A method for systematically evaluating the hemostatic ability of hydrogels in vitro

Abstract

The bursting strength is a key parameter to assess hemostatic ability of tissue sealants. It is associated to mechanical property of the materials, the binding strength of the materials to the tissues as well as the applied conditions of the materials, such as temperature and wound size. Few works have systematically investigated the relationship of the hemostatic ability of hydrogels with the factors listed above. This study introduced a method to systematically investigate the effect of the thickness, covered area and components of hydrogels, and the applied conditions on the bursting strength of hydrogels. The gelatin hydrogel and fibrin glue were used in this study. The method quantitatively investigated the effect of material properties and applied conditions on the bursting strength of materials. It also suggested a minimum dosage of tissue sealant used in both animal study and clinical practice. This study proved that the method we proposed is reliable to assess the bursting strength of materials for the hemostatic application.

Keywords

Hemostatic ability fibrin glue evaluating method in vitro

1. Introduction

Tissue sealants have attracted rapidly growing interest and been used for hemostasis, stopping serous fluid and air leaks [1,2]. In the latest study, three hemostasis mechanisms have been proposed: (1) quickly forming clots by activating the platelets and coagulation factors; (2) absorbing water from blood to concentrate the coagulation factors as well as the platelets at the bleeding sites; (3) adhering to tissue strongly to block the bleeding. Recently, local hemostatic hydrogels have been applied at surgical wounds in order to arrest bleeding from suture holes and cross-sectional surfaces of parenchymatous organs in case wherein the use of cautery, ligature or other conventional hemostatic methods is impractical [3].

Enzyme-mediated in situ cross-linking hydrogels have received attention in tissue engineering because of their tunable mechanical property, rapid gelation time and low toxicity, and the mild cross-linking conditions [4,5]. Specially, fibrin glue is one of the earliest surgical glues used in medical applications [6]. And it is made with a number of components from plasma that enables the adhesive to mimic the final stages of blood clotting. Typically, fibrin glue can take effect in relatively short time, form covalent connections with surrounding tissues via the amidation reaction, and also function as hemostat [7]. However, fibrin glue presents relatively weak tensile and adhesion strengths, especially in wet environments or wounds with abundant amount of body fluids [8].

The bursting strength is a key parameter to assess hemostatic ability of tissue sealants. However, the burst strength of the hemostatic agents was assessed mostly by non-standard devices and not systematically studied [9–12]. It could be also vary with testing conditions such as temperature and wound size. Few works have been done for assessing tissue sealants with these factors. Therefore, it is important to establish an assessment method for the measurement of bursting strength. A method was developed to evaluate the bursting strength of hydrogels in this study. The factors which influence the bursting strength of tissue sealants were studied systematically with this method in vitro. The properties of hydrogel, such as component and cross-linking degree, have been studied and proven to have an impact on the bursting strength [9,13]. Besides the nature of hydrogel, the size of wound on tissue surface was used [11,14,15]. However, the different wound size was not unified and the effect of serial wound size on the hemostatic result was not investigated. And the area of hydrogel applied to wound site was not studied. Furthermore, to our knowledge, very few publications systematically investigated all of the factors mentioned above.

In this study, an effective approach to measure bursting strength of the hemostatic materials in vitro was developed. It was used to investigated the effects of thickness, covered area and concentration of hydrogels and wound size on the bursting strength. The method would be effective to evaluate the biomaterials which could be used as tissue sealant in vivo.

2. Materials and methods

2.1. Materials

Gelatin (from porcine skin, type A) was brought from Kuaikang Co. Ltd, Guangdong, China. Fibrin glue was purchased from Shanghai RAAS Blood Products Co. Ltd, Shanghai, China. Canine small intestines were harvested from adult beagle dogs, which were used in other study.

2.2. Measurement of gelation time

The gelation time was determined using the vial-tilting method [16]. The gelatin solutions was taken out from 60°. At the very first moment to invert the vial, there was no flow observed. The duration was regarded as the gelation time ( $n = 5$ ).

2.3. Bursting strength test

The bursting strength was tested by a custom-made air-pressure testing apparatus (Fig. 1A), which includes an inflatable ball, a pressure gauge and a suction flask and rubber pipes. One end canine small intestine segment was connected with the inlet of rubber pipe, the other end was clamped with hemostatic forceps (Fig. 1B). A leak check was performed at 5 kPa before the bursting test to confirm the system was leak-proof. In the course of bursting test (Fig. 2A), a pore was created on the intestine wall with a syringe needle (step 1); a teflon ring was placed on the intestine, the punctured pore was in the center of the ring (step 2); then a certain volume of hydrogel solution was injected into the ring (step 3) and gelation occurred within minutes (step 4); after solution gelled, the teflon ring was removed (step 5). This system was gradually inflated until the hydrogel bursted. The pressure regarded as the bursting strength of hydrogel was recorded. The canine small intestine was punctured with a 16G, 12G or 8G syringe needle corresponding to the injury diameter of 11.9, 2.16 and 3.43 mm (Fig. 2B), then the gelatin hydrogels were fabricated with varied depth (1, 2, 3, 4 and 5 mm) (Fig. 2C), cover areas (20, 64, 132 mm²) corresponding to their inner diameter of 5, 9 and 13 mm (Fig. 2D), and gelatin concentrations (5%, 10%, 20%, 30% and 40%) (Fig. 2E).

