Cryopreserved Amniotic Membrane Research Articles

Association of Treatment Duration and Clinical Outcomes in Dry Eye Treatment with Sutureless Cryopreserved Amniotic Membrane.

While sutureless, cryopreserved amniotic membrane (cAM) has been shown to significantly improve signs and symptoms of dry eye disease (DED), no studies have assessed the association of cAM treatment duration to the differential response in clinical outcomes. A multi-center, retrospective study was conducted on patients with moderate-to-severe DED who were treated with self-retained cAM (Prokera® Slim) for 2 to 7 days. The primary outcome measure was DEWS severity score assessed at 1 week, 1 month, and 3 months. Secondary outcome measures included ocular discomfort, visual symptoms, corneal staining, and visual acuity. A total of 89 eyes (77 patients) with moderate-to-severe DED (DEWS severity 3.24 ± 0.56) received treatment with self-retained cAM for 2 days (n = 10), 3 days (n = 15), 4 days (n = 12), 5 days (n = 19), 6 days (n = 6), or 7 days (n = 27). DEWS scores significantly improved at 1 week, 1 month, and 3 months for all treatment duration groups, with no significant difference observed between groups at any timepoint. In addition to an improvement in DEWS severity scores, those receiving cAM treatment for 2 days demonstrated a significant improvement in corneal staining, visual symptoms, and ocular discomfort at 1 week, 1 month, and 3 months. This retrospective study suggests that a single placement of self-retained cAM for 2 days can significantly improve signs and symptoms of DED with a lasting benefit observed for up to 3 months.

A novel method to pack cryopreserved amniotic membrane for wound dressing—the pathway through validation of a new biological product

Amniotic membrane (AM) has been widely used as a biological dressing for many pathologies and illnesses worldwide, and products derived from this tissue have been commercially available in several countries. In Brazil, regulatory agencies have recently authorized its clinical use as a non-experimental therapy for burns, diabetic and venous stasis ulcers, and intrauterine adhesions. In this study, we present our pathway through validating the first available service in the country of AM cryopreservation, with a protocol for long-term storage in high-efficiency nitrogen cryogenic freezers and a specific way of packing the tissue for optimal clinical handling and efficient storage space utilization while preserving live cells and the tissue’s biological properties. Using gauze as support, cryoprotectant dimethyl sulfoxide and product presentation as a multilayer roll exhibited the best cell viability results and maintained the tissue integrity and presence of stem/progenitor cells. Essential proteins involved in tissue regeneration and immune and antimicrobial control were detected from the secretome of cryopreserved tissue similar to fresh tissue. Furthermore, immunogenic markers, such as human leukocyte antigens, were detected at very low levels in the tissue, confirming their low immunogenicity. Finally, we demonstrate that the tissue can be kept under refrigerated conditions for up to 7 d for further use, maintaining sterility and considerable cell viability. Our cryopreservation and storage protocol kept the AM viable for at least 20 months. In conclusion, this study enabled us to determine a novel efficient protocol for long-term AM preservation for future clinical applications.

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.

Comparison of Vestibular Depth Relapse and Wound Healing After Reconstructive Preprosthetic Surgery Using Cryopreserved Amniotic Membrane and Acellular Dermal Matrix - A Comparative Study.

Introduction:The significance of membranes as wound dressing in oral surgeries has been reported by previous studies. The aim of the present split-mouth randomized clinical study was to assess and compare the wound dressing properties of acellular dermal matrix (ADM) and cryopreserved human amniotic membrane (AM) after reconstructive preprosthetic oral surgery.Materials and Methods:Twenty-eight patients with complete mandibular edentulism and resorbed alveolar bone were included. After taking mandibular impression, a clear acrylic splint with increased labial flange height was created. In each participant, labial vestibular depth was elevated using the Clark's technique. Subsequently, half of the exposed periosteum was covered with ADM while the other half was covered with cryopreserved human AM. Vestibule depth and relapse in the two sides were measured immediately after vestibuloplasty and at the end of the 1st week, 2nd week, 1st month, and 3rd months with graduations of 0.1 mm. Furthermore, after 3 and 7 days, samples were collected from graft material, and the macrophage population was analyzed by flow cytometry.Results:There was no significant difference in the relapse of vestibule depth between the two grafts at different time intervals. However, the frequency of wound-infiltrating macrophages (CD68+ cells) was significantly higher in areas covered by ADM after 3 and 7 days.Discussion:ADM is as effective as cryopreserved AM in terms of maintaining the postoperative vestibular depth. On the other hand, our results suggested that the onset of healing phase in ADM-covered areas occurs faster compared to the periosteum covered with cryopreserved human AM. This clinical trial showed significantly faster postoperative healing onset when ADM was used than when cryopreserved human AM was applied on the periosteum.

