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.

Lyophilized amniotic membrane graft for primary pterygium surgery: preliminary results.

The aim of this study was to investigate the tolerability, safety and efficacy of new lyophilized amniotic membrane (LAM) presentation for ocular use. A prospective case-series cohort of four patients with primary nasal pterygium which undergone excision and LAM implantation was evaluated for complications and clinical outcomes. Surgical manipulation of LAM was also assessed. LAM was stiff and easy to manipulate as well as no tearing occurred during surgery or suturing. Ocular comfort was checked and similar among those patients with LAM glued or sutured. After 12 months, there were no issues about tolerability or adverse events. Lower cosmetic outcomes (recurrence) were stated in 3 patients. Our study showed that LAM could be an effective alternative to cryopreserved amniotic membrane for graft after pterygium excision surgery. Its main advantage, storage at room temperature, can make it of immediate availability. Further studies comparing clinical outcomes of pterygium surgery with cryopreserved amniotic membrane versus LAM would confirm the benefits of the last.

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.

Pterygium surgery with lyophilized versus cryopreserved amniotic membrane graft.

PURPOSE: To evaluate surgical outcomes (recurrence rate, aesthetics and symptoms) of pterygium surgery with two different amniotic membrane preservation approaches - lyophilized (LAM) and cryopreserved (CAM). METHODS: Primary pterygium patients were randomized to either LAM or CAM surgery. Demographic data, ocular surface disease index (OSDI), aesthetic grading (1 to 4), recurrences and complications were recorded over a 6-month follow-up period. RESULTS: Twenty-nine patients were recruited. Recurrence at month 6 was detected in 11 cases (37.9%) and was more prevalent with CAM grafts, without reaching statistical significance (P=0.196). Aesthetic outcome grading showed no differences between LAM and CAM at month 6 (P=0.124). Aesthetic results were mostly unsatisfactory (grade 3 and 4) without statistical differences between groups (P=0.514). Baseline OSDI was similar in both groups (P=0.888), and it significantly decreased by the last follow-up visit (P<0.001) for both the LAM and CAM groups. This decrease did not significantly differ between amniotic membrane preservation approach surgery groups (P=0.714). CONCLUSION: LAM might be considered a legitimate alternative to CAM, showing no inferiority in outcomes, since clinical and aesthetic outcomes were similar for both groups.

Microbial Keratitis following Self-Retained Cryopreserved Amniotic Membrane.

The aim of this study was to report a severe case of mixed fungal and bacterial microbial keratitis following implantation of a self-retained cryopreserved amniotic membrane, PROKERA® SLIM (Bio-Tissue, Inc) in a patient with history of neurotrophic ulcer secondary to herpetic epithelial keratitis. Despite maximally tolerated topical and systemic therapy, the patient's eye continued to deteriorate and eventually required evisceration. PROKERA implantation might be associated with severe recalcitrant microbial keratitis. Caution is urged when considering implantation especially in monocular patients.

A comparative study of different amniotic membrane orientations during extraocular muscle surgery in rabbits.

PURPOSE: To histopathologically compare the effect of different orientations of cryopreserved human amniotic membrane (AM) transplant during extraocular muscle surgery in rabbits. METHODS: Fifty-two albino rabbit eyes underwent 4-mm resection of the superior rectus. Eyes were randomly divided into four groups. In Group C (Control group, 16 eyes) the muscle was not wrapped with amniotic membrane. In the three AM groups, cryopreserved AM was wrapped around the muscle, oriented with either its stroma (Group S, 15 eyes) or epithelium (Group E, nine eyes) towards the muscle, or folded on itself with the epithelium externally (Group F, 12 eyes). The rabbits were sacrificed and the eyes were enucleated 6 weeks after surgery. Histopathological examination was conducted for periamniotic, foreign body, scleral, and conjunctival inflammation, conjunctival vascularity, adhesions and muscle fibrosis. RESULTS: In all AM eyes, the AM was surrounded by periamniotic inflammation, with no adhesions detected between the muscle and surrounding tissues in the segment where the AM was present, but detected elsewhere. Adhesions were detected in all group C eyes. Foreign body inflammation was significantly less in Group C than in each of the AM groups (p < .05), but was insignificantly different among the three AM groups (p > .05). Scleral inflammation was absent in all specimens. No significant differences were noted among all groups in terms of conjunctival vascularity, conjunctival inflammation, or muscle fibrosis (p > .05). CONCLUSIONS: All AM orientations were equally effective in preventing the development of postoperative adhesions between the extraocular muscle and surrounding tissues.

The Effect of Cryopreserved Human Placental Tissues on Biofilm Formation of Wound-Associated Pathogens.

Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers) clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM) resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT) on biofilm formation of S. aureus and P. aeruginosa, the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.

A Novel Technique for Amniotic Membrane Transplantation in Patients with Acute Stevens-Johnson Syndrome.

Cryopreserved amniotic membrane (AM) transplantation is an emerging technique that is becoming the gold standard for the management of acute Stevens-Johnson syndrome (SJS) and its more severe variant, toxic epidermal necrolysis (TEN). We describe a novel surgical technique utilizing a single, large sheet of AM (5 x 10 cm) and a custom-made forniceal ring, which facilitates AM placement. Our technique is easy to use and minimizes suturing and manipulation of ocular tissues, resulting in decreased operative time. This technique may be applied in the management of multiple ocular surface disease processes, including chemical or thermal burns, severe ocular graft versus host disease (GVHD), and other autoimmune diseases.