Time-Dependent Stability of Growth Factors and Endostatin in Human Amniotic Membrane Eye Drops

Human Amniotic Membrane (AM) contains a variety of beneficial bioactive factors and has low immunogenicity [1]. Clinical observations have shown that AM transplantation promotes corneal epithelial healing [2], reduces scarring [3], suppresses inflammation [4] and inhibits angiogenesis [5,6]. Many of these properties are attributable to the histological structure of the AM, which has a thick basement membrane that acts as a substrate for epithelial growth, and to its biochemical composition, rich in cytokines and growth factors. Recently, AM eye drops have been reported to have satisfactory clinical effects in the treatment of corneal ulcers, obviating the need for invasive surgical procedures [7-10]. In addition, topical application of an amniotic extract has been shown to have an inhibitory effect on corneal neovascularization in vivo [11].


Introduction
Human Amniotic Membrane (AM) contains a variety of beneficial bioactive factors and has low immunogenicity [1]. Clinical observations have shown that AM transplantation promotes corneal epithelial healing [2], reduces scarring [3], suppresses inflammation [4] and inhibits angiogenesis [5,6]. Many of these properties are attributable to the histological structure of the AM, which has a thick basement membrane that acts as a substrate for epithelial growth, and to its biochemical composition, rich in cytokines and growth factors. Recently, AM eye drops have been reported to have satisfactory clinical effects in the treatment of corneal ulcers, obviating the need for invasive surgical procedures [7-10]. In addition, topical application of an amniotic extract has been shown to have an inhibitory effect on corneal neovascularization in vivo [11].
The levels of growth factors in AM preparations depend on a number of factors including: gestational and donor age [12], preservation method applied [13], as well as filtration and centrifugation protocols employed during eye drop preparation. Thus AM eye drop preparation should minimize growth factor depletion, and storage conditions should be designed to minimize protein denaturation. For these reasons, it is important to characterize the composition of AM eye drops in terms of factors which are known to be involved in re-epithelialization, such as bFGF, EGF, HGF, NGF and the endogenous anti-angiogenic factor endostatin, and to determine the best concentration and preservation schedules in order to achieve optimal clinical results.
Consequently, the purpose of this study was (1) to define a protocol for the preparation of AM eye drops, (2) to characterize AM eye drops in terms of levels of a panel of growth factors and endostatin and (3) to examine the stability of AM eye drops over time, thereby evaluating the possibility that this treatment may constitute a new practical and viable alternative to enhance re-epithelialization.

Preparation of human amniotic membrane
Donors were selected among patients of the Department of Obstetrics of the La Paz University Hospital (Madrid, Spain). The criteria applied for patient selection were the same criteria required to donate AM for ocular surface surgery. Inclusion criteria were: age between 18 and 42, seronegativity for human immunodeficiency virus, human hepatitis B, C and syphilis; and full-term pregnancies (38-42 weeks). Exclusion criteria were gestational or fetal diseases.
Human placentas were handled according to the tenets of the Declaration of Helsinki. After obtaining proper informed consent, four human placentas were collected after elective caesarean deliveries and were processed immediately after surgery under sterile conditions. First, they were washed with sterile saline solution to remove blood clots. The AM was then carefully detached from the chorion by blunt dissection and rinsed several times with a saline solution containing antibiotics and antimycotics (penicillin 10,000 U ⁄ ml, streptomycin 50 mg ⁄ ml and amphotericin B 2.5 mg ⁄ ml). Each AM was then introduced into a sterile container with a 1:1 solution of Dulbecco's Modified Eagle's Medium: glycerol (Gibco® DMEM, USA) and stored at -80°C until use.
Evaluation of total protein, growth factor and endostatin content in native amniotic membrane protein quantification kit. Data was expressed in µg/ml. Using ELISA, we similarly measured the levels of Basic Fibroblast Growth Factor (bFGF), endostatin, Hepatocyte Growth Factor (HGF), Nerve Growth Factor (NGF) and Epithelial Growth Factor (EGF). These results were expressed as pg/µg of total protein.

Statistical analysis
All experiments and chemical determinations were performed in duplicate. Data was expressed as mean + SD. To evaluate growth factor stability over time, we performed a statistical analysis using the regression of mixed models. P <0.05 was considered to be statistically significant.

Comparative analysis of peptide levels in intact amniotic membrane and eye drops
Peptide levels in three different non-lyophilized human amniotic membranes were measured by ELISA and these levels were compared with those measured in AM eye drops (two 20% AM eye drops and two 30% AM eye drops) after 6 weeks of re-hydration ( Figure 2).
Endostatin, HGF and NGF levels (pg/µg) were higher in the cryopreserved AM (n=3) than in the 20% (n=2) and 30% (n=2) lyophilized AM eye drops. NGF levels were significantly lower in both eye drop preparations, perhaps due to its lability. Peptide levels were higher in 30% vs 20% eye drops, suggesting that the peptides may be more stable at higher concentrations ( Figure 2).

Growth factor and endostatin levels 6 weeks after reconstitution
Six weeks after reconstitution of lyophilized amniotic membrane, mean bFGF and endostatin levels in AM eye drops presented highest values. The rank order of concentration of the other growth factors was HGF>NGF>EGF. Results are expressed as pg/µg and also as pg/ml in Table 1.