Update on the application of amniotic membrane in immune-related ocular surface diseases.

Immune-related ocular surface diseases, a group of diseases in which immune dysregulation damages the ocular surface, can induce uncontrolled inflammation and persistent epithelial defect, thus leading to the most severe forms of acute keratoconjunctivitis, dry eye disease, epithelial keratitis, stromal ulceration, and corneal perforation. As these diseases are often refractory to treatments, they have a threatening impact on the vision and life quality of patients. This review summarizes the current literature regarding the clinical application of sutured and self-retained cryopreserved amniotic membrane (AM) in treating Stevens–Johnson syndrome/toxic epidermal necrolysis, ocular graft-versus-host disease, Sjögren's syndrome, Mooren's ulcer, and peripheral ulcerative keratitis. Current evidence supports the safety and effectiveness of AM, especially self-retained cryopreserved AM, in decreasing ocular surface inflammation, promoting corneal epithelial and stromal healing, improving visual acuity, and preventing sight-threatening complications. Future studies are still required to validate the above findings and explore the varied application methods of AM to improve the clinical efficacy in maintaining ocular surface health.

Retrospective Chart Review of Cryopreserved Amniotic Membrane for Knee Pain

Objective: Treatment of joint pain with an injection of the amniotic membrane has not been adequately studied. This study retrospectively reviewed Visual Analog Scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and analgesic usage data from patients treated with the injection of cryopreserved amniotic membrane (CAM) in their knees to determine the impact of treatment on patients’ pain, quality of life, and analgesic usage. Methods: Chart review was conducted on 40 patients. Institutional Review Board (IRB) approval was obtained prior to initiation of the project. The membrane was utilized as per the FDA guidance of 21CFR1271. Retrospective data, including demographics, medical history, pain score, quality of life score, analgesic usage and adverse events, were collected from their medical records for each consenting patient through 6 months after CAM injection. Results: A total of 40 patients were considered in the final analysis. Mean VAS for pain level improved from 7.0 to 2.6 (p<0.001). WOMAC daily activity function score improved from a mean score of 52 to 28 (p<0.001). Opioid and non-steroidal anti-inflammatory drug (NSAID) usage decreased from 97% to 25% (p<0.001). No adverse events were reported. Conclusion: Mean values for VAS and WOMAC scores significantly improved at all time points and the number of patients who used analgesics decreased as compared to baseline. CAM injection into painful knee joints decreases pain, improves physical function, and decreases the use of analgesics in the absence of adverse events.

Assessment of Corneal Haze After PRK and the Effect of Sutureless Amniotic Membrane Graft by Corneal Densitometry.

To assess whether densitometry analysis appropriately monitors the development of haze in myopic patients after photorefractive keratectomy (PRK) when compared to subjective slit-lamp haze grade examinations, and whether sutureless cryo-preserved amniotic membrane reduced postoperative haze development when compared to the standard bandage contact lens. In this retrospective cohort at the Center for Refractive Surgery, Walter Reed National Military Medical Center, a secondary analysis of prospectively collected data was performed. In the prospective study, participants underwent PRK for myopia. Postoperatively, a standard bandage contact lens was applied to the dominant eye and a sutureless cryo-preserved amniotic membrane graft to the nondominant eye. Participants were evaluated at 1, 3, and 6 months postoperatively for haze formation and corneal densitometry using slit-lamp biomicroscopy and Scheimpflug imaging, respectively. Densitometry measurements at 6 months postoperatively were positively and significantly associated with the presence or absence of haze as assessed by slit-lamp examination in 39 patients (78 eyes; age range: 21 to 44 years). Eyes with increased densitometry measurements had 2.3 to 3.4 times the odds (P ⩽ .014) of having clinical haze on slit-lamp examination. Eyes with the amniotic membrane graft showed a positive correlation with increased corneal densitometry throughout most layers of the cornea. Densitometry analysis appears to be a useful tool to supplement slit-lamp examination in monitoring haze development after PRK. The amniotic membrane failed to show a reduction in corneal densitometry in myopic eyes after PRK. [J Refract Surg. 2020;36(5):293-299.].

