Effects of lyophilization on human amniotic membrane

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1 Effects of lyophilization on human amniotic membrane M. Teresa Rodríguez-Ares, 1 María J.Lo pez-valladares, 1 Rosario Tourin o, 1 Begon a Vieites, 2 Francisco Gude, 3 María T. Silva 4 and Jose Couceiro 4 1 Department of Ophthalmology, University of Santiago de Compostela (USC) Hospital Complex, Santiago de Compostela, Spain 2 Department of Pathology, USC Hospital Complex, Santiago de Compostela, Spain 3 Department of Clinical Epidemiology, USC Hospital Complex, Santiago de Compostela, Spain 4 Institute of Orthopaedics and Tissue Bank, University of Santiago de Compostela, Santiago de Compostela, Spain ABSTRACT. Purpose: This study aimed to evaluate the effects of lyophilization and cryopreservation on human amniotic membrane (HAM) in terms of histological characteristics and growth factor levels. Methods: Non-preserved, lyophilized and cryopreserved HAM samples from 13 placentas were investigated. The morphological characteristics of HAM were evaluated using light and electron microscopy. Immunohistochemical methods were also applied to assess the distribution of collagen IV in the basement membrane. Total protein amounts were measured in extracts of intact amniotic membrane from non-preserved, lyophilized and cryopreserved samples. An enzyme-linked immunosorbent assay (ELISA) was used to assay growth factor protein levels for epidermal growth factor, fibroblast growth factor basic, hepatocyte growth factor, keratinocyte growth factor, transforming growth factor-b1 and nerve growth factor. Results: Histological examination of lyophilized and cryopreserved human amniotic membrane showed similar results. Immunohistochemistry showed presence of collagen IV throughout the basement membrane, both in cryopreserved and lyophilized samples. Total protein amount was higher in cryopreserved samples, without statistical significance. Growth factors ELISA did not show statistically significant differences except for fibroblast growth factor basic, with higher levels in cryopreserved amniotic membrane. Conclusions: Lyophilization maintains the histological structure of HAM, but seems to cause greater reductions in total protein amount and growth factor concentration than cryopreservation. Key words: amniotic membrane growth factors lyophilization ocular surface Acta Ophthalmol. 2009: 87: ª 2008 The Authors Journal compilation ª 2008 Acta Ophthalmol doi: /j x Introduction Amniotic membrane (AM) is a thin tissue that covers the placenta on the fetal side. Human AM (HAM) has been used for nearly 100 years as a biomaterial for the surgical reconstruction of different tissues and for promoting wound-healing processes (in plastic surgery, skin transplantation, burns etc.) (Davis 1910). The use of HAM in ocular surgery was first reported by de Ro th (1940) and Sorsby & Symmons (1946). Kim & Tseng (1995) reported the use of cryopreserved HAM (C-HAM) for ocular surface reconstruction in a rabbit model. Since then, numerous studies have confirmed the usefulness of AM transplantation (AMT) in ocular surface surgery (Azuara-Blanco et al. 1999; Dua et al. 2004) for conditions such as corneal ulcers (Lee & Tseng 1997; Kruse et al. 1999; Chen et al. 2000; Hanada et al. 2001), perforations (Rodriguez-Ares et al. 2004), conjunctival scarring and reconstruction (Tsubota et al. 1996; Tseng et al. 1997; Shimazaki et al. 1998; Ma et al. 2000; Solomon et al. 2001), chemical burns (Shimazaki et al. 1997; Meller et al. 2000; Gomes et al. 2003) and partial or total limbal stem cell deficiency (Shimazaki et al. 1998; Tseng et al. 1998; Gomes 396

