Growth Factors BDNF and TGF 1 β in Tissue of Cryopreserved Human Placental Amniotic Membrane after Antiglaucomatous Surgery

The effect of cryopreserved human placental amniotic membrane (HPAM) on the release of cell growth factors BDNF and TGF1β into both an environment and eye within the observation period of up to 21 days has been studied. By enzyme immunoassay there was investigated the content of cell growth factors in ocular tissues after use as a coating of the frozen according two-step regimen HPAM in antiglaucomatous surgery. The efficiency of using the frozen-thawed HPAM in respect of releasing the cell growth factors after its application into the post-surgery defect zone has been demonstrated. There was found the dependence of accumulation of growth factors TGF1β and BDNF on time of cryopreserved HPAM application, eye tissue type and tropism of growth factors.

Cryopreservation of HPAM allows to create the stocks of the tissue for future application in antiglaucomatous surgery (AGS) when needed.However, there are no valid data on releasing the biologically active substances and neuroprotectors into an ocular tissue from the covering the wound defect amnion in situ to the 21 st day of post-surgery period.Two-step protocol was used to freeze the human placental tissue previously, providing a high rate of the tissue structure preservation [9].This paper will demonstrate an effective freezing regimen in respect of the release of cell growth factors from the frozenthawed HPAM after application to an ocular tissue.
The research goal was to determine the content of growth factors in eye tissues in case of using in antiglaucomatous surgeries a biological coating of cryopreserved HPAM (cHPAM), frozen according to two-step protocol.Efficiency of the cryopreservation regimen was assessed by release of cell growth factors from cryopreserved HPAM (cHPAM) in eye tissue after surgery, i. e. neurotrophic factor of cerebral origin (BDNF) and transforming growth factor of cells (TGF1β).We planned to procure cHPAM which would have a minimal permeability of these markers after thawing of the tissue and to use such freezing regimen, which would provide the longest release (up to 21 days) of neurotrophic and cellular factors of cHPAM in an eye.

Materials and methods
Experiments were carried out in Chinchilla rabbits (n = 25) kept in the animal house conditions with appropriate light regimen and the standard diet.Adrenaline was administered to simulate eye glaucoma in animals [5,7].
The work was performed in accordance with the General Principles of Experiments in Animals, approved by the 5 th National Congress in Bioethics (Kiev, 2013) and consistent with the statements of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986).
Prior to freezing the HPAM fragments were incubated in dimethyl sulfoxide (DMSO) cryoprotectant solution at a final concentration of 10% (Arterium Corporation, Ukraine) for 18-20 min, then each fragment was placed into a sterile disposable container (Nunc, Germany), sealed and marked.The samples were frozen according to the two-step program: cooling down to -40°C at a rate of 3 to 5 deg/min and further immersion into liquid nitrogen (-196°C).After freezing the containers with the material were transferred to the Low Temperature Bank for storage at liquid nitrogen temperature.
Within 2 months after simulation of glaucoma the laboratory animals were subjected to AGS, during which the cHPAM was applied in a zone of post-surgery defect right after thawing [12].The surgery was performed in any of the eyeball free sectors between the rectus muscles.Near the limb, after a preliminary determination of the projection of Schlemm's canal, we have dissected the deep layers of filter area with a sinus and trabeculae as a triangle.The scleral flap was fixed at its top to the convex part of the sclera by means of one U-shaped suture and interrupted sutures on each side with a simultaneous capture of cHPAM.Conjunctival wound was closed with a continuous suture.
Debris-free homogenate from native and frozen tissue of HPAM, as well as other eye tissues of laboratory animals were prepared as follows.A single dose of amniotic membrane was taken from liquid nitrogen, in some experiments the content of growth factors was determined in the non-frozen amniotic membrane or in healthy tissue (cornea, retina, optic nerve and sclera) from the locus of AGS.The homogenate was derived from 100 mg HPAM tissue or eye tissue which were ground in a 1.5 ml tube using a Potter micro-homogenizer in phosphate based physiological saline pH 7.4, supplemented with 1 mM protease inhibitor phenyl-methylsulfonilfluoride in 1:9 ratio.The resulted homogenate was cleared of debris by centrifugation at 1000g for 10 min.The supernatant was collected into separate tubes for the measurement of cell growth factors and neurotrophins.
To assess the yield of growth factors BDNF and TGF1β the frozen-thawed cHPAM tissue was incubated in a sterile physiological saline.The cHPAM tissue (100 mg) was placed into 1 ml solution at 37°C and after 1, 3, 6, 9 hrs the supernatant was collected for measurement of growth factors.
Content of TGF1β, BDNF growth factors (human) was measured by highly sensitive test systems from DRG (USA; sensitivity 2 pg/ml) and R&D Systems (USA, sensitivity 20 pg/ml), respectively.Measure- The data were statistically processed by the Student test and represented in the Figures as M ± m.The differences were considered as significant at p < 0.05.