Amniotic membrane transplantation for managing dry eye and neurotrophic keratitis.

Neurotrophic keratitis (NK), a degenerative disease caused by damage to the trigeminal nerve, abolishes both tearing and blinking reflexes, thus causing the most severe forms of dry eye disease (DED). Conversely, the increasing severity of DED also leads to progressive loss of corneal nerve density, potentially resulting in NK. Both diseases manifest the same spectrum of corneal pathologies including inflammation and corneal epithelial keratitis, which can progress into vision-threatening epithelial defect and stromal ulceration. This review summarizes the current literature regarding outcomes following sutured and sutureless cryopreserved amniotic membrane (AM) in treating DED as well as epithelial defects and corneal ulcers due to underlying NK. These studies collectively support the safety and effectiveness of cryopreserved AM in restoring corneal epithelial health, improving visual acuity in eyes with NK and DED, and alleviating symptomatic DED. Future randomized controlled trials are warranted to validate the above findings and determine whether such clinical efficacy lies in promoting corneal nerve regeneration.

The use of self-retained, cryopreserved amniotic membrane for the treatment of Sjögren syndrome: a case series.

To determine whether signs and symptoms of ocular surface disease improve after placement of a self-retained, cryopreserved amniotic membrane (CAM) in patients with Sjögren syndrome (SS). The medical records of SS patients who received a self-retained CAM implant (Prokera or Prokera Slim; TissueTech Inc, Doral, FL) for the treatment of ocular surface disease between August 2012 and August 2016 at a single, large academic institution were reviewed retrospectively. Visual acuity, results of slit-lamp examination of the cornea and conjunctiva, and dry eye symptoms, were evaluated before and after CAM insertion. A total of 6 eyes of 6 patients (all female; mean age, 62.5 ± 13.0 years [range, 49-86 years]) were included. All patients were on topical medications at the time of the study and had signs of ocular surface dryness. There were reductions in corneal and/or conjunctival staining in 5 eyes (83%) after the CAM dissolved. All patients who completed therapy (5/5) experienced a relapse in their signs and symptoms within 1 month of removal of the CAM, with an average time to relapse of 24.6 days. Mean follow-up time was 54.5 days. Foreign body sensation and blurred vision were the most common complaints associated with the CAM implant. In this small case series, self-retained CAM implantation was found to be beneficial in SS patients with ocular surface disease that is refractory to standard therapies; however, we found that the effects were temporary. Future larger studies are needed to confirm these benefits.

Cryopreserved Amniotic Membrane (ProKera) Treatment of Anterior Stromal Haze Secondary to HSV Keratitis

Purpose: To review the efficacy of cryopreserved amniotic membrane (CAM) to reduce stromal scarring in a case of refractory anterior stromal haze related to herpes simplex keratitis (HSK). The initial treatment course of conventional topical steroid and oral antiviral was completed followed by the addition of CAM to further address scarring.
 Methods: Clinical presentation of a cornea with stromal haze that was refractory to previous steroid and topical and oral anti-viral therapy were documented with anterior segment smartphone capture. Best corrected visual acuity was documented to contrast clinical observations. Use of the cryopreserved amniotic membrane (CAM) Prokera® Slim was successful in reducing both anterior stromal haze and best corrected acuity. 
 Conclusions: CAM treatments may provide an additional therapeutic agent in the management of anterior stroma infiltration and scarring secondary to HSK. As demonstrated in this case, patients with visually significant stromal haze that is non-responsive to topical therapy may benefit from non-surgical placement of CAM. Due to the accessibility of CAM in private clinical settings, it should be considered as a viable new option for patients who have exhausted other traditional treatments and perhaps be considered in early infectious corneal disease states.

Treatment outcomes in the DRy Eye Amniotic Membrane (DREAM) study.