2 et al. 2003). Other studies have demonstrated the usefulness of AM as a substrate for ex vivo expansion of diverse cell lines such as limbal epithelial cells (He et al. 1999; Koizumi et al. 2000a, 2000b; Tsai et al. 2000; Grueterich & Tseng 2002; Meller et al. 2002) and corneal (Pellegrini et al. 1997; Koizumi et al. 2000a, 2000b, 2000c) and conjunctival epithelial cells (Meller & Tseng 1999). Amniotic membrane has a range of properties that makes it an ideal tissue for ocular surface reconstruction. These properties reside in both its morphological characteristics and the cytokines and growth factors it contains (Hao et al. 2000; Koizumi et al. 2000a, 2000b; Lee et al. 2000). The basement membrane promotes epithelial cell migration and adhesion (Azuara-Blanco et al. 1999; Dua et al. 2004) and the avascular stromal matrix reduces inflammation, neovascularization and scarring (Hao et al. 2000; Lee et al. 2000). The method employed to preserve HAM until surgery must guarantee that such tissue properties are preserved. The ideal method would be one that facilitates transport and prolonged storage without deterioration. Currently, cryopreservation is widely considered the only preservation method to guarantee the maintenance of such properties. This method requires a deep-freezing facility (to ) 80 C), which is expensive and frequently unavailable, especially in underdeveloped countries. Moreover, maintaining the cold chain makes transportation difficult. Lyophilization is a preservation method that consists of removing water from a tissue by sublimation. This results in the inhibition of deleterious chemical reactions that lead to tissue alteration. Lyophilized tissue can then be stored at room temperature for long periods and its transportation is easy, thus resolving the two main disadvantages of cryopreservation. Several studies assessed whether lyophilized HAM (L-HAM) retains the properties necessary for subsequent use in ocular surface reconstruction and cell cultures. The aim of the present study was to investigate the influence of lyophilization and cryopreservation on the characteristics and properties of AM, based on light and electron microscopy studies, immunohistochemistry and enzymelinked immunosorbent assay (ELISA) for measurement of growth factor concentrations. Materials and Methods Preparation of preserved human amniotic membrane Human placentas were handled according to the tenets of the Declaration of Helsinki. After obtaining proper informed consent, 13 human placentas were collected after elective caesarean deliveries from donors seronegative for human immunodeficiency virus types I and II, human hepatitis B and C, and syphilis. The placentas were processed immediately after surgery in sterile conditions, as previously described (Kim & Tseng 1995). 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 U ml, streptomycin 50 lg ml and amphotericin B 2.5 lg ml). The AM was then laid (with the epithelial side up) on a cellulose nitrate filter and cut into patches of approximately 4 4 cm. Patches of AM from each donor (n = 13) were divided into three groups, one for each preservation method and a third that was used as control material (non-preserved HAM [NP-HAM]). Preparation of L-HAM Samples of AM were lyophilized (VIRTIS Genesis freeze-dryer; VirTis, SP Industries, Gardiner, NY, USA) and stored in a closed container at room temperature until processing. Preparation of C-HAM Samples of AM were preserved in Dubbelco s modified Eagle medium (Gibco Ò DMEM, Invitrogen Corp., Carlsbad, CA, USA) and glycerol 1 : 1 and stored at ) 80 C until processing. Samples of L-HAM and C-HAM were processed after 1 month. Before processing, L-HAM samples were rehydrated in saline solution for 15 mins. Samples of C-HAM were thawed at room temperature. Light microscopy Small samples of HAM from every group were fixed in 10% formalin for 24 hours. They were then embedded in paraffin by standard techniques and sectioned with a microtome. The sections (4 lm thickness) were stained with haematoxylin and eosin and periodic acid Schiff (PAS) for light microscope examination. Transmission electron microscopy Amniotic membrane samples were fixed in 2% glutaraldehyde for 2 hours, washed in 0.1 m sodium cacodylate buffer (ph 7.4), and postfixed in 1% aqueous osmium tetroxide. After a 10-min wash in distilled water, the tissue was dehydrated through a graded acetone series and embedded in Spurr s resin (Electron Microscopy Sciences, Fort Washington, PA, USA), following the manufacturer s recommendations. The specimens were cut to obtain semithin sections of 200-nm thickness that were stained with toluidine blue, and ultrathin sections of nm that were collected on copper grids and stained with lead citrate. Section samples were examined with a Zeiss 910 