Preparation and characterization of absorbable hemostat crosslinked gelatin sponges for surgical applications

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Abstract

The aim of this study, is synthesis of an absorbable hemostat gelatin sponge crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). The effects of various gelatin contents and crosslinking agent concentrations, freezing temperature on the mechanical properties, rate of biodegradation in 24 h, hemolytic ability and cell viability of sponges were investigated. The results of scanning electron microscopy (SEM) showed that freezing temperature had a significant effect on the structure and density of sponges. SEM views of the sponges demonstrated that they have pores with mean diameters of 180–280 μm. Fourier transform infrared (FTIR) spectra indicated that there is no residual of the crosslinking agent in the structure of final gelatin sponges because of C–N bonds (urea derivatives) absence. The sample with 1% gelatin and 0.05% EDC freezed at −25 °C (before freeze-drying) showed more than 0.37 cm−1 UV absorption of hemoglobin or blood clotting in half an hour. The compression modulus of the same sample was more than 150 kPa and percentage of degradation was about 28% in 24 h.

Introduction

The molecular and cellular events for platelet and coagulation system activation that occur when blood contacts foreign materials is becoming increasingly well understood [1]. Bioactive hemostats and wound dressings consist of either inherently active materials or act as delivery vehicles which contain such materials [2]. There is a need for an absorbable hemostatic agent that can be successfully used in deep splenic and hepatic injuries. Such an agent must be safe, rapidly effective and have good systemic and local compatibility [3]. Interactions of biomaterials with blood should not damage blood cells or change protein activity. Polymers like gelatin and sodium alginate are biocompatible, biodegradable and bioresorbable [4].

Hemostasis and blood clot formation are primary concern in the surgery. In even simple extractions, rapid clotting helps to prevent infection and initiates the process of tissue repair and healing. Absorbable materials which aid in hemostasis and can be used to accelerate healing have advantages in surgery [5].

Proper handling is essential to control bleeding and only the required amount should be used, even though the hemostat is expected to dissolve promptly [6]. A dry local hemostat absorbs several times of its weight and expands postoperatively. Therefore, when an absorbable hemostatic agent is retained on or near bony or neural spaces, the minimum amount should be left after hemostasis is achieved. Minimum inflammation without strong foreign body reactions or blockade of healing is desirable after the use of local hemostats. Local hemostat can be used to arrest suture hole bleeding and bleeding from the cross-sectional surface of parenchymatous organs such as liver and spleen [5].

One of the earliest bilayer wound dressings, consisted of a silicone membrane attached to an inner layer of collagen/chondroitin-6-sulfate sponge [7]. Similar bilayer wound dressings were later developed [8], [9] by modifying Yannas’s [7] approach.

Recently, gelatin has shown to exhibit activation of macrophages [10] and high hemostatic effect [11]. Gelatin is obtained by a controlled hydrolysis of the fibrous insoluble protein, collagen, which is widely found in nature and is the major constituent of skin, bones and connective tissue.

Being a protein, gelatin is composed of a unique sequence of amino acids. Characteristic features of gelatin are the high content of the amino acids such as glycine, proline and hydroxyproline. Structurally, gelatin molecules contain repeating sequences of glycine-X-Y triplets, where X and Y are frequently proline and hydroxyproline [12]. These sequences are responsible for the triple helical structure of gelatin and its ability to form gels where helical regions form in the gelatin protein chains immobilizing water.

Gelatin is water-soluble, so it should be crosslinked in order to be insoluble in aqueous solutions. The toxicity of the crosslinking agent should be taken into serious consideration while developing biomaterials.

The water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), however, was recently known to be non-toxic, biocompatible because it is not incorporated into the crosslinked sponge structure, but simply changed to a water-soluble urea derivative. The cytotoxity of the urea derivative was found to be quite low compared with that of EDC [13].

Section snippets

Materials

An alkaline-processed gelatin with pH 4–7 and theoretical molecular weight of 100,000 and 1-Ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Merck (Germany).

Preparation of gelatin sponge

Gelatin powder was dissolved in double-distilled water, stirred at 50 °C for 1 h to prepare 1 wt% (w/w) solution and poured into a suitable mold. The hydrogels were frozen at different temperatures for 24 h. The frozen hydrogels placed in Petri dishes and lyophilized at freeze-dryer (Alpha 1-2 LD,

Density measurement

The average densities of dried sponges are listed in Table 2. As the freezing temperature was lowered, the density of freeze-dried sponges increased. Density of freeze-dried sponges obtained by freezing in N2 (l) was almost twice as high as the ones freezed at −25 °C. The results indicated that the densities of samples are strongly dependent on the freezing temperature before freeze-drying [12]. The addition of EDC as crosslinking agent changed density very slightly, but no direct relationship

Conclusion

The strength and stability of gelatin sponges used in surgery must satisfy their intended biomedical needs. Crosslinking of gelatin sponges with EDC provides a means of easily tailoring a sponge biocompatibility and mechanical properties. In general, these techniques may be used to increase ease of handling in general. Based on obtained results, intermediate concentrations of EDC specifically 0.025 wt% or even 0.05 wt% increase gelatin sponge strength and stability while it has no cytotoxicity

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