Cryopreserved amniotic membrane in chronic nonhealing wounds: a series of case reports.

A case series of the use of amniotic membrane (AM) for treating chronic nonhealing wounds. It presents five cases of polymorbid patients with a total of nine chronic nonhealing wounds. The patient group consisted of four men and one woman with various comorbidities, aged 45-72 years. The mean initial wound size was 15.8 cm2, and the mean time from the onset of the wound to the first application of AM was 122 weeks. The wounds were caused by chronic venous insufficiency and/or peripheral arterial disease. Wounds were treated in a standardized protocol. AM was applied weekly in the first month and then every two weeks. Photo documentation of the wound and microbiological colonization was carried out at each visit. In three out of five patients, the AM treatment effectively promoted healing up to complete wound closure. In two cases, the wounds stayed unhealed despite numerous AM applications. Pain relief was noted in all patients. The success of the treatment was closely tied to patient factors, such as adherence to the prescribed treatment regimen and individual patient characteristics. In some cases, treatment failure was observed, possibly due to underlying comorbidities, wound parameters, or poor patient compliance. AM treatment has the potential to become a viable treatment option for these nonhealing wounds. However, the effectiveness of the treatment may be influenced by various patient factors and the underlying cause of the wound. Therefore, it is crucial to have an individualized treatment plan that considers these particular factors.

Inter-placental variability is not a major factor affecting the healing efficiency of amniotic membrane when used for treating chronic non-healing wounds.

This study aimed to evaluate the efficacy of cryopreserved amniotic membrane (AM) grafts in chronic wound healing, including the mean percentage of wound closure per one AM application, and to determine whether the healing efficiency differs between AM grafts obtained from different placentas. A retrospective study analyzing inter-placental differences in healing capacity and mean wound closure after the application of 96 AM grafts prepared from nine placentas. Only the placentas from which the AM grafts were applied to patients suffering from long-lasting non-healing wounds successfully healed by AM treatment were included. The data from the rapidly progressing wound-closure phase (p-phase) were analyzed. The mean efficiency for each placenta, expressed as an average of wound area reduction (%) seven days after the AM application (baseline, 100%), was calculated from at least 10 applications. No statistical difference between the nine placentas' efficiency was found in the progressive phase of wound healing. The 7-day average wound reduction in particular placentas varied from 5.70 to 20.99% (median from 1.07 to 17.75) of the baseline. The mean percentage of wound surface reduction of all analyzed defects one week after the application of cryopreserved AM graft was 12.17 ± 20.12% (average ± SD). No significant difference in healing capacity was observed between the nine placentas. The data suggest that if there are intra- and inter-placental differences in AM sheets' healing efficacy, they are overridden by the actual health status of the subject or even the status of its individual wounds.

The healing dynamics of non-healing wounds using cryo-preserved amniotic membrane.

We evaluated the effect of the application of cryo-preserved amniotic membrane on the healing of 26 non-healing wounds (18 patients) with varying aetiologies and baseline sizes (average of 15.4 cm2 ), which had resisted the standard of care treatment for 6 to 456 weeks (average 88.8 weeks). Based on their average general responses to the application of cryo-preserved AM, we could differentiate three wound groups. The first healed group was characterised by complete healing (100% wound closure, maximum treatment period 38 weeks) and represented 62% of treated wounds. The wound area reduction of at least 50% was reached for all wounds in this group within the first 10 weeks of treatment. Exactly 19% of the studied wounds responded partially to the treatment (partially healed group), reaching less than 25% of closure in the first 10 weeks and 90% at maximum for extended treatment period (up to 78 weeks). The remaining 19% of treated wounds did not show any reaction to the AM application (unhealed defects). The three groups have different profiles of wound area reduction, which can be used as a guideline in predicting the healing prognosis of non-healing wounds treated with a cryo-preserved amniotic membrane.

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2.

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions.

Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.

The comet assay applied to cells of the eye.

The human eye is relatively unexplored as a source of cells for investigating DNA damage. There have been some clinical studies, using cells from surgically removed tissues, and altered DNA bases as well as strand breaks have been measured using the comet assay. Tissues examined include corneal epithelium and endothelium, lens capsule, iris and retinal pigment epithelium. For the purpose of biomonitoring for exposure to potential mutagens in the environment, the eye-relatively unprotected as it is compared with the skin-would be a valuable object for study; non-invasive techniques exist to collect lachrymal duct cells from tears, or cells from the ocular surface by impression cytology, and these methods should be further developed and validated.