transmission electron microscope (TEM). Immunohistochemical staining Immunostaining was performed with the EnVision Plus system (Dako Corporation, Glostrup, Denmark) that uses an automated immunostaining technique (Tech-Mate). Before immunostaining, antigens were exposed by trypsin and heat treatment. The primary antibody used to stain basement membrane was anti-collagen IV (M785; Dako Corporation, Glostrup, Denmark) at a 1 : 10 dilution. Amniotic membrane extracts for total protein assay and growth factors ELISA Samples of HAM from every group were cut and homogenized in saline solution. After centrifugation at r.p.m. ( g) for 15 mins at 4 C, the supernatants were stored at ) 80 C until assayed. Total protein amount was measured using the BCA Protein Assay Kit (Pierce Corporation, Rockford, IL, USA). The same 397

3 amount of protein was used for all the samples to quantify six growth factors. Epidermal growth factor (EGF), fibroblast growth factor basic (bfgf), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and transforming growth factor-b1 (TGFb1) were measured using the ELISA Commercial Kit (R&D Systems, Minneapolis, MN, USA.) according to the manufacturer s instructions. Nerve growth factor (NGF) was measured using the NGF Emax Immunoassay System (Promega Corp., Madison, WI, USA), according to the manufacturer s instructions. The samples were assayed in duplicate. We also assayed the samples of NP- HAM from the same donors (n = 13). These were stored at 4 C in saline solution and processed within hours as control material. Statistical analysis Data are expressed as median (range). A Wilcoxon test for paired data was used for comparisons of total protein amount and growth factor levels for the three groups studied. Significances of differences were Bonferroniadjusted for three comparisons. All statistics were calculated using spss software (SPSS Inc., Chicago, IL, USA). A p-value of < 0.05 was considered statistically significant. Results Morphological characteristics (light microscopy) Normal HAM comprises three layers. The amniotic epithelium consists of a single layer of cuboidal cells that rests upon a thick basement membrane composed of reticular fibres. The stroma, the thickest layer, contains loose, fibrous connective tissue with occasional fibroblasts, and is avascular. Macroscopically, L-HAM appeared as a smooth white membrane after rehydration, similar to C-HAM. Examination of histological C-HAM samples by light microscopy showed that the epithelium maintained its organization as a single layer of cuboidal cells, with minimal vacuolar degeneration and a thick basement membrane (Fig. 1A). In L-HAM samples, the epithelium also maintained its organization as a single layer of cuboidal cells, but with more vacuolar degeneration and some areas of stratification and cell loss, and the stroma was oedematous (Fig. 1B). Staining by PAS indicated a well preserved basement membrane that appeared as a continuous line above the stroma in L-HAM samples (Fig. 2A, B). Electron microscopy Ultrastructural evaluation of AM provided more information about morphological characteristics. Specifically, examination by TEM showed preservation of the amniotic epithelium, with a single layer of cuboidal cells in both C-HAM (Fig. 3A) and L-HAM (Fig. 3B), with more intracytoplasmatic vacuoles in lyophilized samples (Fig. 3B). The cells were linked by desmosomes and attached to the basement membrane by hemidesmosomes in both preparations (Fig. 3A, B). Fig. 4 shows the desmosomas in great detail. The basement membrane was well preserved in all samples, although some areas were thinned in L-HAM (Figs 3B and 4A) compared with C- HAM (Fig. 3A). Immunohistochemistry study Immunohistochemistry showed the presence of collagen IV forming a continuous flat line along the basement membrane, in both C-HAM (Fig. 5A) and L-HAM (Fig. 5B). This was thinner in lyophilized samples. Fig. 1. Examination by light microscopy of human amniotic membrane. In cryopreserved samples, the epithelium maintains its organization as a single layer of cuboidal cells, with a thick basement membrane. In lyophilized samples, the epithelium likewise maintains its organization as a single layer of cuboidal cells, but more vacuolar