PurposeTo evaluate the efficacy of cryopreserved amniotic membrane (CAM) in reducing signs and symptoms of dry eye disease (DED) in a large patient population.MethodsA retrospective chart review at 10 clinical sites was done of patients with refractory DED who received CAM and completed at least 3 months of follow-up. Data collected were demographics; medical history including previous and current ocular treatment, diagnosis, clinical presentations, comorbidity, duration and frequency of treatment with CAM; and concomitant medications. The primary outcome was the change in dry eye workshop (DEWS) score after treatment.ResultsA total of 97 eyes of 84 patients exhibited severe dry eye despite maximal medical treatments including topical artificial tears, cyclosporine-A, serum, antibiotics, and steroids. Patients manifested with superficial punctate keratitis (86%), filamentary keratitis (13%), exposure keratitis (19%), neurotrophic keratitis (2%), and corneal epithelial defect (7%). After CAM treatment for 5.4±2.8 days, 74 (88%) patients demonstrated an improved ocular surface along with a notable reduction of the severity as the overall DEWS score was significantly reduced from 3.25±0.5 at baseline to 1.44±0.6 at 1 week, 1.45±0.6 at 1 month, and 1.47±0.6 at 3 months (p<0.001). Ten eyes (10%) required repeated treatment to complete healing. Apart from discomfort during CAM placement, there were no adverse events.ConclusionPlacement of CAM is promising to enhance the recovery of ocular surface health and reduce signs and symptoms in patients with moderate-to-severe DED.

In vitro anti-angiogenic effects of cryo-preserved amniotic membrane and the role of TIMP2 and thrombospondin

Abstract Background Amniotic membrane (AM) has been used as a biological substrate in ophthalmology and other fields for several years. It has a valuable role in ocular surface burns and non-healing ulcers. It is known that AM is non-immunogenic and has anti-inflammatory and anti-fibrotic properties and supports epithelial cell proliferation and migration. These are supposedly mediated through a host of growth factors and cytokines. Despite its clinically accepted role in reducing corneal vascularisation when applied on the eye, there have been contradicting reports on the effect of AM on angiogenesis. Methods The effect of soluble factors released from clinically prepared cryo-preserved AM on cultured human umbilical vein endothelial cell (HUVEC) proliferation was quantified and angiogenesis assessed on matrigel. Antibody blockage of soluble tissue inhibitors of metalloproteinase (TIMP)2 and Thrombospondin, identified on Searchlight protein arrays, was used to evaluate their role in the effect of cryo-preserved AM. Results AM conditioned medium (AMCM) was found to reduce HUVEC proliferation and angiogenesis. Angiogenesis was reduced by direct application of AM, a gradient effect was observed, where HUVEC closer to the AM failed completely in forming any tubules. In addition, HUVEC failed to survive directly on the AM. Analysis of the soluble factors released by the amnion included high levels of anti-angiogenic factors, thrombospondin and TIMP 1 and 2. Antibody blockage of TIMP2 and thrombospondin prevented the anti-angiogenic effect of AMCM and AM. Conclusion AM in vitro has potent anti-angiogenic properties dependent on secreted TIMP2 and thrombospondin, validating some of the effects observed clinically.

Effect of Cryopreserved Amniotic Membrane on the Mechanical Properties of Skeletal Muscle after Strabismus Surgery in Rabbits

ABSTRACTPurpose: To study the functional recovery of the superior rectus muscle (SRM) after its partial resection in a rabbit model with and without cryopreserved amniotic membrane (AM).Material and methods: Resection of the right and left SRMs of 30 rabbits was performed. On the left eyes, a single sheet of equine cryopreserved AM was placed covering the muscle edge sutured. Active and passive mechanical properties of muscles operated with and without AM were monitored over time at 30 (n = 10), 60 (n = 10), and 90 (n = 10) days after surgery. Muscle samples were extracted and electrically stimulated to register the force exerted by the samples, characterizing its active behavior. They were, then, subjected to stretching test to obtain its resistance to deformation, known as passive behavior. Moreover, right and left eyes of a control group (n = 5) were equally subjected to active and passive tests to characterize the physiological behavior of SRM muscles.Results: On active function examination, statistically significant differences were documented between the following: control vs AM and no AM at 30 days (p = 0.002 and p = 0.04, respectively). All other comparisons were insignificant (p > 0.05). On passive function analysis, significant differences were only found between control vs. no AM at 30 days (p = 0.004) and between AM vs. no AM at 30 days (p = 0.002). Indeed, muscle operated without AM did not recover a normal passive function until 60 days after surgery.Conclusion: Cryopreserved AM is effective in accelerating recovery of SRM passive function in rabbits. Nevertheless, AM produced no significant effect on recovery of SRM active function.