Temporal stability of growth factors in human amniotic membrane eye drops
Growth factor levels were expressed relative to total protein homogenized with phenylmethanesulfonyl fluoride (1 ml) and phosphate buffered saline (10 ml) followed by centrifugation at 3,000 rpm, 5 min at 4ºC. The collected supernatants were aliquoted, passed through a 0.22 µm filter, and stored in a new container.
The Nanodrop and the EZQ methods were used to assess total protein content and data were expressed as µg/ml. bFGF, HGF, NGF, EGF and endostatin were analyzed by ELISA according to the manufacturer's instructions. Data were expressed as pg/µg of total protein.

Lyophilization of amniotic membrane
The four AMs were defrosted. The AMs were pooled together, mechanically disaggregated by grinding and sonication and then subjected to centrifugation (3,000 rpm, 15 min; see Figure 1). The supernatant was extracted and centrifuged again (3,000 rpm, 15 min). The resulting supernatant was dosed (2 ml each) into 40 bottles by a Watson-Marlow pump and lyophilized in three phases as follow: 4 hours prefreeze at -80ºC, followed by 8 hours at -22ºC, and finally a three stage secondary dryness with 0.020 mBar high vacuum, 4 h at -10ºC, 2 h at 0ºC and finally 2 h at 25ºC. The bottles were closed and injected with sterile nitrogen gas. A quality test was performed to check residual humidity (<0.5%). Because this work was performed under sterile and high biological risk conditions (in an ISO 5 room), at a temperature lower than 8ºC, no filtration was required. Three microbiological tests showed negative results and the forty bottles were sent to Bioftalmik (Bizkaia, Spain) to proceed with the quantification of proteins and growth factors (Figure 1).

Growth factor levels in native AM and AM eye drops
We initially evaluated if AM prepared as eye drops contains mitogenic growth factors. Here, we have demonstrated that AM eye drops contain high levels of bFGF, endostatin and HGF, and lower levels of NGF and EGF. The levels of some growth factors, such as HGF, NGF and endostatin, were lower in the lyophilized AM eye drops than in the native AM tissue, whereas other growth factors such as bFGF and EGF maintain similar levels in both native tissue and 30% AM eye drops.
The values which we reported differ in some respects from those reported in previous studies of growth factor concentrations. For example, HGF has been reported to have the highest levels in AM [12] and also in AM suspension [8]. These differences are likely due to differences in AM preparation methods such as the use of fresh vs cryopreserved AM, the use of Dulbecco's Modified Eagle Medium vs sterile water for the dissolution of powdered AM, and whether centrifugation is performed after reconstitution, or before lyophilization [8].
The growth factor levels which we reported here are also different to those reported in other biological eye drops used to treat corneal epithelial diseases. Thus, similar levels of EGF have been reported in autologous serum (410 pg/ml) [14] and plasma rich in growth factors (PRGF) (468.9 +/-97.6 pg/ml) [15], whereas EGF levels were somewhat lower (303.5 pg/ml, 30% AM eye drops) in our study. In contrast, the levels of other growth factors were higher in our eye drops than in PRGF [15]; (our study vs 82.6 pg/ml), HGF (3,966.57 pg/ml vs 149.5 pg/ml) and for example, FGF (52,371.45 pg/ml NGF (714.76 vs 37.7 pg/ml)). Although the autologous PRGF eye drops also have a valuable effect on corneal wound healing, the AM eye drops provide some additional advantages. Since the AM has anti-inflammatory, antiangiogenic and anti-scarring properties, it may well be the treatment of choice for epithelial defects accompanied by inflammation.
The biochemical factors derived from the AM epithelium which are involved in re-epithelialization are known to be bFGF, HGF, NGF, EGF and KGF. However, most useful clinical information has been obtained with NGF which has been applied alone to diseased human corneal tissue. In clinical studies [16][17][18][19][20], topical treatment with purified murine NGF was found to induce complete healing of corneal ulcers, as well as a marked improvement in both corneal sensitivity and visual acuity. This purified murine NGF solution was composed of 100 or 200 µg/ml NGF, which is much higher than that contained in our AM eye drops (0.00071 µg/ml).

Stability of growth factors in AM eye drops
Next, we evaluated if growth factor levels were stable in the AM eye drops. The stability of growth factors in eye drops is important for its clinical application because growth factor denaturation has been reported to occur in other eye drop preparations, such as autologous serum eye drops [21]. The levels of all the growth factors which we studied (bFGF, HGF, NGF and EGF), and of endostatin, were found to be nicely maintained over a period of at least 6 weeks. This biochemical stability over time is an added advantage for the clinical usefulness of AM eye drops, since it can be stored without freezing at 4ºC for at least for 6 weeks, and it can be applied immediately if required or if any contraindication exists for autologous serum. Further biological tests will be required to evaluate if the factors present at 6 weeks post defreezing continue to be functionally efficacious in a clinical context.   Table 2: The concentrations of growth factors and endostatin in both 20% and 30% AM eye drops were invariant over time. P values were derived from the regression of mixed models to determine differences which were statistically significant. All p values > 0.05, which was the limit considered to be statistically significant.