degeneration, and an oedematous stroma are seen. Amniotic membrane extracts for total protein assay and growth factors ELISA Total protein amounts and growth factor levels in L-HAM, C-HAM and NP-HAM samples are given in Table 1 (median [range], n = 13). NP-HAM samples showed the highest total protein amounts, revealing statistically significant differences with L-HAM samples (p = 0.009). C-HAM samples showed higher protein concentrations than L-HAM samples, but this difference was not statistically significant (Table 1). Proteins were not detected in L-HAM samples from five of the 13 placentas (38%) and C-HAM samples from one placenta (8%). In the NP-HAM group, protein levels were detected in all samples. A statistically significant difference was found between NP-HAM and L-HAM. The distribution of total protein amounts in the three groups is shown in Fig. 6. The 398

4 Fig. 2. Haematoxylin and eosin and periodic acid Schiff stainings show a well preserved basement membrane that appears as a continuous line above the stroma in lyophilized human amniotic membrane. Fig. 3. Transmission electron microscopy shows preservation of the amniotic epithelium, with a single layer of cuboidal cells in both cryopreserved human amniotic membrane (HAM) and lyophilized HAM. More intracytoplasmatic vacuoles (white arrows in and black arrows in ) and some thinning of the basement membrane are seen in lyophilized samples. highest growth factor levels in the three studied groups were for HGF. No statistically relevant differences were found between the three groups. The only growth factor to show statistically significant differences in levels between C-HAM and L-HAM was b-fgf, which showed higher levels in C-HAM (p = 0.02). NP-HAM had the lowest levels of EGF, representing a statistically significant difference with both L-HAM and C-HAM (p = in both cases). Discussion This study confirms that lyophilization is able to maintain the main morphological characteristics of HAM, in terms of the single layer of epithelial cells and the basement membrane, with minimal changes. However, total protein amount assay and growth factor levels showed that lyophilization caused a greater loss of protein than cryopreservation in our samples. Currently, most AM employed in ocular surface surgery is cryopreserved using the standard protocol proposed by Kim & Tseng (1995). Other methods for AM preservation and storage have been described, but very few studies have assessed whether these alternative methods guarantee the maintenance of AM properties as effectively as cryopreservation (Dua et al. 2004; Nakamura et al. 2004; Von Versen-Ho ynck et al. 2004). Clearly, any method of AM preservation may potentially alter its characteristics and, consequently, its clinical properties. Kruse et al. (2000) demonstrated that cryopreservation significantly impaired the viability and proliferative capacity of AM and its cells; this was confirmed by Kubo et al. (2000). Recently, Hopkinson et al. (2006) reported the influence of handling on TGF-b1 and HGF content. In the present study, we lyophilized and cryopreserved AM samples from the same placentas and evaluated their histological characteristics and growth factor levels, comparing the results with those in NP-HAM obtained from the same placentas. For these comparisons, morphological tests were employed and protein and growth factor levels were assessed. The selection of the comparison tests was based on the fact that, although the exact mechanism of action of AM remains unknown, the main clinical properties of AM apparently reside in the combination of a thick basement membrane and its extracellular matrix (Dua et al. 2004) and the abundance of growth factors and cytokines contained within the membrane (Hao et al. 2000; Koizumi et al. 2000c; Lee et al. 2000; Touhami et al. 2002; Dua et al. 2004; Shao et al. 2004; Coassin et al. 2005). One of the proposed mechanisms of action of AM transplantation is the release of growth factors that facilitate 399