Short-term in vivo morphological changes of amniotic membrane after fibrin glue-assisted pterygium surgery on anterior segment optical coherence tomography: a case presentation

BackgroundThe evaluations of morphological changes of amniotic membrane (AM), even after successful AM transplantation surgery without complications, may be difficult. Moreover, there was no report regarding morphological changes after fibrin glue-assisted AM transplantation with pterygium excision. Here, we highlight and describe the use of spectral domain optical coherence tomography (OCT) for the evaluation of the morphological features of amniotic membrane (AM) and of associated in vivo structural changes after fibrin glue-assisted pterygium surgery.Case presentationAll three patients underwent cryo-preserved AM transplantation using the permanent inlay technique (epithelial side up) with fibrin glue. In vivo morphological changes of AMs were evaluated using a spectral domain OCT equipped with an anterior segment imaging module (RTVue-100, Optovue, Inc., Fremont, CA, USA). Anterior segment OCT examinations demonstrated morphological changes, that is, re-absorption of fibrin glue or subconjunctival hemorrhage, migration of epithelium, and integration of AM into sclera, of AMs over first postoperative months.ConclusionsAnterior segment OCT might provide additional structural information, including quantitative and qualitative data, on AMs after pterygium surgery as compared with conventional slit-lamp examination.

Grafting with Cryopreserved Amniotic Membrane versus Conservative Wound Care in Treatment of Pressure Ulcers: A Randomized Clinical Trial.

To compare the healing process of pressure ulcers treated with cryopreserved human amniotic membrane allograft and routine pressure ulcer care in our hospital. From January 2012 to December 2013, in a prospective randomized clinical trial (IRCT201612041335N2), 24 patients with second and third stage of pressure ulcers were enrolled in this study. All patients needed split-thickness skin grafts for pressure ulcer-wound coverage. Selected patients had symmetric ulcers on both upper and lower extremities. The patients were randomly divided into two groups: amnion and control. In the amnion group, the ulcer was covered with cryopreserved amniotic membrane and in the control group it was treated with local Dilantin powder application. The duration and success rate of complete healing was compared between the two groups. The study group was composed of 24 pressure ulcers in 24 patients (19 males and 5 females) with a mean age of 44±12.70 years. The demographic characteristics, ulcer area, and underlying diseases were similar in both groups. The early sign of response, such as decrease in wound discharge, was detected 12-14 days after biological dressing. Complete pressure ulcer healing occurred only in the amnion group (p< 0.001). Partial healing was significantly higher in the amnion group (p< 0.03). Healing time in this group was faster than that the control group (20 days versus 54 days). No major complication was recorded with amniotic membrane dressing. Cryopreserved amniotic membrane is an effective biologic dressing that promotes re-epithelialization in pressure ulcers.

Grafting with Cryopreserved Amniotic Membrane versus Conservative Wound Care in Treatment of Pressure Ulcers: A Randomized Clinical Trial

To compare the healing process of pressure ulcers treated with cryopreserved human amniotic membrane allograft and routine pressure ulcer care in our hospital.From January 2012 to December 2013, in a prospective randomized clinical trial (IRCT201612041335N2), 24 patients with second and third stage of pressure ulcers were enrolled in this study. All patients needed split-thickness skin grafts for pressure ulcer-wound coverage. Selected patients had symmetric ulcers on both upper and lower extremities. The patients were randomly divided into two groups: amnion and control. In the amnion group, the ulcer was covered with cryopreserved amniotic membrane and in the control group it was treated with local Dilantin powder application. The duration and success rate of complete healing was compared between the two groups.The study group was composed of 24 pressure ulcers in 24 patients (19 males and 5 females) with a mean age of 44±12.70 years. The demographic characteristics, ulcer area, and underlying diseases were similar in both groups. The early sign of response, such as decrease in wound discharge, was detected 12-14 days after biological dressing. Complete pressure ulcer healing occurred only in the amnion group (p< 0.001). Partial healing was significantly higher in the amnion group (p< 0.03). Healing time in this group was faster than that the control group (20 days versus 54 days). No major complication was recorded with amniotic membrane dressing.Cryopreserved amniotic membrane is an effective biologic dressing that promotes re-epithelialization in pressure ulcers.

Corneal Nerve Regeneration after Self-Retained Cryopreserved Amniotic Membrane in Dry Eye Disease.

