PREPARATION AND EVALUATION OF CROSS LINKED CHITOSAN-POLYURETHANE MESH IN TISSUE REPAIR IN SHEEP

In the present study, an interpenetrating polymer network (cross-linked chitosan-polyurethane mix) was successfully prepared. It was found that the mixing of 70% chitosan and 30% polyurethane gave the best results as far as the mechanical properties of the network are concerned. Toxicological evaluation of the network was done in 15 rabbits, and it was found to be pathologically nontoxic. The possible effects of the network on the liver, kidneys, and skeletal muscles of sheep with clean or infected open wounds were studied through the measurement of plasma aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and acid phosphatase. In sheep with clean open wounds that were implanted with the network, the levels of the enzymes were 11.04 ± 0.54; 30.83 ± 1.20; 36.37 ± 1.52; and 5.52 ± 0.37, respectively. In sheep with contaminated wounds, the levels of the enzymes were 11.29 ± 0.30; 38.58 ± 1.16; 38.20 ± 1.21, and 6.31 ± 0.46, respectively. Statistically significant differences were not encountered in the values of alanine aminotransferase, alkaline phosphatase, and acid phosphatase in sheep with clean wounds and those with contaminated wounds. However, the level of aspartate aminotransferase was higher significantly (P<0.01) in sheep with contaminated wounds than in sheep with clean wounds. Histologically, the healing of the clean and contaminated open wounds proceeded through the same processes as described in the literature. Bacterial infection was not observed in any of the wounds that were implanted with the network. Iraqi Journal of Veterinary Sciences, Vol. 20, No.2, 2006 (145-161) 146 كبشم مييقتو ريضحت يف همادختساو نيثيروي يلوبلاو ايضرع كباشتملا ناسوتياكلا نم ً ا حلاصا نأضلا يف ةنيللا ةجسنلا يديبعلا سابع يرابلا دبع ) ١ ( يدعسلا ميھاربا ظفاح ، ) ٢ ( * مشاھ ركاش حلاص ، ) ٣ ( ةيدان ، سقرم انح ) ٤ ( ، ةيريرسلا تاساردلا عورف ) ١ ( ضارملاا ملعو ) ٢ ( لا مسقو ،يرطيبلا بطلا ةيلك ، ةيلك ،ءايميك ،دادغب ةعماج ،يرطيبلا بطلا ةيلك ،ديلوتلاو ةحارجلا عرفو ةرصبلا ةعماج ،مولعلا * يلاحلا ناونعلا : ،لصوملا ،لصوملا ةعماج ،يرطيبلا بطلا ةيلك ،ضارملاا ملع عرف


INTRODUCTION
Chitin is a linear homopolymer of β (1, 4)-linked N-acetyl-D-glucosamine, and chitosan is a partially deacetylated chitin (1)(2)(3)(4).Chitin is widely distributed in nature as the skeletal material of crustaceans and insects, as well as a component of cell walls of bacteria and fungi, and is the second most abundant polymer occurring in nature, after cellulose.Chitosan lacks irritant or allergic effects and is biocompatible with both healthy and infected human skin (1).When chitosan was administered orally in mice, the LD 50 was found to be in-excess of 16 g/kg, which is higher than that of sucrose (1).The intriguing properties of chitosan have been known for many years and the polymer has been used in the fields of agriculture, industry, and medicine (5)(6)(7).In agriculture, chitosan has been described as a plant antivirus, an additive in liquid multicomponent fertilizers and it has also been investigated as a metal-recovering agent in agriculture and industry (1).Chitosan has been noted for its application as a film-forming agent in cosmetics, a dyebinder for textiles, a strengthening additive in paper and a hypolipidic material in diets (1,8).It has been used extensively as a biomaterial, owing to its immunostimulatory activities, anticoagulant properties, antibacterial and antifungal action and for its action as a promoter of wound healing in the field of surgery (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).In addition, chitosan has a variety of promising pharmaceutical uses and is presently considered as a novel carrier material in drug delivery systems, as indicated by the large number of studies published over the last few years (20)(21)(22)(23)(24).The purpose of this study were to prepare a new interpeneterating polymers networks (chitosan-polyurethane meshs) (IPN) and to evaluate the application of these networks in the treatment of experimentally-induced skin injuries in sheep.