Fig. 1.

Diagrammatic representation of the air-pressure type apparatus (A) and the hydrogel formed on the wound site (B).

Fig. 2.

The steps of test method (A) and photographs of varied diameters of syringe needles (B), thicknesses of hydrogel (C), diameters of teflon rings (D) and concentrations of hydrogels (E).

2.4. Statistical analysis

Data in the study were presented as mean ± standard deviation. The bursting strengths were analyzed using a one-way analysis of variance (ANOVA). Student’s t-test was performed as the post-hoc test to examine the statistical difference between groups. $p < 0.05$ was regarded as significance in statistics.

3. Results

3.1. Gelation time of gelatin hydrogel

The gelation time of gelatin hydrogels at 4, 25 or 37°C were investigated (Table 1). Gelatin hydrogel could not form at 37°C. But at 25°C, the solutions were able to form gel. The gelation time reduced from $90.2 \pm 2.06$ min to $17.3 \pm 2.08$ min with the increase of the gelatin concentration from 5% to 40% (w/v). Furthermore, the gelation time of the gelatin hydrogels was shortened drastically within 10 min at 4°C. It took only $3.3 \pm 0.76$ min to form gel for 40% gelatin solution at 4°C.

Table 1
The gelation time of gelatin hydrogels at 4, 25 and 37°C

Concentration of Gelatin (w/v) 4°C/(min) 25°C/(min) 37°C/(min)

5% 8.9 ± 1.08 90.2 ± 2.06 No gelation

10% 7.5 ± 0.50 59.0 ± 2.65

20% 6.0 ± 0.61 38.0 ± 1.63

30% 5.1 ± 0.65 25.7 ± 1.53

40% 3.3 ± 0.76 17.3 ± 2.08

Concentration of Gelatin (w/v)	4°C/(min)	25°C/(min)	37°C/(min)
5%	8.9 ± 1.08	90.2 ± 2.06	No gelation
10%	7.5 ± 0.50	59.0 ± 2.65
20%	6.0 ± 0.61	38.0 ± 1.63
30%	5.1 ± 0.65	25.7 ± 1.53
40%	3.3 ± 0.76	17.3 ± 2.08

3.2. Bursting strength test

The leak-proof of the system was confirmed as the pressure was stable at 5 kPa for 300 s (Fig. 3).

Fig. 3.

The air-tight test of the air-pressure type apparatus.

The effects of hydrogel thickness, covered area, gelatin concentration and the size of pore on the bursting strength of hydrogels were investigated in this study (Table 2). The bursting strength increased from $16.06 \pm 1.07$ kPa to $30.61 \pm 4.22$ kPa with the increase of thickness of gelatin hydrogels from 1 mm to 5 mm. Significant improvement of burst strength was achieved from 1 and 2 mm to 3 and 4 mm, and from 3 mm to 4 mm of the thickness (Fig. 4). The bursting strength of hydrogels was found to be significantly improved with the increase of the covered area from 20 to 64 and 132 mm² (Fig. 5).

Table 2

Conditions for the bursting strength test

Variable factor	Fixed factors
Hydrogel thickness	12G syringe needle, hydrogels with 20% gelatin, and teflon ring of 5 mm in diameter at 25°C
Diameter of teflon ring	12G syringe needle, and 1-mm-thick hydrogels with 20% gelatin at 25°C
Gelatin concentration	12G syringe needle, 1-mm-thick hydrogels, and teflon ring of 5 mm in diameter at 4°C
Diameter of syringe needle	1-mm-thick hydrogels with 20% gelatin, and teflon ring of 5 mm in diameter at 25°C

Fig. 4.

The bursting strengths of hydrogels (20% gelatin) with varied thicknesses at 25°C ( $^{*} p < 0.05$ ).

Fig. 5.

The bursting strengths of hydrogels (20% gelatin) with varied diameters of teflon circle at 25°C ( $^{*} p < 0.05$ ).

The effect of gelatin concentration on bursting strength was investigated at 4°C (Fig. 6). The bursting strength was significantly influenced by the gelatin concentration. The bursting strength has reached $34.20 \pm 1.98$ kPa for the hydrogel of 40% (w/v) gelatin, and decreased to $3.20 \pm 1.27$ kPa for that of 5% (w/v) gelatin. Moreover, the pore created by syringe needles with diameters of 1.19, 2.16 and 3.43 mm resulted in the burst of hydrogels at $24.23 \pm 2.67$ , $16.06 \pm 1.07$ and $4.83 \pm 0.06$ kPa, respectively (Fig. 7). It demonstrated that an increasing size of wound corresponded to a decreasing bursting strengths of hydrogels.

