Zamyrbek F.Z., Zhusipova D.A., Zulpuhar A., Tastambek K.T., Karibayev E.K., Abdullayeva B.

Al-Farabi Kazakh National University, Almaty, Kazakhstan

IN VIVO INVESTIGATION OF WOUND DRESSINGS BASED ON CARBONIZED RICE HUSK WITH SILVER IONS

Abstract

Sorption properties of the sorbent obtained from carbonized rice husk (CRH) has an important role in the treatment of purulent wounds. In efferent therapy, the practical importance of silver/CRH biocomposite is great. This study demonstrates in vitro studies of antimicrobial activity of silver containing carbonized rice husk (CRH) obtained by high temperature carbonization against the total bacterial count of causative agents of purulent wound. The results showed that the antimicrobial activity of CRH/silver biocomposite become wider as the silver content increases. It was created wound dressings based on complex therapeutic agents, including highly efficient sorbents with nanostructured surface (carbonized rice husk) enriched silver ions for the treatment of purulent wounds. In vivo studied wound-healing activities of wound dressings on the purulent wound of laboratory rats.

Key words: purulent wound, wound dressings, carbonized rice husk (CRH), silver, total bacterial count.

Topical preparations for wound care have been used for centuries. Dressings play a major role in wound management and have developed greatly over the last 50 years from passive to more active types [1]. Dressing selection is a vital part of the successful management of infected wounds and those at risk of infection. The role of silvers as an antimicrobial agent is particularly attractive, as it has a broad spectrum of antimicrobial activity with minimal toxicity toward mammalian cells at low concentrations and has a less likely tendency than antibiotics to induce resistance due to its activity at multiple bacterial target sites. There are already a large number of silver-containing dressings, and the list is growing [2].

The properties of metallic silver is a broad antibacterial spectrum of action, virucidal and fungicidal activity, the complexity of generating protective mechanisms of microorganisms to it, justify promising studies using it in terms of wound infection [3].

Treatment of purulent wound is an extremely complex and multifaceted phenomenon. In a study of its pathogenesis make the main contribution microbiology, biochemistry, cell biology and immunology. That is why it is necessary to develop completely new approaches to the delivery of silver ions to destruction of causative agents of purulent wound. The solution to this problem may be found in new techniques in biotechnology based on the use of immobilized preparations in which silver ions are adsorbed on the surface of a carrier.

Among these of special interest are the carbonized sorbents with nanostructured surface that manifest not only high affinity to bacterial cells but also detoxifying activity. It means that such sorbent will act as carrier for silver ions and at the same time neutralizes various toxins of purulent wound.

Several studies have shown that prospective for practical use are carbon sorbents with a nanostructured surface based on vegetable raw materials [4]. Carbon obtained by carbonization such materials retains its original thinly organized structure. By varying, the conditions of carbonization can produce complex composition of carbon, giving the final product a variety of properties that determine their practical application. Large quantities of plant wastes and agricultural by-products, such as grape seeds, nutshells, corn waste, etc. are produced annually. Meanwhile, excellent renewable raw materials for adsorbent production can be prepared. In most cases, as raw plant materials are commonly used monocyclic crop plants, such as wheat, rice, etc. For instance, rice shell is considered to be excellent renewable raw material for activated carbon production [5].

One of the ways to optimize the problem of improving the quality of treatment of the wounded is the use of dressings [6]. For the treatment of purulent wounds suggested a wide variety of methods and means of the local application. The use of wound dressings on the basis of carbonized rice husk as a sorbent which has been impregnated on its surface with silver in wound treatment procedures have a great relevance. It is also important to study how the exudates from the wound accumulate on the surface of the material and these results in a large reduction in the material's capacity to effect sterilization.

The main purpose of this work is to investigate new wound dressings with carbonized rice husk impregnated with silver ions in vivo and antimicrobial activity of CRH functioned with silver ions against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Proteus spp. in vitro.

MATERIALS AND METHODS

As the materials, it were taken the carbonized rice husk (CRH), AgNO₃, nonwoven materials, laboratory rats, test strains from the collection of Applied Microbiology laboratory: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Proteus spp.

Carbonized rise husk

In Kazakhstan, the carbon materials with nanostructured surface for the first time obtained by high-temperature carbonization from cheap secondary vegetable raw materials. Such as stone fruit, walnut shells and rice husks at the Institute of Combustion Problems led by Professor Z.A.Mansurova [7]. The presence of nanostructural surface of these materials enhances their attachment ability for microbial cells. Especially, the diameter of formed pores in the carbonization process controlled by carbonization temperature.

Silver. The silver ion is bioactive and in sufficient concentration readily kills bacteria in vitro. Silver exhibits low toxicity in the human body, and minimal risk is expected due to clinical exposure by inhalation, ingestion, dermal application. The medical uses of silver include its incorporation into wound dressings, creams, and as an antibiotic coating on medical devices [8].

Nonwoven materials. Nonwovens have specific characteristics that allow them to deliver high-performance across a wide range of applications. Specific functions include absorbency, liquid repellency, resilience, stretch, softness, strength, flame retardancy, washability, cushioning, filtering, bacterial barrier and sterility. Today, innovations in nonwoven fabrics are growing as rapidly as the demand for them, with almost unlimited possibilities for a wide variety of industries [9]. In this work, it was taken surgical robes as nonwoven materials.

METHODS

Preparation of silver/CRH biocomposite

The silver enriched CRH was synthesized by treatment with AgNO₃ at the concentrations of 44; 22 and 0.44 µg/g. These concentrations were chosen because according to the concentration of Actisorb® Silver Antimicrobial Dressing 220 and preliminary experiments done by us. This process is allowed to proceed overnight then washed thoroughly with distilled water.

Calculation the total bacterial count at silver and CRH effect

The pure culture of microorganisms is enlivened in Nutrient broth for one day, and then it is carried out according to the dilution method by Koch. In 10⁴ dilutions in 50 ml is added CRH enriched with silver ions. The flask contents were incubated for 15 minutes and 1 hour at 37⁰C on a shaker at 200 rotations. After 15 minutes and 1 hour, from flask contents is taken out 0.1 ml suspension, then it is distributed on the surface of the selective medium with a sterile spatula. The plates are incubated for 24 hours at 37⁰C. The next day, the total number of grown microorganisms would calculate. Number of cells in 1 ml of the substrate is calculated by the formula:

(1)

where, M - amount of cells in 1 ml, a - average number of colonies present at seeding dilutions, 10 - dilution coefficient, n - number of serial dilutions of the seeding which was done, V - the volume of suspension taken for seeding in ml.

Preparation wound dressings with silver/CRH biocomposite

In the preparation of wound dressings the concentration of CRH enriched silver were performed according to the concentration of Actisorb® Silver Antimicrobial Dressing 220. Within the dressing, there is 22 µg silver per 1g CRH. As nonwoven material it was taken surgical robes. The flasks with silver and CRH were shaken in Shaker for 15 minute at 200 rotations. Then CRH impregnated with silver was filtered and dried under the dry box, then put into the nonwoven material.

RESULTS AND DISCUSSION

1. The separate effect of silver ions on total bacterial count

It was studied the effect of Ag on total bacterial count of test strains. It was added Ag concentrations on 10⁴ suspensions. Initial it was taken 0.1 ml suspension from the flask contents. Taken volume of suspension spread over the surface of the medium with a sterile spatula. The total bacterial count after incubation for 24 hour at 37⁰С in thermostat and the results in the presence of 3 Ag concentrations (22µg/ml, 11µg/ml and 0.22µg/ml) were illustrated in Table 1.