Preparation of chitosan:
To isolate and purify the chitin, the exoskeleton of shrimps were isolated, thoroughly cleansed in water, dried in an electrical oven at 100◌ ْ C, and grinded till a smooth powder was obtained.A hundred grams of the powder was mixed with one liter of 5% HCL and the mixture was agitated continuously for 24 hrs at room temperature (demineralization).The solution was filtered through a clean piece of clothes and the precipitate was cleansed with water for several times to get rid of residues of the acid.A one liter of 50% sodium hydroxide (NaOH) was added to the precipitate and the mixture was agitated continuously at 90◌ ْ C and for 3 hrs to get rid of the protein (deproteinization).The mixture was then left to cool, filtered, rinsed with distilled water for several times to obtain chitin in a ratio of 35% of the quantity used (25)(26).Chitosan was obtained by adding 250 ml of potassium hydroxide (50%) to 20 gm of the isolated chitin.The mixture was then heated at 90◌ ْ C for 6 hrs with continuous agitation.This process was repeated for 3 times, and the precipitate was rinsed and dried.The chitosan was obtained at 75% of the quantity used (25)(26).

Chitosan crosslinking:
Cross linking of chitosan was done through dissolving the chitosan in 0.1 M HCL.The cross linking material (glutaraldehyde) was then added to the mixture at the rate of 8 mg/ml.Neutralization of the mixture was done through the addition of 0.1M sodium hydroxide till a swollen precipitate was obtained which represented the cross linked chitosan (26).

Preparation of the IPN:
The IPN were prepared through the mixing of the cross linked chitosan with polyurethane.The cross linked chitosan was mixed with methylene diphenyl dissocyanate so that the later enter the spaces of the cross-linked chitosan.Polyester polyol was then added to the mixture in order to allow the reaction between the methylene diphenyl dissocyanate and the polyester polyol.The formation of foam and the emission of Co 2 are indications for the occurrence of the reaction.In order to obtain the best results concerning flexibility and homogeno of the prepared IPN, the proportions of chitosan and polyurethane were changed as shown in Table (1).The 70% of chitosan was found to be the most suitable and was used in the experiments.

Physical and chemical evaluation of IPN:
Diagnosis of chitin and chitosan and the IPN were done by the infrared technique (FTIR 84005, Shimadzu Co.) * .The technique includes mixing a quantity of the specimen with potassium bromide (KBr).Following good mixing, the mixture was encysted under the pressure of 15 tons, to obtain a thin disc, which later subjected to examination and measurements.
The tensile strength of the IPN was measured by an Instron apparatus type 1193 * .The specimen which is made into dumbbell shape is placed between the two levers of the apparatus and pulled at a speed of 5mm/second till the specimen is cut, and the tensile strength was read at this point.

Toxicological evaluation of the IPN:
Fifteen adult male rabbits of a local breed and weighing 1.5-2 kg were used in this experiment.Following acclimatization, the rabbits were allocated into 3 equal groups, and the gluteal region of each rabbit was prepared routinely for surgery.Each rabbit was premeditated with 10mg/kg acepromazine maleate ** intramuscularly to induce tranquilization.After 10 minutes, the animal was given intramuscularly a mixture of ketamine hydrochloride *** (35 mg/kg) and xylazine **** (5 mg/kg), (27).The IPN was disinfected with ultraviolet radiation at a wave length of 250-280 nm and for 15 minutes.A full thickness skin piece (3×4cm) was removed, and the IPN was placed in the defect and fixed from the margins and center of the wound using a 3 0 absorbable suture material (28)(29).The animals were then euthanatized at the rate of 5 rabbits at each of the 24, 48, and 72 hrs post-surgery.Tissue specimens were collected from the liver, kidneys, and intestines, and fixed in 10% formalin for 48 hrs, trimmed to suitable sizes, washed, dehydrated, cleared in xylol, embedded in paraffin wax, sectioned at 5-6 μm thickness, stained with hematoxylin and eosin, and examined with a light microscope (30).