Antimicrobial efficiency and stability of two decontamination solutions.

Two decontamination solutions, commercially produced BASE•128 and laboratory decontamination solution (LDS), with analogous content of antibiotic and antimycotic agents, were compared in their antimicrobial efficiency and stability (pH and osmolarity). Both solutions were compared immediately after thawing aliquots frozen for 1, 3 or 6 months. Agar well diffusion method was used to test their antimicrobial efficiency against five human pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli and Enterococcus faecalis. The difference in the inhibition of growth between the two decontamination solutions was mostly not statistically significant, with few exceptions. The most pronounced difference between the LDS and BASE•128 was observed in their decontamination efficacy against E. coli and E. faecalis, where the LDS showed to be more efficient than BASE•128. The osmolarity value of LDS decreased with cold-storage, the osmolarity values of the BASE•128 could not be measured as they were below the range of the osmometer. Slight changes were found in pH of the less stable LDS solution, whose pH increased from initial value 7.36 ± 0.07 to 7.72 ± 0.19 after 6 m-storage. We verified that BASE•128 and LDS are similarly efficient in elimination of possible placental bacterial contaminants and may be used for decontamination of various tissues.

Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review.

The use of amniotic membrane in ophthalmic surgery and other surgical procedures in the fields of dermatology, plastic surgery, genitourinary medicine and otolaryngology is on the increase. Furthermore, amniotic membrane and its epithelial and mesenchymal cells have broad use in regenerative medicine and hold great promise in anticancer treatment. Amniotic membrane is a rich source of biologically active factors and as such, promotes healing and acts as an effective material for wound dressing. Amniotic membrane supports epithelialization and exhibits anti-fibrotic, anti-inflammatory, anti-angiogenic and anti-microbial features. Placentas utilised in the preparation of amniotic membrane are retrieved from donors undergoing elective caesarean section. Maternal blood must undergo serological screening at the time of donation and, in the absence of advanced diagnostic testing techniques, 6 months postpartum in order to cover the time window for the potential transmission of communicable diseases. Amniotic membrane is prepared by blunt dissection under strict aseptic conditions, then is typically transferred onto a nitrocellulose paper carrier, usually with the epithelial side up, and cut into multiple pieces of different dimensions. Amniotic membrane can be stored under various conditions, most often cryopreserved in glycerol or dimethyl sulfoxide or their mixture with culture medium or buffers. Other preservation methods include lyophilisation and air-drying. In ophthalmology, amniotic membrane is increasingly used for ocular surface reconstruction, including the treatment of persistent epithelial defects and non-healing corneal ulcers, corneal perforations and descemetoceles, bullous keratopathy, as well as corneal disorders with associated limbal stem cell deficiency, pterygium, conjunctival reconstruction, corneoscleral melts and perforations, and glaucoma surgeries.

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion.

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.

The effect of culture medium and carrier on explant culture of human limbal epithelium: A comparison of ultrastructure, keratin profile and gene expression.

Patients with limbal stem cell deficiency (LSCD) often experience pain and photophobia due to recurrent epithelial defects and chronic inflammation of the cornea. Successfully restoring a healthy corneal surface in these patients by transplantation of ex vivo expanded human limbal epithelial cells (LECs) may alleviate these symptoms and significantly improve their quality of life. The clinical outcome of transplantation is known to be influenced by the quality of transplanted cells. Presently, several different protocols for cultivation and transplantation of LECs are in use. However, no consensus on an optimal protocol exists. The aim of this study was to examine the effect of culture medium and carrier on the morphology, staining of selected keratins and global gene expression in ex vivo cultured LECs. Limbal biopsies from cadaveric donors were cultured for three weeks on human amniotic membrane (HAM) or on tissue culture coated plastic (PL) in either a complex medium (COM), containing recombinant growth factors, hormones, cholera toxin and fetal bovine serum, or in medium supplemented only with human serum (HS). The expanded LECs were examined by light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) for keratins K3, K7, K8, K12, K13, K14, K15 and K19, as well as microarray and qRT-PCR analysis. The cultured LECs exhibited similar morphology and keratin staining on LM, TEM and IHC examination, regardless of the culture condition. The epithelium was multilayered, with cuboidal basal cells and flattened superficial cells. Cells were attached to each other by desmosomes. Adhesion complexes were observed between basal cells and the underlying carrier in LECs cultured on HAM, but not in LECs cultured on PL. GeneChip Human Gene 2.0 ST microarray (Affymetrix) analysis revealed that 18,653 transcripts were ≥2 fold up or downregulated (p ≤ 0.05). Cells cultured in the same medium (COM or HS) showed more similarities in gene expression than cells cultured on the same carrier (HAM or PL). When each condition was compared to HAM/COM, no statistical difference was found in the transcription level of the selected genes associated with keratin expression, stemness, proliferation, differentiation, apoptosis, corneal wound healing or autophagy. In conclusion, the results indicate that ex vivo cultures of LECs on HAM and PL, using culture media supplemented with COM or HS, yield tissues with similar morphology and keratin staining. The gene expression appears to be more similar in cells cultured in the same medium (COM or HS) compared to cells cultured on the same carrier (HAM or PL).