Purpose To evaluate the efficacy of self-retained cryopreserved amniotic membrane (CAM) in promoting corneal nerve regeneration and improving corneal sensitivity in dry eye disease (DED). Methods In this prospective randomized clinical trial, subjects with DED were randomized to receive CAM (study group) or conventional maximum treatment (control). Changes in signs and symptoms, corneal sensitivity, topography, and in vivo confocal microscopy (IVCM) were evaluated at baseline, 1 month, and 3 months. Results Twenty subjects (age 66.9 ± 8.9) were enrolled and 17 completed all follow-up visits. Signs and symptoms were significantly improved in the study group yet remained constant in the control. IVCM showed a significant increase in corneal nerve density in the study group (12,241 ± 5083 μm/mm2 at baseline, 16,364 ± 3734 μm/mm2 at 1 month, and 18,827 ± 5453 μm/mm2 at 3 months, p = 0.015) but was unchanged in the control. This improvement was accompanied with a significant increase in corneal sensitivity (3.25 ± 0.6 cm at baseline, 5.2 ± 0.5 cm at 1 month, and 5.6 ± 0.4 cm at 3 months, p < 0.001) and corneal topography only in the study group. Conclusions Self-retained CAM is a promising therapy for corneal nerve regeneration and accelerated recovery of the ocular surface health in patients with DED. The study is registered at clinicaltrials.gov with trial identifier: NCT02764814.

Sutureless cryopreserved amniotic membrane graft and wound healing after photorefractive keratectomy

To evaluate the effect of sutureless cryopreserved amniotic membrane (Prokera) on corneal wound healing after photorefractive keratectomy (PRK). Center for Refractive Surgery, Walter Reed Army Medical Center, Washington, DC, USA. Prospective nonrandomized control trial. Patients had PRK for myopia with or without astigmatism. A 20% ethanol solution was used to create a standard 9.0 mm epithelial defect followed by photoablation with the Allegretto Wave Eye-Q 400 Hz laser. After surgery, a high-oxygen-transmissible bandage contact lens (Acuvue Oasys) was applied on the dominant eye and cryopreserved amniotic membrane on the nondominant eye. The postoperative regimen was otherwise identical for both eyes. Postoperatively, patients were evaluated daily until complete corneal reepithelialization occurred in both eyes and then at 2 weeks and 1, 3, 6, and 12 months. Reepithelialization was assessed daily with slitlamp examination, fluorescein staining, and photography. Secondary outcome measures included adverse effects, ocular comfort, visual outcomes, and corneal haze. Forty patients were enrolled. The amniotic membrane graft sped corneal reepithelialization 1 day after PRK but was not better than the bandage contact lens in hastening complete reepithelialization of the cornea. Visual outcomes, corneal clarity, and optical quality of the cornea were comparable between the amniotic membrane graft eyes and bandage contact lens eyes. Although the amniotic membrane graft was reasonably well tolerated with few significant adverse effects, the role of amniotic membrane in modulating wound healing after PRK remains speculative.

Accelerated Restoration of Ocular Surface Health in Dry Eye Disease by Self-Retained Cryopreserved Amniotic Membrane

PurposeTo evaluate the clinical efficacy of self-retained cryopreserved amniotic membrane in treating dry eye disease. MethodsRetrospective review of 10 patients treated with self-retained cryopreserved amniotic membrane (PROKERA® Slim [PKS], Bio-Tissue, Miami, FL) for moderate-to-severe dry eye refractory to conventional maximal medical treatments. Patients' symptoms, use of medications, conjunctival inflammation, corneal staining, and visual acuity were compared before and after treatment. ResultsPKS was placed in 15 eyes of the 10 patients for 4.9 ± 1.5 days. All patients experienced symptomatic relief for a period of 4.2 ± 4.7 months (P<.001). Such improvement was accompanied by reduction of OSDI scores (P<.001), use of topical medications (P<.001), conjunctival hyperemia (P<.001), corneal staining (P<.001), and improvement of the visual acuity (P=.06). Linear regression analysis estimated that the optimal duration of PKS placement was 5 days to achieve an average symptom-free duration of 4 months in patients with dry eye. Surprisingly, PKS placement also generated improvement in the contralateral eyes. ConclusionThis pilot study suggests that self-retained cryopreserved amniotic membrane via PKS can be used to treat moderate dry eye diseases and warrants further prospective controlled studies.

A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.