Efficacy of Chitosan-Based Dressing for Control of Bleeding in Excisional Wounds

Determining timing and dosage

The initial step was to determine the suitable dosage of heparin to produce significant bleeding from excisional wounds. Heparin was administered according to a previously published protocol.¹¹ Two 12-mm circular full-thickness wounds were made on the dorsal dermis of 5 rats. Five dosing regimens were compared. The regimens consisted of 400 IU/kg of heparin administered 1 hour prior to surgical dermal excision, 400 IU/kg of heparin administered 2 hours prior to surgical dermal excision, 800 IU/kg of heparin administered 1 hour prior to surgical dermal excision, 800 IU/kg of heparin administered 2 hours prior to surgical dermal excision, and, finally, no heparin administered. Gauze dressings were applied. The dressings were weighed before and after application (Fig 1). Blood loss was quantified by the difference in weight before and after blood cessation. The 800 IU/kg dosage at 2 hours was chosen, as it produced significant bleeding. In addition, this highest dosage was considered to be an appropriate challenge for the chitosan dressing.

Figure 1. Comparison of blood loss of 800 IU/kg of heparin versus 400 IU/kg of heparin versus no heparin.

For the comparison of chitosan dressing and the woven gauze dressing, all animals were administered 800 IU/kg of heparin via intraperitoneal route 2 hours prior to surgical dermal excision. Animals were housed in single cages. Six paired, 12-mm circular, full-thickness cutaneous wounds, 3 wounds on either side, were made on the dorsal dermis of each rat (Figs 2 and 3).

Figure 2. Visual representation of full-thickness wounds on rat model for part one 1 and part 2.

Figure 3. Photographic examples showing comparison of blood loss of gauze versus Opticell.

Dynamic assessment of bleeding (part 1)

For this part, 4 rats were used. Chitosan dressing was applied on one side (3 wounds) and gauze on the other (3 wounds). Gauze and the chitosan dressing were removed and instantly replaced using consistent, brief, 3-second gentle pressure every minute until bleeding cessation. The pressure applied over skin was the same for both sides. The dressings were weighed before and after application. The increase in weight was quantified as blood loss per minute (mg/min).

Total bleeding evaluation (part 2)

Two rats were used in this part. Six paired, 12-mm circular, full-thickness wounds, 3 wounds on either side, were made on the dorsal dermis of each rat. Chitosan dressing was applied on one side and woven gauze on the other. They were left in place until cessation of bleeding. Complete bleeding cessation occurred after 10 minutes in both groups. Gauze and chitosan dressing were weighed before and after bleeding cessation, with the difference representing blood loss. Blood loss was expressed in mg/10 min.

Determination of heparin dosing

As shown in Figure 1, the animal administered 800 IU/kg of heparin 2 hours prior to excisional wound bled 300 mg/10 min. Bleeding seen with the other dosages were as follows: 800 IU/kg administration for 1 hour was 56 mg/10 min, 400 IU/kg administration for 2 hours was 47 mg/10 min, and 400 IU/kg administration for 1 hour was 157 mg/10 min. These results indicate that 800 IU/kg of heparin 2 hours prior to excisional wound stimulates a sufficient amount of blood loss to challenge the hemostatic capability of chitosan dressing versus standard gauze.

Dynamic assessment of bleeding

All animals for part 1 and part 2 were housed in single cages for 2 hours after administration of heparin. All six 12-mm circular full-thickness wounds were created after the 2-hour period. The dressings were weighed before and after in contact with the bleeding wound. Blood loss was assessed immediately after every minute until bleeding cessation. The average blood loss in the chitosan dressing group, in the consecutive span of 10 minutes, was 91, 54, 58, 88, 27, 20, 11, 25, 17, and 27 mg. The average blood loss in the gauze group was 183, 140, 110, 97, 85, 57, 60, 80, 49, and 103 mg (Fig 4). On the basis of the average blood loss values, analysis of variance with the Tukey posttest was used to evaluate differences in mean blood loss for significance. Significant differences in blood loss were seen with the chitosan dressing at minutes 1, 2 and 3 (^***P = .002, ^**P = .004, ^*P = .045) (Fig 5). The results from part 1 indicate that the efficacy of chitosan-based dressing for bleeding cessation was superior to the standard gauze.