Identification of six novel mutations in ZEB1 and description of the associated phenotypes in patients with posterior polymorphous corneal dystrophy 3.

Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584*) and c.1717_1718del; p.(Val573Phefs*12) in two Czech families, c.1176dup; p.(Ala393Serfs*19), c.1100C>A; p.(Ser367*), c.627del; p.(Phe209Leufs*11) in three British families and a splice site mutation, c.685-2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs*3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5-52.7 diopters, steep keratometry 48.1-54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation.

Detailed assessment of renal function in a proband with Harboyan syndrome caused by a novel homozygous SLC4A11 nonsense mutation.

BACKGROUND/AIMS: To identify the underlying molecular genetic cause of disease in a patient with Harboyan syndrome and to perform a detailed assessment of her renal function. We also assessed the influence of the SLC4A11 mutation identified on the corneal endothelium in the heterozygous state. METHODS: A 55-year-old female was examined ophthalmologically, audiologically and nephrologically including 24-hour urine collection. The coding region of SLC4A11 was directly sequenced. Specular microscopy was performed in the proband's 21-year-old daughter. RESULTS: The proband had bilateral iridectomy at the age of 3 months because of an initial diagnosis of congenital glaucoma and since the age of 12 years she underwent several keratoplasties in each eye. Nephrological examination did not reveal any abnormalities. Moderate bilateral sensorineural hearing loss was confirmed by audiometry. A novel homozygous mutation predicted to lead to a premature stop codon at the protein level, c.2188C>T; p.(Arg730*), was identified in SLC4A11. No changes in corneal endothelial cell morphology or density were observed in the heterozygous daughter. CONCLUSION: In contrast to the Slc4a11(-/-) mouse, no abnormalities in daily renal ion excretion or polyuria were observed in the Harboyan syndrome patient. The mutation identified does not affect corneal endothelial cell morphology or density in the heterozygous state.

Molecular and biochemical expression of TLRs in human amniotic membrane: a comparative study of fresh and cryopreserved specimens.

BACKGROUND: To assess the expression of toll-like receptors (TLRs) in human amniotic membrane (AM) specimens and compare this expression with those of AMs undergoing the standard preservation procedure (handling) for ocular surgery. METHODS: Human fresh (n = 10; five spontaneous and five cesarean) or handled (n = 5) AMs were analyzed for TLR gene and protein expression. Two pieces were obtained from each specimen, and subjected to molecular or biochemical analysis. Relative real-time PCR and SDS-PAGE were carried out according to standard procedures. The REST-ANOVA coupled analysis was used to compare the molecular and biochemical data. RESULTS: The fresh membranes expressed all the TLRs (TLR1-10), with different gene expression as detected/evidenced by the Ct values, the intra-fresh group analysis showing that there was a variation of TLR expression whichvaried within the fresh membranes. The handled AMs retained the TLR expression after standard processing and preservation, but with a particular pattern which included a high TLR3/TLR4 and low TLR6 expression, when compared to the fresh membranes. The molecular data were confirmed by Western blot analysis. CONCLUSIONS: AM is routinely used in several ophthalmic surgical procedures, and notwithstanding its preservation procedure, AM is reported to favour wound healing and exert anti-angiogenic, anti-inflammatory, anti-scarring as well as anti-bacterial activities. The presence of TLRs in handled AM would imply that TLRs might be preserved in AMs used in ocular surgery. The findings herein described provide additional data concerning the presence of TLRs in cryopreserved AM, and suggest a possible contribution of AM in ocular surgery, via the innate immune response.