Application of the network for treatment of clean cutaneous
wounds in sheep: Twenty four adult Awasi sheep of both sexes were used in this study.The gluteal region of each rabbit was prepared for aseptic surgery.Each animal was given a mixture of ketamine HCL (4 mg/kg) and xylazine (0.05 mg/kg), as well as diazepam (2 mg/kg) intramuscularly.Following induction of anesthesia, a full thickness skin piece (5× 6 cm) was excised and replaced by the IPN which was fixed in situ by an 3 0 absorbable suture material.Biopsy specimens were collected from the wounds at 7, 15, 30, 60, 90 and 120 days post operation.The tissue specimens were fixed in 10% formalin for 48 hrs, processed, and examined as mentioned previously.
Blood samples were collected from all sheep and preserved in plastic test tubes devoid of anticoagulant and left in oblique position at room temperature.The blood serum was separated by centrifuging the tubes at 3000 rpm for 15 minutes, and the serum was preserved at -20◌ ْ C. till analysis and measurements of the enzymes which included AST (aspartate aminotransferase), ALT ( alanine aminotransferase), alkaline phosphatase (ALP), and acid phosphatase (ACP).A Biomaghreb test kit was used to measure the enzymes following the Cecil colorimetric method (French made apparatus) at a wavelength of 490-520 nm (Clinical Pathology Unit, the Gurna General Hospital).

Application of the network for treatment of contaminated wounds
in sheep: Twenty four adult Awasi sheep of both sexes were used in this study.Full thickness skin piece (5× 6 cm) was excised aseptically from the gluteal region of each rabbit and the wounds were left open for 24 hrs.The defect was then replaced by the IPN as described in the previous group.Collection of biopsies, fixation, processing, and examination were done as described in the previous group.Similarly, AST, AIT, ALT, and ACP were determined as described in the first group.8. Statistical Analysis: Results of mechanical properties and measurements of enzymes were analyzed statistically using a one-way analysis of variance.When a statistically significant difference (P < 0.05) was obtained, Tukey-HSD test was then performed (31).

RESULTS
Results of infrared analysis of the chitosan and the network prepared from it are shown in figures 1 sand 2, respectively.From data presented in these figures, the sites of frequencies of the functional groups were ascertained and are presented in table (2).The frequency of absorption of the NH 2 group appeared as two bands in the range of 3300-3400 cm -1 (Fig. 1).In figure 2, the NH 2 group disappeared and replaced by OH group at the site 3400 cm -1 .Similar absorption bands of CH2, C-O-C, and C-C groups are evident in figures 1 and 2. The frequency of absorption of urethane group occurred at the sites of 1300 and H 1700 cm -1 , (Fig. 2).This frequency is not seen in figure 1 and this means the occurrence of a reaction between the chitosan and the polyurethane.Table (3), presents results of mechanical evaluation of the IPN (Cross-linked chitosan-polyurethane) as compared to cross-linked chitosan alone.From these data it became obvious that the IPN is more flexible and withstand more stress than the cross-linked chitosan alone.Furthermore, the possible force inflicted on the surface area of the cross section of the polymeric network depends on strength, flexibility, strain, and elasticity as shown in figure 3 which illustrates the types of stress-strain curve for some elastic polymers.Gross and microscopic pathological lesions were not encountered in the viscera of rabbit in which the IPN were implanted for 24, 48, and 72 hrs.Table (4), presents the serum values of ALT, AST, ALP, and ACP in animals with clean wounds and animals with contaminated wounds.Statistical analysis of these values have indicated that the levels of SGOT, ALP, and ACP in the first group of sheep were 11.04 ± 0.54, 30.83 ± 1.20, 36.37 ± 1.52, and 5.52 ± 0.37, respectively.In the second group of sheep, these values were 11.29 ± 0.30, 38.58 ± 1.16, 38.20 ± 1.21, and 6.31 ± 0.46, respectively.Statistically significant differences were not encountered between the ALT, ALP, and ACP values of sheep with clean wounds and those with contaminated wounds.However, the AST level was higher significantly (P<0.01) in sheep of group 2 ( with contaminated wounds) than in sheep of the first group (with clean wounds).Histological examination of the clean wounds that have been induced in sheep of group (1) revealed increased thickness of the epidermis close to the wound line.The wound defect was filled with a dense mature granulation tissue that consisted of large numbers of fibroblasts and bundles of collagen fibers (Fig. 4).Although the fibroblasts were haphazardly arranged in the wound defect, the majority of them were horizontally arranged in relation to the wound line.Similarly, the collagen fibers were arranged as bundles horizontally oriented relative to the wound line.Few newly-formed blood vessels were seen in the granulation tissue.Large numbers of IPN crystals were also seen in the granulation tissue (Fig. 4).These crystals were surrounded by numerous mononuclear cells (lymphocytes, plasma cells, and macrophages) and foreignbody giant cells (Fig. 5).At the 15 th postoperative day, the histology of the wounds was similar to that seen in the 7 days old wounds.Additionally, a large number of mononuclear cells and foreign-body giant cells were seen around the IPN crystals (Fig. 6).At this period, many of the IPN crystals disappeared from the wound tissue.At the 30 th , 60 th , and 90 th postoperative days, a gradual decrease of the IPN crystals was seen (Fig. 7).At 120 days following infliction of the wounds, it was difficult to recognize the wound line and the IPN crystals disappeared completely.
In contaminated wounds at the 7 th postoperative day, the wound defect was filled with mature granulation tissue that was arranged horizontally relative to the wound line (Fig. 8).Few IPN crystals surrounded by large numbers of erythrocytes were seen in the granulation tissue.At the 15 th postoperative day, large numbers of mononuclear cells and foreign-body giant cells appeared in the granulation tissue particularly around the IPN crystals (Fig. 9).Many of these crystals were degraded into small fragments.At the 30 th postoperative day, the wound defect was filled with mature granulation tissue that contained mononuclear and foreign-body giant cells arranged around IPN crystals (Fig. 10).The IPN crystals were not seen at the 60 th postoperative day.At the 90 th postoperative day, the site of the wound has healed completely and the tissue regained its normal shape and structure.At 120 days following infliction of the wounds, the site of the wound was normal histologically.

DISCUSSION
In the present study, the aims of adding polyurethane to the cross-linked chitosan were to give the chitosan network flexibility and to increase its tensile strength and porosity through the liberation of Co 2 .Infrared examination of the IPN was done to ascertain the mechanical properties of the network flexibility, homogenicity, and status of the polymer.This technique has been also used by others and for the same purpose (2,17).Results of infrared examination of the IPN showed that this network was better than the cross-linked chitosan alone.Chitosan is a biodegradable compound and biodegradation is accomplished through hydrolysis with lipase and chitosanase (32).The 70% of chitosan used in the IPN in this study was chosen due to the good results it gave in wound healing as compared to other concentrations.This finding is in accordance with that of others (33) who also found that using 50% chitosan has lead to disruption of the network and its withdrawal to one of the sides of the wound.
Results of the toxicological pathology in rabbits have shown that the IPN did not have any deleterious effects on the tissues (liver, kidneys, and intestines).This indicates that the IPN was biocompatible.This finding was in agreement with that of other workers (2,24,34,35) and did not differ from that of others who used high dose of chitosan orally for the treatment of inflammations of the urinary tract (15).
As far as measurements of AST, ALT, ALP, and ACP are concerned, significant differences were not encountered between the values of ALT, ALP, and ACP in sheep with clean wounds and in sheep with contaminated wounds.This finding indicates that the IPN did not have any deleterious effect on the liver, kidneys, and the skeletal muscles of sheep with clean wounds and in those with contaminated wounds.However, the values of AST were higher significantly (P<0.01) in sheep with infected wounds than in sheep with contaminated wounds.This difference could be due to the fact that AST has the function of transferring the α amine group from aspartic acid to ketonic acid and that the amine groups of the chitosan could be intermingled with those present in aspartic acid.It could be also due to the influence of wound pH since contaminated wounds contain higher quantity of tissue debris than the clean wounds.These findings and postulations were in accordance with those of others (16,36,37,38).
Results of histological examination of the healing clean wounds that were implanted with IPN in sheep were similar to those reported in the literature (15)(16)(39)(40)(41)(42)(43)(44).However, an additional complication was the presence of the IPN crystals in the healing wounds.These crystals have elicited a mononuclear cell reaction and the encircling of the crystals with foreign-body giant cells.This means that the IPN crystals were inert but still capable of eliciting a foreign -body granulomatous reaction.In clean wounds, the IPN crystals disappeared completely from the healing wounds after the 60 th postoperative day.Healing of contaminated wounds occurred in a similar way and at the same rate as that of clean wounds but the IPN crystals disappeared at an earlier period (before the 60 th postoperative day).Furthermore, bacterial infection was not encountered in the contaminated wounds.This finding could be explained on the basis of results of previous research findings that chitosan has antimicrobial actions (7,45,46).It has been found that chitin-chitosan inhibits in vitro growth of microorganisms including Candida and in vivo has a protective effect on Candida infection (45).When chitosan was dissolved in saline, distilled water, or laboratory media, it exhibits antimicrobial activity against some strains of filamentous fungi (47,48), yeasts (47,49), and bacteria (50).The mode of chitosan antimicrobial action is still obscure (7).That the IPN crystals disappeared earlier from contaminated wounds than from clean wounds could be attributed to the ability of many types of bacteria to produce chitosan-degrading enzymes (chitosanase and lysozyme) (7).Additionally, it has been found that the rate of disappearance of the crystals of chitosan depends on the degree of polymerization, mechanical properties, molecular weight, and structural chemistry of the implant as well as temperature and pH of the medium in which the implant is present (51).
In the present study, the healing of the clean and contaminated open wounds in sheep proceeded with a good rate that coincided with that of clean open wounds (without implant).This finding is in agreement with that of others who found that chitosan has enhancing effects on wound healing (2,6,13,15,18,37,38,52).These effects could be related to the biocompatibility and biodegradability of the chitosan implant, the hemostatic and antimicrobial actions of chitosan, as well as the enhancement of collagen production and stimulation of fibroblasts by chitosan.
From results of the present study it could be concluded that (1) a crosslinked chitosan-polyurethane mesh was successfully prepared and proved useful in supporting existing tissue or in replacing lost tissue; (2) a 70% chitosan was the most suitable addition to the network to improve its biodegradability and tensile strength; (3) the IPN was non-toxic and could be used safely in animals; (4) chitosan has a beneficial effects on the process of wound healing of both clean and contaminated open wounds; and (5) chitosan has an antibacterial effect in healing wounds.