Results and discussion
The findings allowed to establish that to days 7, 14, and 21 after application of cHPAM, frozen-thawed by two-step program, to the site of post-surgery suture the ocular tissue recovered more quickly and a soft suture formed.
One of the most important indices of HPAM tissue structure preservation after freeze-thawing could be the ability to keep the intracellular substances.Quite common is the measurement of intracellular substances release from a tissue, which concentration is usually determined immediately after thawing of biological object.However we believe such a methodical approach could not to provide a true information, since it has remained unclear how cHPAM application in the post-surgery suture zone could affect its safety in a considerable time after thawing.In order to more objectively assess the structural integrity of cHPAM and to simulate the real conditions of its being in an eye after thawing the tissue was placed in a physiological saline, and afterwards the release of growth factors BDNF and TGF1β into a supernatant was measured .
After using two-step protocol of HPAM freezing in 10% DMSO solution the yield of growth factors within one hour was considerably lower than that with no cryoprotectant used during freezing.The findings suggested the higher preservation rate of the cells and tissues as well as their structural integrity, which were supported by the data of morphological studies [6].
After one-hour incubation of unfrozen HPAM tissue (control) in a physiological saline an insignificant release of BDNF and TGF1β growth factors was observed, after freezing according to two-step protocol a moderate yield was found (in average by 20% higher than the control) and a significant loss of cell contents was noticed in negative control (freeze-thawing with no cryoprotectant).Incubation of HMAP in physiological saline could simulate the coating of a wound surface of conjunctiva with the HPAM, i. e. we can conclude a release of the growth factors during healing (Fig. 1).Therefore, the effective healing of postsurgery suture wound will be largely determined by the durable release of biologically active substances from the cHPAM into an eye.
Содержание BDNF пг/мл BDNF content, pg/ml Содержание TNF1β, пг/мл TNF1β content, pg/ml Время инкубации, ч Incubation term, hrs Время инкубации, ч Incubation term, hrs freezing by immersion into liquid nitrogen unlike the two-step protocol of freezing in 10% DMSO solution, when the integrity of HPAM cells was preserved and the loss of cell growth factors was minimal after thawing of cHPAM tissue.
Afterwards it was of an importance to assess the delivery and distribution of growth factors in different ocular tissues when using cHPAM after the AGS.To do this we investigated the tissues of cornea, retina, optic nerve and sclera in the site of surgery.At different post surgery terms we measured the content of growth factors BDNF and TGF1β in these tissues.Fig. 3 shows that there was more intense release of growth factors from the cHPAM into the corneal tissue to days 1 and 3 if compared with unfrozen one, that might be associated with releasing of intracellular content from some damaged cells of the frozen HPAM.The index was higher than 60% if compared to the control.At the same time period (e.g. during the first 24 hrs) the entering of BDNF growth factor into retina and optic nerve tissue exceeded by 100% this index for an unfrozen HPAM.The same tendency was found in the optic nerve, wherein to day 7 the growth factor BDNF content was 30% higher than in unfrozen HPAM.It should be noted that the release of BDNF neurotrophin into retinal and corneal tissues was similar to the one in unfrozen HPAM.For sclera there was found a poor BDNF release from the cHPAM tissue both at initial terms and after days 7, 14, 21 of observation period.
Major amount of the cell growth factor TGF1β has been found to be accumulated in the corneal tissue  within the first 24 hrs of cHPAM application (Fig. 4), an insignificant content was found in the ophthalmic nerve, retina and sclera.Herewith the action of cryopreserved and unfrozen HPAM was similar [3,11].At late observation terms the TGF1β content, exceeding the control, was observed only in cornea, and no statistically significant differences were found in retinal tissue, ophthalmic nerve and sclera after using cHPAM if compared with unfrozen HPAM.However, there was a tendency to decrease the TGF1β content in sclera tissue.
that experimental glaucoma was accompanied primarily with the death and atrophy of small and medium sized ganglion cells of retina.Number of large cells was significantly decreased, but their proportion in the total number of cells increased [6,10].Therefore we believe the largest amount of biologically active substances to enter the cells mostly damaged by glaucoma.There is likely a qualitative and quantitative dependence of cell growth factors accumulation, determined by the type of an ocular tissue and growth factor tropism.Since the very in the damaged by glaucoma ocular tissues there are likely defective receptor and ganglion nerve cells, whereto from cHPAM in considerable amounts the BDNF having a pronounced neuroprotective effect is released [8].

Conclusions
Thus, application of HPAM, frozen-thawed according to the two-step program in 10% DMSO solution in the AGS zone resulted in an increased content of BDNF and TGF1β growth factors in ocular tissues (up to 7 days' observation term) and stable level over the postoperative observation period (up to 21 days observation term).An effective freezethawing regimen for HPAM provided a sustained release of BDNF and TGF1β growth factors in an eye from the site of post-surgery defect.

Fig. 3 .
Fig. 3. Content of BDNF growth factor in various tissues of eye in different terms after applying the HPAM during antiglaucomatous surgery:control;freeze-thawing in 10% DMSO.

Fig. 4 .
Fig. 4. Content of TGF1β growth factor in various tissues of eye in different terms after applying the HPAM during antiglaucomatous surgery:control;freeze-thawing in 10% DMSO.