Proteomic Comparison of Amnion and Chorion and Evaluation of the Effects of Processing on Placental Membranes

Abstract

Objective.

The purpose of this study is to compare the growth factor and cytokine content found within the amnion and chorion layers and to determine the effects of dehydration on them.

Materials and Methods.

Placentas were collected from 5 to 6 consented donors following elective cesarean section, and 1-cm² sections of either amnion or chorion were immediately stored at −80°C or dehydrated prior to −80°C storage until proteomic analysis. Signaling molecules from tissue samples were evaluated using quantitative multiplex proteomics microarrays, and data were analyzed based on a per cm² basis and also on pg/mg of extracted protein for potency.

Results.

Fresh chorion contained more of some signaling molecules per cm² compared with amnion. Specifically, the chorion contained significantly higher levels of adiponectin, APN, ANG-2, bFGF, EG-VEGF, HGF, IGF-1, PDGF-AA, PDGF-BB, TIMP-2, and TIMP-4. When samples were dehydrated, a significant drop in total growth factor and cytokine content was observed in both amnion and chorion samples with a loss of 51.1% ± 20.2% and 55.5% ± 37.3%, respectively. When evaluating the potency of fresh amnion and fresh chorion, there were similar levels of signaling molecules found with some exceptions. Amnion had significantly higher GAL-7, TGF-β1, and IL-1F5, and chorion had significantly more EG-VEGF, PDGF-BB, and TIMP-2.

Conclusion.

The processing of placental membranes can have a dramatic effect on the total growth factor and cytokine load found within these tissues.

Amniotic membranes have a long history of use for the treatment of wounds, dating back to 1910.¹ In their fresh state, amniotic membranes contain various multipotential cells, growth factors, and extracellular matrix proteins that support healing. These membranes are especially suited for chronic wound healing, because they are known to naturally address many of the contributing factors of chronic wound development, including suppressing dysregulated/uncontrolled inflammatory responses, increasing levels of matrix metalloproteinase inhibitors in the wound environment, stimulating proliferation and migration of important cell types, and promoting angiogenesis.^2–4 Most commercially available membrane products are processed by dehydration or lyophilization, which alter those characteristics to varying degrees, and the layers included in these grafts vary with most containing a double-layer amnion, amnion and chorion, or the amnion layer alone.

The purpose of this study is to compare the growth factor and cytokine content found within the amnion and chorion layers and to determine the effects of dehydration on these signaling molecules.

Materials and Methods

Placentas were collected following elective cesarean sections from 6 donors who gave written informed consent. The placental disk was removed and amnion and chorion layers were separated via blunt dissection, with a total of 6 donors used for amnion analysis and 5 donors for chorion. Membranes were washed in sterile saline and cut into 1-cm² sections. To compare the structural differences between the fresh and dehydrated samples, tissue was paraffin embedded, sectioned, and stained with H&E. For proteomic assays, 1-cm² sections were either immediately stored at −80°C or dehydrated using standard techniques prior to storage at −80°C until analysis. Of note, all sections (fresh and dehydrated) were deep frozen for a short period of time to equally preserve protein content until analysis of all donors and groups. Growth factor and cytokine content were assessed using a quantitative multiplex enzyme-linked immunosorbent assay (ELISA) proteomics microarray (RayBiotech, Inc, Norcross, GA). Signaling molecules evaluated in this study are thought to be relevant to wound healing and have previously been identified within placental-derived tissues.^2,4,5 Tissue samples were first homogenized using a Retsch CryoMill (Verder Scientific Inc, Newtown, PA). After cryomilling, the tissue was incubated overnight in a total protein extraction buffer with a protease inhibitor cocktail (EMD Millipore, Billerica, MA) at 4°C with agitation. Following incubation, the supernatant was removed and loaded into the microarray chambers and the assay carried out per the manufacturer’s instructions. The slides were imaged using a GenePix 4000B Microarray Scanner (Molecular Devices, Sunnyvale, CA), and scanned images were imported and analyzed using GenePix Pro 7 Software (Molecular Devices, Sunnyvale, CA). Total growth factor and cytokine content were then represented as pg/cm². To compare the potency of the signaling molecules within each membrane, the extracted protein was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), and the growth factor and cytokine loads were normalized to the total extracted protein from either amnion or chorion. For this study, growth factors and cytokines have been categorized into general functional areas (Table). A Student’s t-test was used to determine significance between the groups, and an asterisk was used to indicate P < .05.

Table.

Relevant growth factor and cytokine categories

Angiogenic
aFGF	PlGF
ANG	SDF-1
ANG-2	TSP-1
bFGF	VEGF
EG-VEGF	VEGF-D
PDGF-AA	ANGPTL4
PDGF-BB
Regenerative
APN	IGF-1
EGF	IGF-2
GAL-7	TGF-α
HGF	TGF-β1
IGFBP-1	TGF-β3
IGFBP-5
Immune Modulating
IL-1F5	IL-10
il-1ra	TIMP-1
IL-4	TIMP-2
IL-6	TIMP-4
IL-8	TNF-α

Results

Qualitative analysis of the H&E tissue samples indicated that dehydration of the membranes resulted in a thinner, more condensed structure, with a loss of visible porosity (Figure 1). In general, both unprocessed amnion and chorion had similar growth factor and cytokine compositions; however, there were some differences in distribution (Figure 2). Fresh chorion contained more growth factors and cytokines per cm² compared with amnion, likely due to the overall increased thickness compared with amnion. Specifically, fresh chorion contained significantly higher levels of APN, ANG, ANG-2, bFGF, EG-VEGF, HGF, IGF-1, PDGF-AA, PDGF-BB, TIMP-2, and TIMP-4 (data not shown). When samples were dehydrated, a significant drop in total growth factor and cytokine content was observed in both amnion and chorion samples with a loss of 51.1% ± 20.2% and 55.5% ± 37.3%, respectively (Figure 3). When comparing the potency of amnion and chorion membranes (pg/mg extracted protein), the investigators found the membranes were similar in overall composition with some exceptions. Amniotic membranes had significantly higher levels of GAL-7, TGF-β1, and IL-1F5, and chorion membranes had significantly higher levels of EG-VEGF, PDGF-BB, and TIMP-2 (Figure 4).

Figure 1.

Histological assessment of effects of processing. (A) H&E of unprocessed human amnion/chorion; (B) H&E of dehydrated human amnion/chorion. Yellow and blue outlines indicate amnion and chorion, respectively. During dehydration, removal of moisture content results in a collapse of the extracellular matrix, as shown by the overall reduction of thickness (scale bar: 100μm) and removal of white space (or pores) throughout the matrix shown in unprocessed membranes (A).

Figure 2.

Relative levels for all growth factors and cytokines measured within unprocessed (A) human amnion and (B) chorion in pg/cm².

Figure 3.

Effect of dehydration on amnion and chorion membranes. Growth factor and cytokine levels for amnion (n=6; A-D) and chorion (n=5; E-H) were measured with Multiplex ELISA proteomic microarray. (A-D) White bars represent fresh amnion and black bars represent dehydrated amnion. Data shown include (A) group totals, (B) angiogenic, (C) regenerative, and (D) immune modulating signaling molecules. (E-H) White bars represent fresh chorion and stripped bars represent dehydrated chorion. Data shown include (E) group totals, (F) angiogenic, (G) regenerative, and (H) immune modulating signaling molecules. Average results are shown. Error bar represents 1 standard deviation.

^a significant difference: P<.05

Figure 4.

Potency comparison between unprocessed/fresh amnion (n=6) and chorion (n=5). White bars represent amnion values and black bars represent chorion for all graphs. (A) Group totals, (B) angiogenic, (C) regenerative, and (D) immune modulating signaling molecules. Bars represent the average ± standard deviation.

^a significant difference: P<.05

Discussion

While previous studies have evaluated the cytokine content within the placental membrane for specific commercial products^6–9 and have investigated the effects of processing on the amnion,^10,11 there is a lack of literature comparing the cytokine content of the amnion and chorion and evaluating how these tissues are altered by dehydration. Likely related to the overall thicker nature of the chorion, the investigators found a greater growth factor and cytokine load within the fresh chorion compared with the fresh amnion (pg/cm²). The levels of growth factors within both membranes were significantly lower following dehydration (compared with fresh tissue). Previous studies that have compared dehydrated amnion with either fresh or cryopreserved amnion have found significant changes to the structure of the membrane, and with the exception of Paolin et al,¹¹ other groups have also observed a decrease in cytokine content after dehydration.^8,9,11 Researchers hypothesize the observed drop in growth factor content following dehydration may be due in part to perturbations of the protein structure known to occur during the dehydration process.¹² When comparing the potency of the amnion and chorion by normalizing growth factor concentration to the quantity of extracted protein, the investigators found that overall both membranes had similar compositions in terms of potency; however, there were some significant differences between the composition of the membranes in regards to specific growth factors.

Placental membranes have been shown to be effective in treating difficult-to-heal chronic wounds^13–15; however, the results presented herein indicate both processing and composition of amniotic grafts play an important role in their content. When evaluating amniotic products for clinical use, it is important to consider which tissue layers these grafts contain and how these grafts are processed (ie, dehydrated, cryopreserved, or fresh).

Limitations

Limitations for this study include the number of donors (N = 5–6), the cytokines evaluated, and the evaluation of only nonsterilized grafts (while most commercial grafts are terminally sterilized). All samples were snap frozen prior to cytokine and growth factor analysis; while this is an appropriate technique for protein preservation, there is the possibility of some sample degradation. In addition, this study evaluated signaling molecules by whether they were present and did not address protein denaturing and how that may affect bioactivity. Future studies will focus on the evaluation of other growth factors/cytokines of interest, the use of mechanistic studies on key cell types to evaluate the effect of processing and sterilization on the bioactivity of growth factors and cytokines within membranes, and the clinical relevance of these findings.

Conclusion

Fresh chorion contained more of some signaling molecules per cm² compared with amnion, and both dehydrated samples saw a significant drop in total growth factor and cytokine content (51.1% ± 20.2% for amnion; 55.5% ± 37.3% for chorion). When evaluating the potency of fresh amnion and fresh chorion, there were similar levels of signaling molecules found with some exceptions. In conclusion, the processing of placental membranes can have a dramatic effect on the total growth factor and cytokine load found within these tissues.

Abbreviation Key

aFGF

acidic fibroblast growth factor

ANG

angiopoietin

ANGPTL4

angiopoietin-like 4

APN

adiponectin

bFGF

basic fibroblast growth factor

BMP

bone morphogenetic protein

EG-VEGF

Endocrine gland-derived vascular endothelial growth factor

EGF

endothelial growth factor

ELISA

enzyme-linked immunosorbent assay

GAL

galectin

H&E

hematoxylin & eosin

HGF

hepatocyte growth factor

IGF

insulin-like growth factor

IGFBP

insulin-like growth factor-binding protein

IL

interleukin

IL-1RA

interleukin-1receptor antagonist

IL-1F5

interleukin 36 receptor antagonist/interleukin 1 family member

OPG

osteoprotegerin

OPN

osteopontin

PDGF

platelet-derived growth factor

PIGF

placental growth factor

SDF

stromal cell-derived factor

TGF

transforming growth factor

TIMP

tissue inhibitor of metalloproteinase

TNF

tumor necrosis factor

TSP

thrombospondin

VEGF

vascular endothelial growth factor