Figure 1 :
Figure 1: Infrared analysis of the cross-linked chitosan.The frequency of absorption of NH 2 group appeared as two bands in the range of 3300-3400 cm -1 .

Figure 2 :
Figure 2: Infrared analysis of the IPN.Note that the NH 2 group disappeared and replaced by OH group at the site 3400 cm -1 .

Figure 4 :
Figure 4: Cross section of a clean open skin wound in sheep at the 7 th postoperative day.The wound defect is filled with a mature granulation tissue.Note the presence of fragments of the IPN in the granulation tissue (arrows).H & E. 40X.

Figure 5 :
Figure 5: Cross section of a clean open skin wound in sheep at the 7 th postoperative day.Crystals of the IPN are surrounded by foreign-body giant cells (G).H & E. 160X.

Figure 6 :
Figure 6: Cross section of a clean open skin wound in sheep at the 15 th postoperative day.Mononuclear and foreign-body giant cells could be seen around remnants of the IPN.H & E. 160X.

Figure 7 :
Figure 7: Cross section of a clean open skin wound in sheep at the 15 th postoperative day.Many of the crystals of the IPN have disappeared and the rest are degraded and surrounded by foreign-body giant cells.H & E. 160X.

Figure 8 :
Figure 8: Cross section of a contaminated open skin wound in sheep at the 7 th postoperative day.Note a dense mature granulation tissue is filling the wound defect.H & E. 40X.

Figure 9 :
Figure 9: Cross section of a contaminated open skin wound in sheep at the 15 th postoperative day.Note the accumulation of mononuclear cells in the granulation tissue and the present of some giant cells around the IPN crystals.H & E. 160X.

Figure 10 :
Figure 10: Cross section of a contaminated open skin wound in sheep at the 90 th postoperative day.Note the presence of mature granulation tissue infiltrated by mononuclear cells by few giant cells particularly around IPN crystal.H & E. 160X.

Table 1 .
Percentages of mixing of chitosan and polyurethane.

Table 2 .
Sites of frequencies and bonds and the functional groups of the chitosan and the IPN.

Table 3 .
Results of examination of the tensile strength of the IPN and the chitosan alone.
Figure 3: The types of stress-strain curves for some elastic polymers.Table4:The levels of enzymes in sheep with clean wounds (group 1) and sheep with contaminated wounds (group 2).* * SGOT was higher significantly (P<0.01) in the second group than in the first group.No significant differences were encountered in the rest of the parameters.