Rapid cooling of the amniotic membrane as a model system for the vitrification of posterior corneal lamellae.

To vitrify human amniotic membrane specimens so that the maximum of epithelial cells survives in order to develop a procedure for the eventual vitrification of posterior corneal lamellae without using cryoprotective agents. To assess different methods of tissue sample preparation preceding vitrification. In group 1, the amniotic membrane specimens were stretched on nitrocellulose support. In group 2, mechanical pressure was used to remove the excess culture medium between the support and the membrane. The samples were frozen in liquid ethane (-183 °C) and stored in liquid nitrogen. The specimens in the control group were not vitrified. Re-warming was performed at 40 °C. The epithelial cell survival rate was assessed after 1, 3 and 7 days of storage following re-warming using calcein and ethidium homodimer-1 fluorescence. A wide range of values was observed among the different groups and among individual specimens within the groups. Resulting average survival rate was 41 % for group 1 and 53 % for group 2; in several samples the cell survival rate exceeded 70 %. The storage period did not significantly affect the survival rates. The results of the rapid cooling of amniotic membranes in liquid ethane indicate that significant percentage of epithelial cells remain viable after the re-warming.

Electrospun poly(l-lactide-co-dl-lactide) nanofibrous scaffold as substrate for ex vivo limbal epithelial cell cultivation.

Ultrathin electrospun poly (l-lactide-co-dl-lactide) nanofibrous membranes coated with fibronectin were explored as scaffolds for the ex vivo cultivation of limbal epithelial cells (LECs) for the treatment of limbal stem cell deficiency. The developed scaffolds were compared with the "gold-standard" fibrin gel. The resulting membranes composed of nanofibers possessed a very low thickness of 4 µm and allowed very good optical transparency in the wet state. The fibronectin-coated nanofibrous scaffolds demonstrated LEC expansion and successful cultivation similar to that on fibrin gel. Unlike the regular cobblestone epithelial cell morphology on the fibrin gel, the nanofibrous scaffold presented a mostly irregular epithelial morphology with a shift to a mesenchymal phenotype, as confirmed by the upregulation of profibroblastic genes: ACTA2 (p = 0.023), FBLN1 (p < 0.001), and THY1 (p < 0.001). Both culture conditions revealed comparable expression of stem cell markers, including KLF4, ΔNp63α and ABCG2, emphasizing the promise of polylactide-based nanofibrous membranes for further investigations.

The impairment of lysyl oxidase in keratoconus and in keratoconus-associated disorders.

Keratoconus (KC) is an eye disease characterized by the progressive thinning and protrusion of the cornea, which results in the loss of visual acuity. This disorder remains poorly understood, although recent studies indicate the involvement of genetic and environmental factors. Recently, we have found that the distribution of the cross-linking enzyme lysyl oxidase (LOX) is markedly decreased in about 63 % of keratoconic specimens. Similarly, LOX activity is significantly reduced by 38 % compared to control tissue. Nearly 70 systemic disorders have been reported in association with KC, most of them affecting the extracellular matrix. In this review we attempted to ascertain whether any KC-associated diseases exhibit signs that may reflect LOX impairment. We hypothesized that very similar changes in the extracellular matrix, particularly at the level of collagen metabolism, including LOX impairment in mitral leaflets, may reflect an association between KC and mitral valve prolapse. Moreover, this putative association is supported by the high frequency of Down syndrome in both diseases. Among other disorders that have been found to coincide with KC, we did not find any in which the LOX enzyme may be directly or indirectly impaired. On the other hand, in cases where KC is present along with other connective tissue disorders (Marfan syndrome, Ehlers-Danlos syndrome and others), KC may not arise as a localized manifestation, but rather may be induced as the result of a more complex connective tissue disorder.

Endothelial quality of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S): two-year outcome of manual preparation in an ocular tissue bank.