5 Fig. 4. Lyophilized human amniotic membrane sample showing intracytoplasmatic vacuoles (black arrows) and cells linked by desmosomes and attached to the basement membrane by hemidesmosomes. Desmosomes seen in great detail. Fig. 5. Immunohistochemistry shows collagen IV forming a continuous flat line along the basement membrane in cryopreserved human amniotic membrane (HAM) and lyophilized HAM, in which the basement membrane is thinner. corneal epithelial healing and reduce corneal scarring and inflammation (Hao et al. 2000; Koizumi et al. 2000a, 2000b, 2000c; Lee et al. 2000; Touhami et al. 2002; Klenker & Sheardown 2004; Shao et al. 2004; Coassin et al. 2005). We assayed six growth factors: bfgf, EGF, HGF, KGF, NGF and TGF-b1. Studies on C-HAM demonstrated the expression and significant levels of all of them, in both intact and denuded HAM (Koizumi et al. 2000a, 2000b, 2000c; Lee et al. 2000; Touhami et al. 2002; Coassin et al. 2005). Moreover, these growth factors are particularly involved in the wound-healing process (Koizumi et al. 2000a, 2000b, 2000c; Touhami et al. 2002; Klenker & Sheardown 2004). The facilitation of wound healing is one of the most important properties of AM and determines most of its clinical indications (Lee & Tseng 1997; Tseng et al. 1997; Shimazaki et al. 1998; Azuara- Blanco et al. 1999; Kruse et al. 1999; Chen et al. 2000; Ma et al. 2000; Hanada et al. 2001; Solomon et al. 2001; Dua et al. 2004; Rodriguez-Ares et al. 2004). Recently, Nakamura et al. (2004) published a study on L-HAM without epithelial cells and sterilized with c-radiation. In the present study, intact AM (with amniotic epithelial cells) was used for all tests and no sterilization method was applied to the tissue. In fact, it remains unclear whether stripping the epithelial layer is beneficial or not prior to preservation of AM for clinical use. The fact that most cytokines and growth factors are present at higher concentrations in the epithelium, however, would support the maintenance of this layer (Koizumi et al. 2000a, 2000b, 2000c). Furthermore, destruction of the amniotic epithelium with vacuolic degeneration and dissolution of the connective tissue layers into single fibre bundles in HAM preserved by different methods and sterilized by irradiation has been reported (Von Versen-Ho ynck et al. 2004). Light and electron microscopy showed the typical structure of HAM in all the preparations studied (Bourne 1960; Van Herendael et al. 1978; Dua et al. 2004). In L-HAM sections, areas of stratification in the epithelium were observed. Rama et al. (2001) observed stratification in about 5% of epithelium in HAM cryopreserved in liquid nitrogen without cryoprotectants such as glycerol used in the processing. Stratification of the epithelium in cryopreserved samples was very low and affected only some areas in very few sections. Maintenance of the basement membrane is essential for applications in ocular surface surgery (Azuara- Blanco et al. 1999; Dua et al. 2004). In our examinations, we found the basement membrane remained intact in all samples under both light and electron microscopy. Therefore, the lyophilization procedure that we 400

6 Table 1. Total protein amounts and growth factor levels. Data are expressed as median (range). applied for the preservation of AM proved effective. Collagen type IV has been identified in amniotic basement membrane and stromal amnion (Aplin et al. 1985; Yurchenco & Ruben 1987; Modesti et al. 1989). Immunohistochemical examination showed immunoreactivity for collagen IV forming a continuous line along the basement membrane in all samples, in a similar distribution to those previously reported (Modesti et al. 1989; Fukuda et al. 1999; Nakamura et al. 2004). Other authors have reported changes in the thickness of AM depending on the sterilization and preservation methods used (Nakamura et al. 2004; Von Versen-Ho ynck et al. 2004). Macroscopically, our lyophilized samples appeared to be NP-HAM L-HAM C-HAM Total protein amount* (mg ml) 0.30 ( ) 0.13 ( ) 0.12 ( ) EGF* (ng mg) 0.00 ( ) 0.24 ( ) 0.47 ( ) bfgf* à (ng mg) 0.92 ( ) 0.11 ( ) 1.44 ( ) KGF* (ng mg) 0.13 ( ) 0.00 ( ) 0.04 ( ) HGF (ng mg) 7.22 ( ) 0.09 ( ) 3.87 ( ) NGF (ng mg) 0.44 ( ) 0.31 ( ) 0.69 ( ) TGF-b1 (ng mg) ( ) 0.10 ( ) 0.03 ( ) * P < 0.05 for differences between the NP-HAM and L-HAM groups. P < 0.05 for differences between the NP-HAM and C-HAM groups. à P < 0.05 for differences between the L-HAM and C-HAM