Fig. 6.

The bursting strengths of hydrogels with varied gelatin concentrations at 4°C ( $^{*} p < 0.05$ ).

Fig. 7.

The bursting strengths of hydrogel (20% gelatin) treated the wounds with varied sizes at 25°C ( $^{*} p < 0.05$ ).

Fibrin glues were investigated at the same experimental conditions as described above. The bursting strengths of fibrin glues were less than 3 kPa, which were much lower than the corresponding values of 20% gelatin. There were significant improvement with the decrease of the diameters of pore. It indicated that the size of wound obviously affect the hemostasis of fibrin glues (Table 3).

Table 3

The bursting strengths of fibrin glues in various conditions at 25°C ( $^{*} p < 0.05$ )

4. Discussion

The purpose of this study was to establish a facile and reliable method to evaluate the hemostatic ability of hydrogels. The results of bursting test indicated that the increase of wound size would lead a significantly decrease of bursting strength. It was consistent with the finding in clinic that hemostasis would be more difficult for a larger wound. The increase of thickness, covered area and concentration of hydrogel resulted in the increase of the bursting strength. Hydrogels with thickness of 1 or 2 mm were prone to failure with lower bursting strengths. The greater bursting strength could be achieved with the increase of thickness of the hydrogels to 3 mm. Significant improvement of bursting strength was observed from thicknesses of 1 and 2 mm were increased to those of 3 and 4 mm. However, the increase of thickness to 5 mm did not significantly improve the bursting strength of hydrogels. It implied that the thickness of 3 mm was good enough to achieve hemostasis for the small wound on small intestine. Similar trends were found in the bursting test with varied covered area of hydrogels and gelatin concentration (Fig. 6). It indicated the method could be used to screen biomaterials for the desirable tissue sealants in vitro. Based on this method, for a certain wound size, the results could recommend a minimum thickness and covered area of hydrogel to resist blood pressure.

It has been reported that fibrin glues could successfully stop bleeding in animal models [3], but the bursting strengths differed considerably in the studies. This may result from varied thickness, covered area, components of fibrin glue hydrogel used in the experiments. In this study, the bursting strength of fibrin glue with varied thickness, covered area as well as the wounds with different sizes were assessed. The highest bursting strength of fibrin glue was $2.80 \pm 1.67 kPa$ , which was remarkably lower than that of 20% gelatin hydrogel ( $> 25 kPa$ ). Moreover, the 1-mm-thick fibrin glue with a covered area of 20 mm² was unable to seal the pore on the wall of small intestine. It indicated the sufficient thickness and covered area must be reached for fibrin glue to serve as tissue sealant in clinic. Meanwhile, the better products are necessary to be developed for the clinical practice.

5. Conclusions

We proposed a method to evaluate the effect of material properties and applied conditions on the bursting strength. The results from gelatin hydrogels and fibrin glue confirmed that this method was reliable. This work demonstrated that the method may facilitate the development of hemostatic products.

Footnotes

Acknowledgements

This work was funded by “Hundred Talents Program” of Chinese Academy of Sciences, Shenzhen Research Programs (GJHZ20170314154914747 and GJHS20160331171605415) and Open Project of Guangxi Key Laboratory of Regenerative Medicine (201701).

Conflict of interest

The authors have no any conflict of interest to report.

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A method for systematically evaluating the hemostatic ability of hydrogels in vitro

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Measurement of gelation time

2.3. Bursting strength test

3. Results

3.1. Gelation time of gelatin hydrogel

Table 1 The gelation time of gelatin hydrogels at 4, 25 and 37°C Concentration of Gelatin (w/v) 4°C/(min) 25°C/(min) 37°C/(min) 5% 8.9 ± 1.08 90.2 ± 2.06 No gelation 10% 7.5 ± 0.50 59.0 ± 2.65 20% 6.0 ± 0.61 38.0 ± 1.63 30% 5.1 ± 0.65 25.7 ± 1.53 40% 3.3 ± 0.76 17.3 ± 2.08

5. Conclusions

Footnotes

Acknowledgements

Conflict of interest

References

Table 1
The gelation time of gelatin hydrogels at 4, 25 and 37°C

Concentration of Gelatin (w/v) 4°C/(min) 25°C/(min) 37°C/(min)

5% 8.9 ± 1.08 90.2 ± 2.06 No gelation

10% 7.5 ± 0.50 59.0 ± 2.65

20% 6.0 ± 0.61 38.0 ± 1.63

30% 5.1 ± 0.65 25.7 ± 1.53

40% 3.3 ± 0.76 17.3 ± 2.08