm. The carbon of aromatic chain extender and diisocyanates resonate between 100-149ppm. Urethane carbon resonates at 157-162ppm. The carboxylic carbon resonates at 166 ppm. The chemical shifts of proton located in IPDI units of PEG are at 1.88ppm, 3.77ppm, 1.11ppm, 2.84ppm and 0.99ppm. It is noted that the chemical shifts of 0.99ppm in IPDI molecule is located at 3.1ppm and shifted to 2.84ppm in IPDI unit of PEG after polymerization reaction, which indicate that the NCO group attached to methane is turned into urethane after polymerization reaction.

Particle size analysis:

PU ionomers are mainly used in coatings; particle size and viscosity are important parameter in deciding the type of coating requirements. For surface coating, large particle size is preferred to ensure faster drying. It penetrates into the substrate is required smaller particle size. Suitable viscosity range is required to avoid sagging (in case of low viscosity) and practical difficulty in application (encountered with high viscosity). In general ionic content is inversely proportional to molecular weight and is directly proportional to particle size. In this case, there is an increase in molecular weight and PU ionic content is decreased. Hence it is interesting to see the dual effect of the ionomer content on the particle size of dispersion as shown in the figure 4. If ionic content plays a major role then the particle size is expected to decrease in PU content.

Figure 4 PARTICLE SIZE ANALYSIS OF GSA 1

Gel permeation chromatography:

The molecular weights (number average and weight average) of the entire polymer were determined by gel permeation chromatography. The GPC data obtained for the five samples of polyurethane is given below.

Table 1: GPC DATA for 5 GSA samples

S.NO	SAMPLE CODE	Mn	Mw	MP	Poly dispersity
1	GSA1	25848	37920	2005	1.46
2	GSA2	28822	74403	17492	2.58
3	GSA3	29678	122082	16213	4.11
4	GSA4	27450	92904	17055	3.38
5	GSA5	27885	60404	16420	2.17

Mn = Number average molecular weight.

Mw = weight average molecular weight.

Poly dispersity = Mw/Mn

To be a bioadhesive it is necessary that the polymer should have high molecular weight. Among the above five samples GSA3 and GSA4 seem to have high molecular weight. Hence it is concluded that they may act as a good bioadhesive.

Haemocompatibility:

Thrombogenecity:

Haemostasis, the spontaneous arrest of bleeding from ruptured blood vessels’ is a broad physiological process of which blood coagulation system is just one part. A graph of tabulated value was drawn taking time (min) in X-axis and OD (540nm) in Y-axis which is presented below:

Figure 5 THROMBOGENICITY OF POLYURETHANES

From the figure 5, it is observed that as the clotting time increases, the thrombus formation also increases. This indicates the synthesized polyurethane is highly thrombogenic. Since polyurethane would be applied in diffuse surfaces with capillary bleeding its haemostatic character can be of great importance. Hence we can suggest that this material can act as a haemostatic agent, improving coagulation and therefore help the cicatrisation process^5,6 of the wound.

Haemolysis index:

The haemolysis index represents the extent of red blood cells broken by the sample contacting with blood. In the greater the haemolytic index value, the more the number of red blood cells broken and the smaller the haemolytic index value, the better the blood compatibility of the biomaterial. So for a biomaterial the haemolytic index value should be below 5%. According to the ASTM F 756-00 materials can be classified as follows:

A corresponding graph of haemolytic index values is drawn as shown below:

Figure 6 HAEMOLYTIC INDEX OF POLYURETHANES

From the observation and figure 6, it is concluded the polyurethanes synthesized is non haemolytic in the untreated form since its haemolytic is lower than 2%. But the treated samples are slightly haemolytic and the PBS extraction solution is highly haemolytic. So it is better to use the sample in untreated form than that of treated form.

Platelet adhesion test:

The platelet activation and adhesion depend on the characteristics of artificial surface and protein adsorption¹. Initially, the blood flows through the artificial surfaces; the plasma proteins such as Alb, IgG and FN are adsorbed on the surface, which depends on the characteristics of the polymers themselves. The polymers are less attractive to proteins than cellulosic materials. Platelets are extremely sensitive cells that may respond to minimal stimulation. Activation causes platelets to become sticky and change in shape to irregular spheres with spiny pseudopods, accompanied by internal contraction and extrusion of the storage granule contents into the extracellular environment⁷. These secreted platelet products stimulate other platelets, cause irreversible platelet aggregation, and lead to the formation of fused platelet plugs. Subsequently, the platelets release some materials such as adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin and platelet factor 4 (PF4), beta-thromboglobulin (bTG), FN, vWFand fibronectin, and then activated arachidonic acid to produce thromboxane A2 (TXA2). Then ADP and TXA2 induce more platelet aggregation on the surface and result in more plugs. Followed by, Hagemen factor (factor XII), which is activated to induce the intrinsic pathway, meanwhile, the white blood cells, release thromboplastin to induce the extrinsic pathway and common pathway. Finally, the system leads to the formation of thrombin, a non-soluble fibrin network, or, thrombus⁸.

(a)

(b)

Figure 7 Optical micrograph of platelets adhesion on polyurethane samples

Poussarda et al., had reported that the platelet adhesion in the presence of plasma proteins decreases gradually with the increasing surface wettability. However, plasma protein adsorption on a wettability gradient surface increased with the increasing surface wettability in the absence of plasma protein. More plasma protein adsorption on the hydrophilic surface caused less platelet adhesion, probably due to platelet adhesion inhibiting proteins, such as high-molecular-weight kininogen, which preferably adsorbs onto the surface by the so-called Vroman effect. Although both the presence of plasma proteins and surface wettability play important roles for platelet adhesion and activation, the porous and surface roughness should also be taken into consideration. In our work we found that GSA 7 and GSA 8 (fig 7) show better adherence for platelet than that of GSA 10.

CONCLUSIONS:

We successfully prepared the segmented polyurethane ionomers, based on different isocyanates (TDI, MDI, HMDI and IPDI), PEG, and chain extender based on Dihydroxybenzoic acid salts. We found certain PUI films could significantly improve the haemocompatibility. We investigated the bulk characteristics of the polymers investigated by FT-IR, FT-NMR, Particle analysis and GPC measurements. FT-IR shows no peak at 2276 cm^_1revels that all the isocyanates are completely reacted and formed polymers. The molecular weight of the polymer is in the range of Mn 37920-122082. The haemocompatibility in vitro with human blood was viewed by optical microscopy. We found that fewer platelets adhere to the polyurethane ionomers surfaces. The samples GSA 7 and GSA 8 are the best haemocompatible PU samples. With these results the PU samples were further analyzed for its application in dentistry as a filling material for tooth cavities.

ACKNOWLEDGEMENT:

The author would like to acknowledge Mr. M. Seenuvasan, Madha Engineering College and Dr. Jai Sankar, CLRI for their guidance to finish this project

REFERENCES:

1. C.C.Chu, J.Anthony van Fraunhofer, Howard P.Greisler. Tissue Adhesives. Wound Closure Biomaterial and devices. 1997: 317-345.

2. Joseph Jagur-Grodzinski. Polymers for tissue engineering, medical devices, and regenerative medicine. Concise general review of recent studies. Polym. Adv. Technol. 2006; 17: 395–418

3. Ting-Yu Liu, Wen-Ching Lin, Li-Ying Huang, San-Yuan Chen and Ming-Chien Yang. Surface characteristics and hemocompatibility of PAN/PVDF blend membranes. Polym. Adv. Technol. 2005; 16: 413–419.

4. Poussarda, F. Burela, J.-P. Couvercellea, Y. Merhib, M. Tabrizianc, C. Bunela. Hemocompatibilty of new ionic polyurethanes: influence of carboxylic group insertion mode. Biomaterials 25; 2004: 3473–3483.

5. Maricel Cabanlit, Duncan Maitland, Thomas Wilson, Scott Simon, Theodore Wun, M. Eric Gershwin, Judy Van de Water. Polyurethane Shape-Memory Polymers Demonstrate Functional Biocompatibility In Vitro. Macromol. Biosci. 7; 2007: 48–55.

6. P. Ferreira, R.Pereira. J.F.J. Coelho, Antonio F.M. Silva, M.H. Gil. Modification of the biopolymer castor oil with free isocyanate groups to be applied as bioadhesive. Int. J. of Biological Macromolecules, 2006, 1-9.

7. Ian C. Bonzani, Raju Adhikari, Shadi Houshyar, Roshan Mayadunne, Pathiraja Gunatillake and Molly M. Stevens. Synthesis of two-component injectable polyurethanes for bone tissue engineering. Biomaterials. 28(3); 2007: 423-433.

8. Mark J. Ernsting, Geneviève C. Bonin, Meilin Yang, Rosalind S. Labow and J. Paul Santerre. Generation of cell adhesive substrates using peptide fluoralkyl surface modifiers’, Biomaterials. 26(33); 2005: 6536-6546.

9. J.P. Santerre, K. Woodhouse, G. Laroche and R.S. Labow. Understanding the biodegradation of polyurethanes: From classical implants to tissue engineering materials. Biomaterials, 26(35), 2005: 7457-7470.

10. Jianjun Guana, Kazuro L. Fujimoto, Michael S. Sacks, William R. Wagner. Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications. Biomaterials. 26; 2005: 3961–3971.

Received on 25.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 143-148

Synthesis, Characterization and Hemocompatibility Evaluation of Polyurethane Ionomers

FT-IR Spectrophotometer:

Haemocompatibility:

S.NO

Mn

Mw

MP