To assess the quantitative and qualitative parameters of pre-cut posterior corneal lamellae for Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S) prepared manually in the Ocular Tissue Bank Prague. All 65 successfully prepared pre-cut posterior corneal lamellae provided for grafting during a 2-year period were analyzed retrospectively. The lamellae, consisting of a central zone of endothelium-Descemet membrane surrounded by a supporting peripheral stromal rim, were prepared manually from corneoscleral buttons having an endothelial cell density higher than 2,500 cells/mm(2). The live endothelial cell density, the percentage of dead cells, the hexagonality and the coefficient of variation were assessed before and immediately after preparation as well as after 2 days of organ culture storage at 31 °C. Altogether, the endothelium of 57 lamellae was assessed. Immediately after preparation, the mean live endothelial cell density was 2,835 cells/mm(2) and, on average, 1.8 % of dead cells were found. After 2 days of storage, the cell density decreased significantly to 2,757 cells/mm(2) and the percentage of dead cells to 1.0 %. There was a significant change in the mean hexagonality and the coefficient of variation after lamellar preparation and subsequent storage. The amount of tissue wasted during the preparation was 23 %. The endothelial cell density of posterior corneal lamellae sent for DMEK-S was higher than 2,700 cells/mm(2) in average with a low percentage of dead cells; 65 pre-cut tissues were used for grafting during a 2-year period.

Distribution of an analgesic palmitoylethanolamide and other N-acylethanolamines in human placental membranes.

BACKGROUND: Human amniotic and amniochorionic membranes (AM, ACM) represent the most often used grafts accelerating wound healing. Palmitoylethanolamide, oleoylethanolamide and anandamide are endogenous bioactive lipid molecules, generally referred as N-acylethanolamines. They express analgesic, nociceptive, neuroprotective and anti-inflammatory properties. We assessed the distribution of these lipid mediators in placental tissues, as they could participate on analgesic and wound healing effect of AM/ACM grafts. METHODS: Seven placentas were collected after caesarean delivery and fresh samples of AM, ACM, placental disc, umbilical cord, umbilical serum and vernix caseosa, and decontaminated samples (antibiotic solution BASE 128) of AM and ACM have been prepared. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used for N-acylethanolamines analysis. RESULTS: N-acylethanolamines were present in all studied tissues, palmitoylethanolamide being the most abundant and the anandamide the least. For palmitoylethanolamide the maximum average concentration was detected in AM (350.33 ± 239.26 ng/g), while oleoylethanolamide and anandamide were most abundant in placenta (219.08 ± 79.42 ng/g and 30.06 ± 7.77 ng/g, respectively). Low levels of N-acylethanolamines were found in serum and vernix. A significant increase in the levels of N-acylethanolamines (3.1-3.6-fold, P < 0.001) was observed in AM when the tissues were decontaminated using antibiotic solution. The increase in decontaminated ACM was not statistically significant. CONCLUSIONS: The presence of N-acylethanolamines, particularly palmitoylethanolamide in AM and ACM allows us to propose these lipid mediators as the likely factors responsible for the anti-hyperalgesic, but also anti-inflammatory and neuroprotective, effects of AM/ACM grafts in wound healing treatment. The increase of N-acylethanolamines levels in AM and ACM after tissue decontamination indicates that tissue processing is an important factor in maintaining the analgesic effect.

Outcome of Application of Cryopreserved Amniotic Membrane Grafts in the Treatment of Chronic Nonhealing Wounds of Different Origins in Polymorbid Patients: A Prospective Multicenter Study.

To compare the therapeutic efficacy of cryopreserved amniotic membrane (AM) grafts and standard of care (SOC) in treating nonhealing wounds (NHW) through a prospective multicenter clinical trial, 42 patients (76% polymorbid) with 54 nonhealing wounds of various etiologies (mainly venous) and an average baseline size of 20 cm2 were included. All patients were treated for at least 6 weeks in the center before they were involved in the study. In the SOC group, 29 patients (36 wounds) were treated. If the wound healed less than 20% of the baseline size after 6 weeks, the patient was transferred to the AM group (35 patients, 43 wounds). Weekly visits included an assessment of the patient's condition, photo documentation, wound debridement, and dressing. Quality of life and the pain degree were subjectively reported by patients. After SOC, 7 wounds were healed completely, 1 defect partially, and 28 defects remained unhealed. AM application led to the complete closure of 24 wounds, partial healing occurred in 10, and 9 remained unhealed. The degree of pain and the quality of life improved significantly in all patients after AM application. This study demonstrates the effectiveness of cryopreserved AM grafts in the healing of NHW of polymorbid patients and associated pain reduction.

Quantification of Analgesic and Anti-Inflammatory Lipid Mediators in Long-Term Cryopreserved and Freeze-Dried Preserved Human Amniotic Membrane.

The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators-palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)-in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM's analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3-5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts.