Microbiological viability of bovine amniotic membrane stored in glycerin 99% at room temperature for 48 months

Submitted on June 12 , 2015 and accepted on December 19 , 2016. 1 Faculdade de Ciências Biológicas e da Saúde, Viçosa, Minas Gerais, Brazil. kellycpontes@yahoo.com.br; ramalho.vet@hotmail.com 2 Universidade Federal de Viçosa, Department of Veterinary, Viçosa, Minas Gerais, Brazil. andrea@ufv .br; digosepulveda@hotmail.com; renato_ufv@hotmail.com *Corresponding author: kellycpontes@yahoo.com.br Microbiological viability of bovine amniotic membrane stored in glycerin 99% at room temperature for 48 months


INTRODUCTION
Sterility maintenance is a primary concern regardless of the method for preserving a biomaterial (Slatter & Dietrich, 2007).Thus, a number of media have been studied for storing biological membranes and glycerin has shown good results (Okamoto et al., 2000).Glycerin has antiseptic properties, acts as fixative and dehydrating agent (Chirife et al., 1982;Amendola, 2007), is capable of preserving the ionic concentration and cellular integrity of the tissue (Pigossi, 1967), and is indicated for the preservation of numerous biological materials.Its low cost and easy handling also encourage its use as storage medium (Pigossi, 1967;Amendola, 2007;Pontes et al., 2008;Pontes et al., 2011).
On the basis of the authors' clinical and experimental experience, the amniotic membrane has already been used in the treatment of superficial and penetrating experimental corneal ulcers in rabbits (Pontes et al., 2008;Pontes et al., 2014), corneal ulcers refractory to clinical treatment in dogs, and after resection of feline corneal sequestrum (Pontes et al., 2010) and dermoid cyst in dogs (Rios et al., 2014).In veterinary medicine, it has already been used in the treatment of penetrating corneal ulcers (Barros et al., 1998), resection of corneal and scleral tumors, in symblepharon treatment (Barros et al., 2005), limbic cell deficiency (Cremonini et al., 2007), and chemical burns (Choi et al., 2011).This type of membrane can be used fresh or preserved in some type of medium.The amniotic membrane is harvested during cesarean section of term fetuses, making it impracticable to apply it fresh in surgeries without previous planning.In this context, when the amniotic membrane is necessary in a certain surgical procedure, it should be available in a tissue bank and stored in a medium that prevents bacterial and fungal growth.It is, therefore, important the creation of amniotic membrane banks.Besides, it is not difficult to acquire amniotic membranes, especially in the veterinary context, which makes it an attractive biomaterial for use in surgeries.
Currently, the use of amniotic membrane has become common in surgeries, but there are no reports in the literature on the suitability of glycerin 99% at room temperature as a microbiological conservation medium for this type of biomaterial.
Thus, the objective of this study was to evaluate the use of glycerin 99% for preserving bovine amniotic membrane at room temperature for 48 months.If its efficiency was demonstrated during this period, banks of this biomaterial could be created, allowing its use over time in several types of surgical procedures, without having to plan a caesarean section for harvesting the amniotic membrane at the time coinciding with its use in fresh.

MATERIAL AND METHODS
This research project was approved by the Research Ethics Committee of the Faculdade de Ciências Biológicas e da Saúde -FACISA / UNIVIÇOSA, number 00113/2012.
The amniotic membrane was prepared according to the literature (Kim & Tseng, 1995); it was harvested under sterile conditions from a pregnant female bearing a full-term fetus during elective caesarean section at the Veterinary Hospital of the Universidade Federal de Viçosa.In a surgical environment, the amniotic membrane, with the chorion, was first washed in 0.9% sterile saline solution at room temperature to remove the clots, then washed three times with sterile phosphate buffer solution containing 1000 IU/ ml penicillin G, 20 mcg/ml streptomycin, and 2.5 mcg/ml amphotericin B. The amnion was manually separated from the chorion (Figure 1A) (Kim & Tseng, 1995), mounted, epithelial face upwards, onto nitrocellulose paper (Figure 1B), and cut into the size of 1.0 cm 2 .Subsequently, the fragment was placed in a sterile glass vial and covered with sterile glycerin 99% (Figure 1C) (Pontes et al., 2014).
A total of 80 flasks containing amniotic membrane in glycerin 99% were randomly distributed into four equally sized groups.Each group contained 20 bovine amniotic membrane fragments and were all maintained at room temperature.Thirty days after harvest, group 1 was microbiologically tested (Barros et al., 1998, Pontes et al., 2008, Pontes et al., 2014).Group 2 was subjected to the same test after 6 months, group 3 after 12 months, and group 4 after 48 months.
Membrane fragments were aseptically collected using sterile tweezers in a laminar flow cabinet, triturated with 0.85% sterile saline solution and inoculated by surface spreading on plates containing blood agar (5% sheep's blood) and plates containing Sabouraud dextrose agar.To isolate bacteria, the samples inoculated on blood agar and incubated at 37 ºC for 48 hours and to monitor fungal growth, the samples were inoculated on Sabouraud agar at 25 ºC for 5 to 10 days (Quinn et al., 2005).

RESULTS AND DISCUSSION
Fungal growth was observed in two samples of G4.Therefore, a new microbiological analysis was performed in all the samples and none of them showed fungal or bacterial growth.This result suggests that there was contamination during the handling of the membranes in the first microbiological analysis, corroborating with Gioso et al. (2002), who found that the simple handling of bones, however careful, could introduce contaminating microorganisms into the samples.
The amniotic membrane was microbiologically evaluated only after 30 days, because according to literature, the membrane should remain for at least 30 days in glycerin for the loss of the ability to stimulate immune reaction to take place (Pigossi, 1967;Daleck et al. al., 1992;Costa Neto et al., 1999).
Additionally, elimination of bacteria may require up to 27 days to occur and the tissue should remain for at least 30 days in glycerin, according to a study on the effects of glycerin on tendons inoculated with gram negative and gram positive bacteria (Krauspenhar et al., 2003).
Although the amniotic membrane has been properly prepared according to the literature (Kim & Tseng, 1995), we can attribute to glycerin 99% the absence of fungal and bacterial growth during storage of bovine amniotic membrane at room temperature for up to 48 months.This result was important, since glycerin had never been microbiologically evaluated as a medium for preserving the amniotic membrane at room temperature.Glycerin has been studied for storing pericardium (Brun et al., 2002a) and bone tissue (Gioso et al., 2002), which in the long term showed no significant growth of microorganisms.
Amniotic membrane preservation has been reported in glycerin at 4 °C (Kim & Tseng, 1995) and at -80 °C (Dekaris & Gabriae, 2009), but our objective was to evaluate the microbiological efficiency of glycerin 99% in preserving the tissue at room temperature.A number of authors found that glycerin in association with a cell culture medium MEM (Minimal Essential Medium), at -80 ºC, acted to guarantee the tissue integrity and the preservation of growth factors and cytokines in human amniotic membrane (Pena et al., 2007), but others concluded that glycerin used as a cryoprotectant may impair the function of human amniotic membrane as a substrate for limbus cell culture (Shortt et al., 2008).Thus, the other effects that glycerin 99% used as storage medium can cause in the amniotic membrane is still controversial.
Because our group frequently uses the amniotic membrane in research and in the hospital routine (Pontes et al., 2011;Pontes et al., 2014), it was important to evaluate this medium aiming at creating a bank of this tissue to be used in the long term.Some other factors were also taken into account to evaluate glycerin 99% as a preservation medium, including low cost and easy handling (Pigossi, 1967;Amendola, 2007;Pontes et al., 2011;Pontes et al., 2014) and acquisition (Pontes et al., 2008), as well as abolishing other high-cost media of difficult transportation (Alvarenga, 1992) such as keeping at -80 °C.
The ability of glycerin 99% to act against some specific bacterium and fungus was not evaluated, since it was not the objective of this study.However, some studies have confirmed its bactericidal action on Staphylococcus aureus, Streptococcus faecalis, Escherichia coli and Pseudomonas aeruginos and its inability to prevent the growth of Clostridium botulinium and Clostridium perfringes (Pigossi et al., 1971;Amendola, 2007).Other studies reported that glycerin has no action on some more resistant sporulated forms (Pigossi et al., 1971) or viruses (Coronado et al., 1998), while some authors reported that glycerin is only effective against type I herpes virus and poliovirus when at 98% (Marshall et al., 1995).The same authors stated that to be effective against these viruses, the tissue must be maintained in 98% glycerin for at least 4 weeks at up to 20 °C.
By analyzing the various media and methods of preservation for biological material in the literature, it can be suggested that glycerin 99%, at room temperature, shows greater advantages when used to store the bovine amniotic membrane under the described conditions.This finding makes it an ideal medium to be used in the creation of a bank of this type of membrane, with the possibility of its microbiological conservation at room temperature for long periods.This fact may facilitate the use of bovine amniotic membrane in the hospital routine, since it is a biomaterial of easy acquisition and handling, reducing discarding because of the impossibility of using it in fresh.

CONCLUSIONS
Glycerin 99% is an efficient preservation medium for bovine amniotic membrane previously prepared as described, with respect to microbiology, keeping the tissue free of microorganism growth in the media kept at room temperature for up to 48 months.

Figure 1 :
Figure 1: Pictures showing bovine amniotic membrane preparation.A -Amnion being manually separated from the chorion.B -Amniotic membrane spreading onto nitrocellulose paper with epithelial face upwards.C -Amniotic membrane fragments in a flask containing sterile glycerin 99% to be stored at room temperature.