Table 1. The separate effect of silver ions on total bacterial count

Test strains	Initial	Ag exposure time (min) and concentrations (µg/ml)
		22 µg/ml		11 µg/ml		0.22 µg/ml
		15 min	60 min	15 min	60 min	15 min	60 min
E. coli	5.7×10^7	-	-	-	-	10^5	-
S. aureus	2.3×10^7	-	-	-	-	-	-
P.aeruginosa	3.5×10^7	-	-	-	-	-	-
Proteus spp.	3.1×10^6	-	-	-	-	2×10^5	-

2. The effect of CRH enriched with silver ions on total bacterial count

It was considered the effect of CRH impregnated with silver on total bacterial count. Initial it was taken 0.1 ml suspension from the flask contents. Taken volume of suspension spread over the surface of the medium with a sterile spatula. The total bacterial count after incubation for 24 hour at 37⁰С in thermostat and the results in the presence of 3 concentrations of CRH enriched with silver (0.5g CRH + 22 µg Ag, 0.5g CRH + 11 µg Ag and 0.5g CRH + 0.22 µg Ag) were demonstrated in Table 2.

Table 2. The effect of CRH enriched with silver ions on total bacterial count

Test strains	Initial	CRH enriched Ag exposure time (min) and concentrations (g/µg)
		0.5g CRH + 22 µg Ag		0.5g CRH + 11µg Ag		0.5g CRH + 0.22 µg Ag
		15 min	60 min	15 min	60 min	15 min	60 min
E. coli	5.7×10^7	-	-	4×10^5	-	6×10^6	-
S. aureus	2.3×10^7	-	-	-	-	4.5×10^5	-
P.aeruginosa	3.5×10^7	-	-	-	-	-	-
Proteus spp.	3.1×10^6	3×10^5	-	2.7×10^5	-	2×10^7	-

We found complex silver/CRH biocomposites have antimicrobial, anti-inflammatory, anti-toxic activities. Therefore, almost at all concentrations of biocomposites were active against test strains. Obtained results show the highest effects of biocomposites were against E. coli, S. aureus, P. aeruginosa even in 15 minute shaking. Definitely, after one hour shaking the total bacterial count of all microorganisms was completely inhibited by the effect of silver/CRH biocomposites.

3. Wound-healing properties of wound dressings to the purulent wound of lab rat.

The laboratory rats were adapted on the condition during the experiment. After the adaptation, it was made wound on the lab rat skin with surgical instruments. Injured zones of skin were treated with causative agents of purulent wound Staphylococcus aureus. The wound of rats treated during 1-2min with the cotton impregnated with bacterial suspension. After one day, it was conducted wound healing procedures with wound dressings (Fig. 1).

Fig. 1. Medical dressing on the rat skin

It was counted the total bacterial count of microflora of purulent wound every second day of treatment with silver impregnated CRH dressings. In control variant of wounds was steady decrease of colony forming units of in 10³ suspensions of microflora (Table 3). The healing of wound lasted 10 days (Fig. 2).

Control (without dressing):

2 day 4 day 6 day 8 day 10 day

Treatment with silver impregnated CRH dressings:

2 day 4 day 6 day 8 day 10 day

Fig. 2. The healing of wounds (in days).

However, in the case of treatment with silver impregnated CRH dressings the healing of wound reduced for 2 days in comparison with control. The purulent wound healed in 6-7 days. In sixth day of healing the number of colony forming units of in 10³ suspensions of microflora has been completely reduced (Table 3).

Table 1. The change of number of microflora during the wound treatment

Wound dressings	CFU of microflora in 10^3 suspension
	2 day	4 day	6 day	8 day	10 day
Control	13,5×10^5 CFU/ml	8,5×10^5 CFU/ml	1,5×10^4 CFU/ml	5×10^3 CFU/ml	-
Ag +CRH experiment	7×10^4 CFU/ml	10^4 CFU/ml	-	-	-

CONCLUSION

The greatest inhibitions of total bacterial count in the presence of silver ions suggest the wide effective, antimicrobial activities against a broad range of microorganisms. In this study, CRH with silver ions were tested using a proposed standard in vitro method for evaluating the antimicrobial activity of this material.

The wound-healing properties of medical dressing, which was created by us showed high effectiveness. In the presence of CRH/silver biocomposite the wound treatment procedures shortened for 2-3 days in comparison with control due to the anti-inflammatory and antimicrobial activities of CRH/silver biocomposite.

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