Figure 4. Average blood loss after 2 hours of heparin administration in 4 male retired breeder rats.

Figure 5. Significant differences between Opticell and gauze (***P = .002, **P = .004, *P = .045).

Total bleeding evaluation

Complete bleeding cessation in both groups was noted after 10 minutes. The dressings were weighed before and after application. The difference between the weights before and after application represents blood loss. The results for part 2 showed that postsurgery, the total blood loss was 247 ± 47 mg/10 min in the chitosan dressing group versus 627 ± 47 mg/10 min in the gauze group. The lower blood loss over 10 minutes indicates more effective hemostatic properties of the chitosan dressing compared with the standard woven gauze (Fig 6).

Figure 6. Difference between Opticell and gauze (*P = .002).

Mode of action of heparin

Because of prolonged bleeding time in patients using anticoagulants, even a small trivial wound could be daily concern. Heparin is a classic anticoagulant that inhibits the coagulation pathway wherein prothrombin is converted to thrombin, leading to the production of fibrin from fibrinogen. Fibrin then forms the blood clot. The drug's anticoagulation activity is due to its binding to a protease inhibitor, antithrombin II (ATII) (a pentasaccharide sequence). Heparin induces a conformational change to the binding site of ATII.^13,14 The conformational change allows for the reactive site to change, enabling the clotting enzyme to bind to ATII. Thus, the clotting enzyme is prevented from adhering to the surface of the wound, inhibiting coagulation of red blood cells.^13,14 Heparin and the fractionated heparin (enoxaparin) must be given by injection. New orally active anticoagulants have recently been developed to interfere with the clotting system by binding directly or indirectly to Factor Xa (dabigatran, rivaroxaban, apixaban, and edoxaban) and blocking the conversion of prothrombin to thrombin.

These new products are easier to manage for patients because they can be taken orally and because their predictable effects do not require blood test monitoring of the original drugs. The fact that heparin can be easily injected intraperitoneally makes it preferable in the rat model to an oral medication. The anticoagulation these new oral drugs produce is similar to that produced by heparin and therefore the heparin model remains suitable for this assessment.

The initial step before proceeding to this study was to confirm that standard rodent dosing of heparin at 800 U/kg intraperitoneally would provide suitable anticoagulation in our rat wounding model.^11,12Figure 1 displays results of the preliminary experiment, indicating that heparin does indeed prevent the coagulation of blood to a suitable degree in this model.

Mode of action of chitosan

Patients taking anticoagulants have prolonged blood coagulation that results in impairment of the healing process. Our quantified results demonstrate the effectiveness of the chitosan-based dressing immediately upon application (significant during the first 3 minutes).

The mechanism of action of chitosan is still elusive. On the basis of available data, a plausible explanation is that the negatively charged red blood cell membranes allow the protonated amine groups on the chitosan to attract these red blood cells, resulting in hemagglutination.^15,16 A previous study has shown that chitosan encourages platelet adhesion and activation via protein adsorption and orientation, which, in turn, affect cellular responses to proteins.¹⁷ Chitosan's ability to enhance platelet aggregation by adsorbing plasma proteins and fibrinogen can be an important feature of its action.⁴ Chitosan subsequently leads to more platelet aggregation because it enhances the expression of glycoprotein IIb/IIIa (GPIIb/IIIa), a glycoprotein.⁴ Chitosan signals for thrombin, clotting promoter, to form, which then stimulates for the activation of GPIIb/IIa.¹⁸ Fibrinogen and GPIIb/IIIa bind to the platelet and activate it; inhibiting the GPIIb/IIIa receptor prevents the binding of fibrinogen to the platelets.¹⁸ Chitosan-based dressing (Opticell) has an efficient mechanism for controlling bleeding. However, the true mechanism of action of chitosan is yet to be fully understood.

In summary, the results from the bleeding timeline indicate that the chitosan-based dressing is superior to standard gauze dressing for control of bleeding from excisional wounds in heparinized rats. Bleeding was reduced immediately in the first 3 minutes after application of the chitosan dressing; the total bleeding group showed a greater than 50% reduction in blood loss in the chitosan-based dressing group compared with the standard gauze group. Chitosan dressing has hemostatic effects that could be a useful tool to control bleeding associated with wound debridement in patients with a bleeding diathesis.