groups. HAM = human amniotic membrane; NP-HAM = non-preserved-ham; L-HAM = lyophilized HAM; C-HAM = cryopreserved HAM; EGF = epidermal growth factor; bfgf = fibroblast growth factor basic; KGF = keratinocyte growth factor; HGF = hepatocyte growth factor; NGF = nerve growth factor; TGF-b1 = transforming growth factor-b NP-AM L-AM C-AM Fig. 6. Distribution of total protein amounts in the three groups of human amniotic membrane (HAM) samples. NP-HAM = non-preserved HAM samples; L-HAM = lyophilized HAM samples; C-HAM = cryopreserved HAM samples. thinner and more fragile than the cryopreserved material. We did not measure the thickness of AM and did not apply any biomechanical test to evaluate the two preparations studied, but a recent biomechanical characterization of different AM preparations, including cryopreserved and denuded dehydrated samples, showed that frozen preparations have more elasticity and require greater force to be broken (Chuck et al. 2004). These differences may affect surgical preferences and uses because of ease of handling and resistance. Lyophilization caused greater loss of protein in our samples than cryopreservation, as five of 13 samples of L-HAM had no detectable soluble protein levels and cryopreserved samples contained more protein than lyophilized samples. Lyophilization causes more protein denaturation than cryopreservation (Jiang & Nail 1997) because the protein damage caused by drying must be added to that caused by the previous freezing phase. This denaturation of protein results in changes to its structure, which may impair the results of quantification tests as this test is designed to recognize a specific structure. These changes in the structure of the protein may also affect the protein function. This effect may be especially important when the donor placenta has low protein levels as these levels may be reduced to nil by lyophilization. Most of the cytokines which have been related to AM clinical effects are proteins; therefore, if interdonor variation is not differentiated before use, a lower amount of protein in L-HAM could impair clinical results. The most abundant growth factor in the three groups was HGF. Both EGF and TGF-b1 showed low levels in the three assayed groups, as previously reported (Koizumi et al. 2000c). In our samples, the growth factors with the lowest concentrations were KGF in the preserved samples and EGF in NP-HAM. The higher EGF concentrations in L-HAM and C-HAM may be attributable to cellular destruction caused by the preservation method and the subsequent release of peptides and proteins (Hopkinson et al. 2006). We noted a wide range of variation in growth factor and protein levels in samples from different placentas, as shown in Fig. 6. These results are consistent with previous reports (Koizumi et al. 2000a, 2000b, 2000c), although our standard deviation is higher. This may be explained on the basis of interdonor variations and the influence of handling (Hopkinson et al. 2006). Cryopreservation preserves proteins and growth factors better than lyophilization, which is especially important when the donor has low protein levels. We did not conduct clinical tests in the present study. However, a recent report showed acellular lyophilized AM to have excellent biocompatibility when used on bare scleral defects after excision of pterigya (Nakamura et al. 2006). In conclusion, the present results demonstrate that lyophilization maintains the histological structure of AM, although it causes more alterations than cryopreservation. However, it causes greater protein loss. Cryopreservation better maintains the AM properties evaluated here. It is, therefore, the best preservation method for HAM. However, L-HAM maintains some of the most important characteristics of AM and thus could be useful when the necessary infrastructure for the transport and storage of cryopreserved tissue is not available. Further study into the clinical application of L-HAM is required, particularly for ocular surface diseases in which growth factors may play a more important role. Moreover, interdonor variation must be taken into consideration, as must its influence on the clinical effects of HAM. 401

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